Characterisation of the Shigella flexneri · Abstract iii Abstract Shigella flexneri is the major causative agent of shigellosis that account for ~14000 deaths annually in Asia. The

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Characterisation of the Shigella flexneri

O Antigen Polymerase Wzy

Pratiti Nath M.Sc, B.Sc (Honours)

Submitted for the degree of Doctor of Philosophy

Department of Molecular and Cellular Biology

School of Biological Sciences

The University of Adelaide

Adelaide, South Australia, Australia

May, 2015

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This thesis is dedicated to my parents Mr Pradip Kumar Nath and Ms Sumita Nath, my

husband Dr Arindam Dey, and my brother Prabreesh for their love, support, and

encouragement

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Declaration

i

Declaration

I certify that this work contains no material which has been accepted for the award of any

other degree or diploma in my name, in any university or other tertiary institution and, to the

best of my knowledge and belief, contains no material previously published or written by

another person, except where due reference has been made in the text. In addition, I certify

that no part of this work will, in the future, be used in a submission in my name, for any other

degree or diploma in any university or other tertiary institution without the prior approval of

the University of Adelaide and where applicable, any partner institution responsible for the

joint-award of this degree.

I give consent to this copy of my thesis when deposited in the University Library,

being made available for loan and photocopying, subject to the provisions of the Copyright

Act 1968.

The author acknowledges that copyright of published works contained within this

thesis resides with the copyright holder(s) of those works.

I also give permission for the digital version of my thesis to be made available on the

web, via the University’s digital research repository, the Library Search and also through web

search engines, unless permission has been granted by the University to restrict access for a

period of time.

_________________________

Pratiti Nath

May, 2015

ii

Abstract

iii

Abstract

Shigella flexneri is the major causative agent of shigellosis that account for ~14000 deaths

annually in Asia. The O antigen (Oag) component of S. flexneri lipopolysaccharide (LPS) is

important for virulence and a protective antigen. It is synthesised by a Wzy-dependent

mechanism. S. flexneri Wzy (WzySf) has 12 transmembrane (TM) segments and two large

periplasmic loops (PL). The modal chain length of the Oag is determined by chromosomally

encoded WzzSf and pHS-2 plasmid encoded WzzpHS2. Although WzySf was identified 20 years

ago, there is a lack of knowledge about its functional amino acid residues as WzySf has low

expression and poor detection. WzySf is thought to interact with WzzSf however, there is no

direct evidence on how these two proteins are associated.

A wzySf-gfp expression construct (pWaldo-wzySf-TEV-GFP or pRMPN1) was made; it

successfully expressed WzySf-GFP and complemented a wzySf mutant (!wzy). To identify

functionally important amino acid residues in WzySf, random mutagenesis was performed on

the wzySf in pRMPN1, followed by screening with colicin E2. Analysis of the LPS conferred

by mutated WzySf proteins in the !wzy strain identified 4 different mutant classes, with

mutations found in PL1, 2, 3, and 6; TM2, 4, 5, 7, 8, and 9, and cytoplasmic loop (CL) 1 and

CL5. The association of WzySf and WzzSf was investigated by transforming these mutated

wzySf plasmids into a wzySf and wzzSf deficient (!wzy !wzz) strain. Comparison of the LPS

profiles in the !wzy and !wzy !wzz backgrounds identified WzySf mutants whose

polymerisation activities were WzzSf dependent. Colicin E2 and bacteriophage Sf6c

sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the

WzySf-GFP mutants in the !wzy and !wzy !wzz backgrounds identified a role for WzzSf in

WzySf stability. Hence, in addition to its role in regulating Oag modal chain length, WzzSf also

affects WzySf activity and stability.

Site-directed mutagenesis was performed on wzySf in pRMPN1 to alter Arg residues in

WzySf’s two large PLs (3 and 5) to Ala. Analysis of the LPS profiles conferred by mutated

WzySf proteins in the !wzy strain identified residues that affect WzySf activity. The importance

of the guanidium group of the Arg residues was investigated by altering the Arg residues to

Abstract

iv

Lys and Glu, which generated WzySf mutants conferring altered LPS Oag modal chain

lengths. The dependence of these WzySf mutants on WzzSf was investigated by expressing

them in the !wzy !wzz strain. Comparison of the LPS profiles identified a role for the Arg

residues in the association of WzySf and WzzSf. Comparison of the expression levels of

different mutant WzySf-GFPs with the wild-type WzySf-GFP showed that certain Arg residues

affected production levels of WzySf in a WzzSf-dependent manner.

WzySf-GFP-His8 was purified by affinity chromatography and I propose that WzySf

may form dimers. The negative dominance study suggested that the dimer formation may be

not essential for functioning of WzySf. In vivo crosslinking was performed in a !wzy strain

carrying plasmids encoding His-tagged WzySf and untagged WzzSf. In vivo crosslinking was

followed by affinity purification of WzySf, and Western immunoblotting with WzzSf antibody

detected the co-purification of WzzSf. This was also supported by mass spectrometry analysis

and provided the first report of complex formation between WzySf and WzzSf. The WzySf

mutants (WzySfR164A, WzySfV92M, WzySfY137H, and WzySfR250K) having Wzz-

dependent activity were still able to form complexes with WzzSf which suggested that

although their activity is Wzz-dependent, the mutational alterations do not affect the

interaction of WzySf with WzzSf. Thus the interaction may involve many regions of WzySf.

This thesis identified and characterised functionally important amino acid residues of

WzySf, identified several novel LPS phenotypes conferred by the WzySf mutants, and found

that WzzSf affects the functioning and stability of WzySf, both positively and negatively. The

work also first time identified direct physical interaction of WzzSf and WzySf, and developed a

purification method for WzySf. Finally, I proposed a model of Wzy-dependent Oag

polymerisation through the interaction of Wzy with Wzz.

Acknowledgements

v

Acknowledgements

First and foremost, I would like to thank my supervisor Associate Professor Renato Morona. I

was fortunate to have him as my mentor. I would like to appreciate him for giving me such a

wonderful project, and I really enjoyed all the challenges and opportunities came on my way.

He not only helped me to understand the research field, but also supported me throughout the

candidature. He has been a great source of inspiration. I have learned a lot from you Renato

and thank you for everything.

I would like to thank the University of Adelaide and School of Biological Sciences for

hosting me and supporting me with the scholarships and other facilities, which helped me to

conduct my research.

Throughout my doctoral study I have been fortunate to work with a number of talented

colleagues in Morona Laboratory. I would like to thank Dr Elizabeth Tran for answering

many questions, for sharing your knowledge on Shigella, and expertise on laboratory

techniques. I am thankful to Dr Stephen Attridge, Dr Alistair Standish, Matthew Doyle,

Jonathan Whittall, and Brad Qin for their help in numerous ways during my doctoral study. I

would also like to thank past and present members of Paton, Mcdevitt, and Kidd laboratories

for their support and making it such a great place to work.

I would like to take the opportunity to express my gratitude to all the teachers who

guided me from my primary school to the Masters degree and prepared me for this doctoral

study, especially Ms Mukti Basu, Mr. Rajen Mandal, Mr Amit Kumar Ghosh, Mr. Satipoti

Moitra, Dr Sharmila Chakraborty, Dr Saroj Kanti Das, and Dr Prajna Mandal. Their guidance,

care, and enthusiastic discussion helped me to pursue a research career.

I am grateful to all my friends from my childhood to now for their love, support, and

encouragement. Tania, Lipika, Mou, Santa, Jhimli, Aditi, Suhas, Tapashree, Swatilekha,

Sreetama, and Suman; thanks for making my life enjoyable and fun packed. My friends in

Adelaide have great impact on my PhD life. Without their constant company it would have

been hard to overcome the last two years of my PhD when my husband moved to interstate

for his work. Thanks to Sanchita for your company and the delicious meals cooked by you.

Thanks to Indrajit for all the parties. I would like to thank all my friends in the MLS building:

Acknowledgements

vi

Min, Mabel, Zuleeza, Donald, Alex, Long, Paul, Zarina, and Rethish. Thanks to Min, Mabel,

and Zuleeza for the dinner dates and shopping sprees. I will miss the weekend outing with

you girls. Thank you Donald for all the delicious cakes and snacks. Alex and Long thanks for

the friendly chatting after a stressful day whic helped me a lot. Paul and Mabel, you used to

annoy me by your dance moves but now I really miss it. Although I still don’t think it was

actually a dance. I had a great time in MLS building with all of you and hope I will see you all

soon.

I would like to thank my family in India; without their constant support and

encouragement it would have been very difficult for me to survive on a foreign land. My

mother was my first teacher and she did a lot of sacrifices to give me a better life. I can’t

imagine a moment when I needed her and she was not there. In any difficult time my dad is

always my strength, his belief in me and encouragement mean so much to me. He always

taught me to be a good human being. They are the best parents in this world and I think

saying thanks to them will be an immense disrespect; I owe my life to you. I would like to

thank my little brother Rick for bringing all the happiness and joy in my life. He was the best

entertainment for me in his childhood (and my teenage). Although he is naughty sometimes

but I can forgive him for all the fun we had together and the lovely memories we share. I

would like to thank my brother Biswajit for all his support, love, and guidance; and my

grandma for her friendship, care, and love. I would also like to thank my uncles (especially

Sejda), cousins, father-in-law, and brother-in-law for their love and support.

And Finally, I would like to thank the man of my life Dr Arindam Dey, my best

friend, my husband, and my partner in crime. It is hard to express your contribution in my life

in words. I will never forget that you used to fly more than 3 hours from Cairns to Adelaide

every fortnight and sometimes every week to spend few hours with me. Whenever I was

stressed you came to see me and you became financially broke due to the expensive flight

tickets. You are the best man for keeping a long distance relationship and without your

unconditional support it would have been hard for me to continue my study. Not only in

personal life, you also did a lot professionally for me. Thanks for proofreading all my drafts,

and your experience as a PhD student always guided me in my study. I am looking for an

exciting future together with challenges, learning, arguments, achievements, and

unconditional love.

Publications

vii

Publications

Nath, P. & Morona, R. (2015). Detection of Wzy/Wzz interaction in Shigella flexneri.

Microbiology (In press) Published online 09 July, 2015, doi:10.1099/mic.0.000132

(Chapter 5)

Nath, P. & Morona, R. (2015). Mutational analysis of the major periplasmic loops of Shigella

flexneri Wzy: identification of the residues affecting O antigen modal chain length control,

and Wzz-dependent polymerization activity. Microbiology 161, 774-785.

(Chapter 4)

Nath, P., Tran, E. N. & Morona, R. (2015). Mutational Analysis of the Shigella flexneri O-

Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants. J Bacteriol 197,

108-119.

(Chapter 3)

viii

Contents

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Contents

Declaration …………………………………………………………………….. i

Abstract………………………………………………………………………… iii

Acknowledgements…………………………………………………………….. v

Publications…………………………………………………………………… vii

Abbreviations…………………………………………………………………. xix

Chapter 1: Introduction ...................................................................................... 1

1.1 Indroduction ...................................................................................................................... 2

1.2 Shigella spp. ...................................................................................................................... 2

1.3 Shigellosis and epidemiology ........................................................................................... 3

1.4 Pathogenesis ...................................................................................................................... 4

1.5 Virulence factors ............................................................................................................... 4

1.5.1 Large virulence plasmid (VP) .................................................................................... 6

1.5.2 Mxi-Spa type III secretion system (TTSS) ................................................................. 6

1.5.3 Effector proteins secreted by TTSS ............................................................................ 7

1.5.4 IcsA protein ................................................................................................................ 8

1.6 Lipopolysaccharide (LPS) ................................................................................................. 9

1.6.1 Morphology .............................................................................................................. 10

1.6.1.1 Lipid A………………………………………………………………………10

1.6.1.2 Core sugar…………………………………………………………………...13

1.6.1.3 O antigen (Oag)……………………………………………………………...13

1.6.2 LPS as a virulence factor .......................................................................................... 19

1.7 LPS biosynthesis and export ........................................................................................... 22

1.7.1 Lipid A and core biosynthesis .................................................................................. 23

1.7.2 O antigen biosynthesis .............................................................................................. 24

1.7.3 LPS export ................................................................................................................ 26

1.8 Oag polymerisation protein Wzy .................................................................................... 29

1.8.1 S. flexneri Wzy (WzySf) ............................................................................................ 30

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1.8.2 Wzy proteins of different bacterial species .............................................................. 30

1.8.3 Comparison of WzySf with other Wzy proteins ....................................................... 33

1.9 Oag chain length regulator Wzz ...................................................................................... 34

1.9.1 S. flexneri Wzz ......................................................................................................... 34

1.9.2 Chain length and virulence ....................................................................................... 37

1.10 Association of the proteins of the Oag biosynthesis pathway ...................................... 38

1.11 Aims and hypotheses .................................................................................................... 39

1.12 Thesis Organisation ....................................................................................................... 40

Chapter 2: Materials and Methods .................................................................. 43

2.1 Bacterial strains and plasmids ......................................................................................... 44

2.2 Bacterial growth media and growth condition ................................................................ 44

2.2.1 Liquid growth media ................................................................................................ 44

2.2.2 Solid growth media .................................................................................................. 44

2.3 Chemicals and reagents ................................................................................................... 53

2.4 Antibodies and antisera ................................................................................................... 53

2.5 Nucleic acid methods ...................................................................................................... 53

2.5.1 Isolation of plasmid DNA and DNA preparation ..................................................... 53

2.5.2 Quantitation of DNA ................................................................................................ 54

2.5.3 Restriction enzyme digestion ................................................................................... 54

2.5.4 Agarose gel electrophoresis ...................................................................................... 54

2.5.5 DNA sequencing ...................................................................................................... 54

2.5.5.1 Sample preparation…………………………………………………………..54

2.5.5.2 Capillary separation DNA sequencing……………………………………….55

2.5.5.3 Sequencing analysis………………………………………………………….55

2.6 Polymerase chain reaction (PCR) ................................................................................... 55

2.7 DNA purification ............................................................................................................ 58

2.7.1 DNA gel extraction .................................................................................................. 58

2.7.2 Purification of PCR products ................................................................................... 58

2.8 Ligation of DNA fragments into cloning vectors ........................................................... 58

2.9 Transformation ................................................................................................................ 59

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2.9.1 Preparation of chemically competent cells ............................................................... 59

2.9.2 Heat shock transformation ........................................................................................ 59

2.9.3 Preparation of electrocompetent cells ...................................................................... 59

2.9.4 Electroporation ......................................................................................................... 60

2.10 Mutagenesis .................................................................................................................. 60

2.10.1 Random mutagenesis .............................................................................................. 60

2.10.2 Site-directed mutagenesis ....................................................................................... 61

2.11 Characterisation of mutants ........................................................................................... 61

2.11.1 Colicin sensitivity assay ......................................................................................... 61

2.11.1.1 ColE2 swab assay…………………………………………………………61

2.11.1.2 ColE2 spot assay………………………………………………………….61

2.11.2 Bacteriophage sensitivity assay .............................................................................. 62

2.12 Protein Techniques ........................................................................................................ 62

2.12.1 Preparation of whole cell lysate ............................................................................. 62

2.12.2 Preparation of whole membrane fraction ............................................................... 63

2.12.3 SDS-PAGE ............................................................................................................. 63

2.12.4 In-gel fluorescence ................................................................................................. 64

2.12.5 Coomassie blue staining ......................................................................................... 64

2.12.6 Western immunoblotting ........................................................................................ 64

2.12.7 Over-expression of WzySf-GFP-His8 ..................................................................... 65

2.12.8 Purification of His-tagged membrane protein ........................................................ 65

2.12.9 In vivo chemical crosslinking ................................................................................. 66

2.12.10 Co-purification or pull down ................................................................................ 66

2.12.11 Proteomics analysis .............................................................................................. 67

2.13 Lipopolysaccharide (LPS) Techniques ......................................................................... 68

2.13.1 Preparation of LPS samples ................................................................................... 68

2.13.2 Analysis of LPS by silver-stained SDS-PAGE ...................................................... 68

Chapter 3: Mutational analysis of the Shigella flexneri O antigen polymerase Wzy;

Identification of Wzz-dependent Wzy mutants ................................................ 71

Title page…………………………………………………………………………………..72

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Statement of authorship……………………………………………………………………73

3.1 Abstract ........................................................................................................................... 74

3.2 Introduction ..................................................................................................................... 74

3.3 Materials and Methods .................................................................................................... 77

3.3.1 Bacterial strains and plasmids .................................................................................. 77

3.3.2 Growth media and growth conditions ...................................................................... 77

3.3.3 Construction of expression vector and cloning of wzySf ........................................... 82

3.3.4 WzySf expression in Lemo21(DE3) and In-gel fluorescence ................................... 83

3.3.5 LPS method .............................................................................................................. 83

3.3.6 Random mutagenesis ................................................................................................ 84

3.3.7 Construction of the strain RMA4437 ("wzy "wzz) .................................................. 84

3.3.8 Detection of WzySf expression in S. flexneri ............................................................ 85

3.3.9 Colicin sensitivity assay ........................................................................................... 85

3.3.9.1 Colicin swab assay…………………………………………………………...85

3.3.9.2 ColE2 spot assay……………………………………………………………..86


3.4 Results ............................................................................................................................. 87

3.4.1 Construction of a WzySf-GFP expression plasmid ................................................... 87

3.4.2 Complementation of wzySf deficiency ...................................................................... 87

3.4.3 Random mutagenesis of wzySf .................................................................................. 89

3.4.4 LPS phenotype conferred by WzySf mutants ............................................................ 93

3.4.5 WzzSf dependence .................................................................................................... 93

3.4.6 ColE2 and bacteriophage Sf6c sensitivities ............................................................. 96

3.4.7 WzySf expression level ............................................................................................. 98

3.5 Discussion ....................................................................................................................... 98

3.6 Acknowledgements ....................................................................................................... 105

3.7 Supplementary information ........................................................................................... 106

3.7.1 Construction of pWaldo-wzySf-GFP-His8 ............................................................... 114

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Chapter 4: Mutational analysis of the major periplasmic loops of Shigella flexneri

Wzy: identification of the residues affecting O antigen modal chain length control, and

Wzz dependent polymerisation activity ........................................................... 119

Title page……………………………………………………………………………….….120

Statement of authorship…………………………………………………………………..121

4.1 Abstract ......................................................................................................................... 122

4.2 Introduction ................................................................................................................... 122

4.3 Materials and Methods .................................................................................................. 125

4.3.1 Bacterial strains and plasmids ................................................................................ 125

4.3.2 Growth media and growth conditions .................................................................... 125

4.3.3 LPS method ............................................................................................................ 125

4.3.4 Site-directed mutagenesis ....................................................................................... 128

4.3.5 Detection of WzySf expression in S. flexneri .......................................................... 128

4.3.6 Colicin sensitivity assay ......................................................................................... 129


4.4 Results ........................................................................................................................... 129

4.4.1 Site-directed mutagenesis of Arg residues in PL3 and PL5 of WzySf .................... 129

4.4.2 LPS phenotype conferred by the WzySf mutants .................................................... 131

4.4.3 WzzSf dependence and polymerisation activity ...................................................... 135

4.4.4 ColE2 sensitivity of strains with WzySf mutants .................................................... 136

4.4.5 Bacteriophage Sf6c sensitivity of strains with WzySf mutants ............................... 139

4.4.6 Protein expression levels of the WzySf mutants ..................................................... 140

4.5 Discussion ..................................................................................................................... 143



Chapter 5: Detection of Wzy/Wzz interaction in Shigella flexneri ............. 153

Title page…………………………………………………………………………… 154

Statement of authorship…………………………………………………………….. 155

5.1 Abstract ......................................................................................................................... 156

5.2 Introduction ................................................................................................................... 156

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5.3 Materials and Methods .................................................................................................. 158

5.3.1 Ethics Statement ..................................................................................................... 158

5.3.2 Bacterial strains, growth media and growth conditions ......................................... 158

5.3.3 Plasmids and DNA methods ................................................................................... 159

5.3.4 Purification of Wzy from Lemo21(DE3) strain ..................................................... 161

5.3.5 In vivo protein cross-linking ................................................................................... 161

5.3.6 Isolation of Wzy from S. flexneri strains ................................................................ 162

5.3.7 SDS-PAGE and Western Immunoblotting ............................................................. 162

5.3.8 In-gel fluorescence ................................................................................................. 163

5.3.9 Liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-

ESI-MS/MS) .................................................................................................................... 163

5.4 Results ........................................................................................................................... 164

5.4.1 Purification of Wzy-GFP-His8 from Lemo21(DE3) ............................................... 164

5.4.2 Detection of complex formation between Wzy and Wzz by immunoblotting ....... 166

5.4.3 MS analysis of protein samples following DSP cross-linking ............................... 169

5.4.4 Analysis of the Wzy mutants with Wzz dependent properties ............................... 173

5.5 Discussion ..................................................................................................................... 175



5.7.1 Optimisation of WzySf-GFP-His8 purification ...................................................... 179

5.7.1.1 Cell fractionation…………………………………………………………...179

5.7.1.2 Optimisation of whole membrane fraction solubilisation………………….179

5.7.1.3 Metal affinity putification of WzySf-GFP-His8………………………….....181

5.7.2 Negative dominance ............................................................................................... 181

Chapter 6: Conclusion .................................................................................... 185

6.1 Introduction ....................................................................................................................... 186

6.2 Residues important for polymerisation function of WzySf ........................................... 187

6.3 Purification of WzySf ..................................................................................................... 188

6.4 Understanding the association of the Oag biosynthesis proteins .................................. 190

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6.5 Mechanism of the association of Wzz and Wzy during the O antigen polymerisation in

S. flexneri ............................................................................................................................ 192

6.6 Conclusion and future work .......................................................................................... 195

Bibliography…………………………………………………………………... 197

Contents

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List of Figures

Figure 1.1 S. flexneri pathogenesis ............................................................................................. 5!Figure 1.2 LPS structural types ................................................................................................ 11!Figure 1.3 S. flexneri Lipid A structure .................................................................................... 12!Figure 1.4 S. flexneri 2a core .................................................................................................... 14!Figure 1.5 Chemical composition of the Oag of different S. flexneri serotypes ...................... 16!Figure 1.6 Synthesis of R3 type LPS core sugar ...................................................................... 25!Figure 1.7 Organisation of S. flexneri Oag biosynthesis genes ................................................ 27!Figure 1.8 S. flexneri Y serotype Oag biosynthesis .................................................................. 28!Figure 1.9 S. flexneri Wzy (WzySf) .......................................................................................... 31!Figure 1.10 S. flexneri modal chain length ............................................................................... 35!Figure 3.1 Complementation of wzySf deficiency by WzySf-GFP ............................................. 88!Figure 3.2 Locations of mutations on the topology map of WzySf ........................................... 90!Figure 3.3 LPS phenotypes conferred by different WzySf mutants expressed in PNRM6

[!wzySf (pAC/pBADT7-1)] ....................................................................................................... 94!Figure 3.4 Comparison of the LPS phenotypes conferred by the WzySf mutants expressed in

the !wzy and !wzy !wzz backgrounds ..................................................................................... 95!Figure 3.5 Protein expression levels of the mutated WzySf-GFP compared to the positive

control ....................................................................................................................................... 99

Figure 3.S1 WzySf expression……………………………………………………………….110

Figure 3.S2 LPS phenotype conferred by the class D WzySf mutants expressed in PNRM6

[!wzySf (pAC/pBADT7-1)]………………………………………………………………….111

Figure 3.S3 Comparison of the LPS phenotype conferred by the class D WzySf mutants

expressed in the !wzy and !wzy !wzz backgrounds……………………………………… 112

Figure 3.S4 Construction of pWaldo-wzySf-GFP-His8 (pRMPN1) plasmid……………… 115

Figure 3.S5 DNA and predicted amino acid sequence of the inserted WzySf sequence in

pWaldo-TEV-GFP plasmid…………………………………………………………….… 116

Figure 4.1 Location of the mutations constructed in this study on the topology map of WzySf

................................................................................................................................................ 130!

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Figure 4.2 Comparison of the LPS phenotype conferred by the WzySf mutants expressed in the

"wzy and "wzy "wzz backgrounds ......................................................................................... 132!Figure 4.3 Protein expression level of the WzySf-GFP mutants ............................................. 141!Figure 5.1 Purification of Wzy-GFP-His8 .............................................................................. 165!Figure 5.2 In vivo cross-linking with DSP .............................................................................. 167!Figure 5.3 Analysis of protein bands by MS .......................................................................... 170!Figure 5.4 Chemical cross-linking of WzySf mutants ............................................................. 174!Figure 5.S1 Topology map of WzySf ...................................................................................... 178

Figure 5.S2 Optimisation of the whole membrane solubilisation………………………….. 180

Figure 5.S3 Negative domonance………………………………………………………….. 182

Figure 6.1 “Activation and inactivation” mechanism ............................................................. 194!

Contents

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List of Tables

Table 1.1 Antigenic determinants of various S. flexneri serotypes .......................................... 18!Table 1.2 Overview of the chapters in this PhD thesis ............................................................. 41!Table. 2.1 Bacterial strains used in this study ........................................................................ 45!Table 2.2 Plasmids used in this study ...................................................................................... 50!Table 2.3 Differenet primers made in this study ...................................................................... 56!Table 3.1 Bacterial strains and plasmids used in this study ..................................................... 78!Table 3.2 ColE2 and bacteriophage Sf6c sensitivities and WzySf-GFP expression of controls

and different classes of mutants ................................................................................................ 91

Table 3.S1 Different primers made in this study…………………………………………....106

Table 3.S2 Colicin E2 (ColE2) sensitivity (performed by swab assay) of different WzySf

mutants during screening……………………………………………………………………107

Table 3.S3 Nucleotide base change in the WzySf mutants………………………………….108

Table 3.S4 Verifications of WzySf topological model using topological prediction

programs…………………………………………………………………………………….109

Table 4.1 Bacterial strains and plasmids used in this study ................................................... 126!Table 4.2 LPS profiles of different WzySf mutant phenotypic classes ................................... 134!Table 4.3 ColE2 and bacteriophage Sf6c sensitivities, and WzySf-GFP expression levels .... 137

Table 4.S1 Primers used in this study……………………………………………………….149

Table 4.S2 Periplasmic loop (PL)3 and PL5 of WzySf……………………………………...151

Table 5.1 Bacterial strains and plasmids used in this study ................................................... 160!Table 5.2 Detected peptides by MS analysis .......................................................................... 172!Table 6.1 Comparison of WzySf and WzyPa ............................................................................ 189!Table 6.2 LPS profiles of the Wzz-dependent WzySf mutants in the presence and absence of

WzzSf ....................................................................................................................................... 191!

Abbreviations

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Abbreviations

Abbreviations

~ Approximately

ABC ATP-binding cassette

ABM actin based motility

ACN Acetonitrile

ACP acyl carrier protein

Amp Ampicillin

"-Me "-mercaptoethanol

Bp base pairs

CL cytoplasmic loop

Cm Chloramphenicol

ColE2 colicin E2

DDM n-Dodecyl-"-D-maltopyranoside

DNA deoxyribonucleic acid

dNTP deoxynucleoside triphosphate

DSP dithiobis(succinimidyl propionate)

dTDP-Rha deoxythymidine diphosphate rhamnose

DTT dithiothreitol

EDTA ethylenediaminetetraacetic acid

FA formic acid

Gal galactose

Abbreviations

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xx

GFP green fluorescent protein

GI gastrointestinal

Glc glucose

GlcN glucosamine

GlcNAc N-acetylglucosamine

GlcNAc-1-P GlcNAc-phosphate

GMMA generalised modules for membrane antigens

h hour (s)

Hep L-glycero-D-manno-heptose

His8 8x histidine

HRP horseradish-peroxidase

Iap inhibitor of # polymerase

IL interleukin

IM inner membrane

IPTG isopropyl-"-D-thiogalactopyranoside

IS insertion sequence

kb kilobase pairs

kDa kilodaltons

Kdo 3-deoxy-D-manno-oct-2-ulosonic acid

Km kanamycin

LB lysogeny broth

LC-ESI-MS/MS Liquid chromatography-electrospray ionisation tandem

mass spectrometry

Abbreviations

!

xxi

LDAO Lauryldimethylamine-oxide

LPS lipopolysaccharides

Lpt LPS transport

M cells membranous epithelial cells

min minute (s)

MS mass spectrometry

Mxi-Spa membrane expression of Ipa proteins and surface

presentation of antigens

NEB New England Biolabs

Ni-NTA nickel-charged agarose

N-WASP neural Wiskott-Aldrich syndrome protein

Oag O antigen

OD optical density

OD600 OD at 600 nm

OM outer membrane

PAI (s) pathogenicity island (s)

PBS phosphate buffered saline

PCP polysaccharide co-polymerase

PCR polymerase chain reaction

PEtN phosphoethanolamine

p.f.u. plaque-forming units

PLs periplasmic loops

PMNs polymorphonuclear cells

Abbreviations

!

xxii

Rha rhamnose

Rif rifampicin

R-LPS rough LPS

RNA ribonucleic acid

RNase ribonuclease

rpm revolutions per minute

RT room temperature

RU(s) repeat unit(s)

Sarkosyl sodium lauroyl sarcosine

SDS sodium dodecyl sulphate

SDDS sodium dodecanoyl sarcosine

SDS-PAGE SDS polyacrylamide gel electrophoresis

Sec seconds

S-LPS smooth LPS

SR-LPS semi rough LPS

S-type short type

TBE tris-borate-EDTA

TBS tris buffered saline

TEMED N,N,N',N'-Tetramethyl-ethylenediamine

Tc or Tet tetracycline

TMs transmembrane segments

Tris tris (hydroxymethyl) aminomethane

TTBS tween tris buffered saline

Abbreviations

!

xxiii

TTSS type III secretion system

UDP uridine diphosphate

Und-P undecaprenol phosphate

V volts

VL-type very long type

VP virulence plasmid

WM whole membrane

WT wild type

WzyEc Escherichia coli O86 Wzy protein

wzyFt Francisella tularensis LVS wzy gene

WzyFt F. tularensis LVS Wzy protein

WzyPa Pseudomonas aeruginosa PAO1 Wzy protein

WzySf Shigella flexneri Wzy protein

wzySf S. flexneri wzy gene

WzzpHS2 pHS2 plasmid encoded S. flexneri Wzz protein

WzzSf Chromosomaly encoded S. flexneri Wzz protein

X-Gal 5-bromo-4-chloro-3-indolyl-"-D-galactoside

!

xxiv

1

Acknowledgements

Chapter 1

Introduction

Introduction

2

Introduction

1.1 Indroduction

Shigella spp. are well adapted human pathogens and are responsible for the disease

Shigellosis. The resistance of Shigella spp. to antibacterials is prevalent and rising (Fulla et

al., 2005). There is no available vaccine against Shigella spp. (Stagg et al., 2009).

Lipopolysaccharide (LPS) is one of the important virulence factors of Shigella and contains O

antigen (Oag), the serotype determinant and protective antigen (Raetz & Whitfield, 2002;

Yethon & Whitfield, 2001). Immunity against Shigella is serotype specific (Liu et al., 2008;

Wang et al., 2010a; Yethon & Whitfield, 2001). Oag is synthesised by a Wzy-dependent

pathway (Woodward et al., 2010). Alarmingly, new serotypes of the bacteria are emerging

(Sun et al., 2012). Understanding the mechanism behind the synthesis of Oag will be helpful

to achieve control over Shigella infection by vaccine and drug development.

This chapter reviews S. flexneri LPS and Oag as a bacterial virulence determinant, and

the roles of Wzy and other proteins in the Oag biosynthesis pathway.

1.2 Shigella spp.

Shigella spp. are Gram-negative facultative intracellular bacteria and are members of the

family Enterobacteriaceae. They are bacilli in shape, and the causative agent of the bacillary

dysentery which is also called shigellosis (Sansonetti, 2001). There are four identified species

of Shigella: S. dysenteriae (Group A), S. flexneri (Group B), S. boydii (Group C), and S.

sonnei (Group D). Each species is further subdivided due to the variation in the chemical

composition of the Oag (Lan & Reeves, 2002; Larue et al., 2009; Parsot, 2005; Simmons,

1993). Comparative genomic analysis showed that sequence divergence between S. flexneri

and Escherichia coli K-12 is roughly 1.5% and Shigella is a subtype of E. coli (Lan &

Reeves, 2002).

The convergent evolution in Shigella spp. occurred by loss of catabolic genes, flagella,

fimbriae, and acquisition of a large 200 kb virulence plasmid (VP), pathogenicity islands

(PAIs), and genes essential for modifying the LPS Oags (Al Mamun et al., 1996; Buchrieser

et al., 2000; Ingersoll et al., 2002; Jin et al., 2002; Maurelli et al., 1998; Pupo et al., 2000;

Introduction

3

Rajakumar et al., 1997; Tominaga et al., 2005; Yang et al., 2005). Loss of catabolic genes,

flagella and fimbriae occurred to facilitate the adaptation to an intracellular lifestyle (Al

Mamun et al., 1996; Tominaga et al., 2005; Yang et al., 2005) and the intracellular movement

and cell-to-cell spreading of Shigella are facilitated by actin-based motility (ABM) that

exploits host factors.

1.3 Shigellosis and epidemiology

Shigellosis is an invasive disease of the lower gastrointestinal tract (GI) and it is characterised

by several symptoms including short term watery diarrhoea, bloody diarrhoea with mucoid

faeces, fever, fatigue, headaches, intestinal cramps, and severe inflammatory bowel disease

(Ashkenazi et al., 1990; Sansonetti, 2001). Shigellosis is generally a self-limiting disease but

sometimes it can create life-threatening conditions (Koster et al., 1978; Niyogi, 2005). The

rate of incidence of shigellosis is higher in developing countries (von Seidlein et al., 2006). A

recent review of literature (1990-2009) by Bardhan et al. (2010) indicated that nearly 125

million shigellosis cases occur annually in Asia, with approximately 14,000 fatalities; the

majority of deaths occur in children under 5 years of age (Bardhan et al., 2010).

Overcrowding, lack of potable water, malnutrition, cost of antibiotics, and poor sewage

management in developing countries are mainly responsible for the high incidence of

shigellosis (Hale, 1991; Jennison & Verma, 2004). S. dysenteriae is mainly responsible for

epidemic shigellosis but in endemic areas infection by any subtype of Shigella can be fatal

(Bennish, 1991). However, S. flexneri is the predominant subtype in endemic areas. In the

developing countries, the predominant serotypes of S. flexneri are 1b, 2a, 3a, 4a, and 6. In the

industrialised countries, the predominant serotype of S.flexneri is 2a (Jennison & Verma,

2004). However, new serotypes are emerging. Emergence of new serotype 1c has been

reported from Bangladesh (Talukder et al., 2003). New serotype Xv appeared in Henan

province China in 2001, and in 2002-2006 it replaced serotype 2a and became the most

prevalent serotype. It also became the most prevalent serotype in Shanxi province in 2006-

2007, and in Gansu, Anhui, and Shanghai in 2007 (Sun et al., 2012).

Introduction

4

1.4 Pathogenesis

S. flexneri infection is transmitted by a faecal-oral route and ingestion of as few as 10-100

organisms is sufficient to cause the disease (DuPont et al., 1989; Waterman & Small, 1996).

S. flexneri uses the membranous epithelial cells (M cells) as a threshold to cross the epithelial

barrier and to propagate (Fig. 1.1). It invades the M cell epithelium and is released into the

intra-epithelial pocket where resident macrophages engulf the bacteria. However, the bacteria

evade the killing mechanism of the macrophage and induce pyroptosis of the macrophage. As

a result, a large amount of interleukin 1 (IL-1) is released which initiates inflammation and

recruits polymorphonuclear cells (PMNs) at the site of infection. The influx of PMNs disrupts

the integrity of the epithelium and facilitates the surge of the lumenal bacteria to the

submucosa (Jennison & Verma, 2004; Kraehenbuhl & Neutra, 2000; Sansonetti & Phalipon,

1999). After reaching the basolateral side the bacteria are able to invade the epithelial cells

through bacterial induced endocytosis, resulting in the engulfment of the bacteria within an

endocytic vacuole. Lysis of the endocytic vacuoles releases the bacteria into the cytoplasm of

the host cell (Blocker et al., 1999; Jennison & Verma, 2004). Within the host cell cytoplasm

Shigella multiplies and polymerises actin filament at one pole by use of the IcsA (VirG)

protein. ABM propels the bacteria through the cytoplasm of the host cell. Bacteria reach the

cell membrane and form a protrusion which is taken up by the adjacent cell. The protrusion is

then pinched off to form a double membrane-bound vacuole with the bacterium inside. S.

flexneri is able to lyse this vacuole, and is released into the cytoplasm of the adjacent cells

(Goldberg, 2001; Monack & Theriot, 2001; Prevost et al., 1992; Schuch et al., 1999; Suzuki

et al., 1996; Suzuki et al., 1998; Suzuki & Sasakawa, 2001). The cycle then repeats.

1.5 Virulence factors

S. flexneri has several virulence factors such as VP encoded Mxi-Spa type III secretion system

(TTSS), proteins secreted by TTSS, IcsA protein, and LPS. LPS is described in detail in

Section 1.6.

Introduction

5

Figure 1.1 S. flexneri pathogenesis S. flexneri are taken up by M cells epithelium then released into the intra-epithelial pocket,

where the bacteria evade the killing mechanism of the resident macrophages and induce

pyroptosis of the macrophages. The bacteria reach the basolateral side, invade the epithelial

cells, release into the cytoplasm, and replicate. ABM propels the bacteria through the

cytoplasm of the host cell. Then the bacteria penetrate the cell membrane, release into the

adjacent cell, and repeat the cycle.

Introduction

6

1.5.1 Large virulence plasmid (VP)

The pathogenicity of S. flexneri is characterised by penetrating epithelial cells (Labrec et

al., 1964) and the ~230 kb VP in the bacteria is required for this ability (Sansonetti et al.,

1982; Sasakawa et al., 1993). VPs pWR100 in S. flexneri 5, pMYSH6000 in S. flexneri 2a,

and pSS120 in S. sonnei and those in other Shigella strains are determinants for invasiveness

and disease causing ability and are collectively termed pINV plasmids (Lan et al., 2001). The

complete nucleotide sequencing of S. flexneri 2a strain 301 VP was performed by Zhang et al.

(2003) which showed that it is 221618 bp with 272 open reading frames, and insertion

sequence (IS) elements are 68 kb in length and cover 30% of the complete genome (Zhang et

al., 2003). The VP contains a 31 kb PAI which is required to elicit the invasion property and

contains the gene encoding TTSS. PAI of S. flexneri contains 28 genes bracketed by several

IS elements (Buchrieser et al., 2000; Galan & Collmer, 1999; Jouihri et al., 2003; Schroeder

& Hilbi, 2008).

1.5.2 Mxi-Spa type III secretion system (TTSS)

TTSSs are evolutionary related to the bacterial flagellar secretion system (Schroeder et al.,

2007). TTSS is a needle like structure with a basal structure spanning bacterial inner

membrane (IM) and outer membrane (OM), a hollow needle, and a hydrophobic translocator

complex that inserts into the target eukaryotic membranes (Hong et al., 2012; Schroeder et

al., 2007). TTSS is a protein transport device and translocates the effector molecules from

bacterial cytoplasm to the membrane and cytoplasm of the host cell (Galan & Collmer, 1999;

Hong et al., 2012; Jouihri et al., 2003; West et al., 2005). S. flexneri employs TTSS to invade

epithelial cells and to kill macrophages (Cossart & Sansonetti, 2004) and it is encoded by mxi

and spa genes on the VP (Blocker et al., 1999; Blocker et al., 2001; Buchrieser et al., 2000;

Cornelis, 2006; Schroeder et al., 2007; Tamano et al., 2000). Secretion by the Mxi-Spa

system is triggered after contacting the host cells (Schroeder et al., 2007; Shen et al., 2010).

Introduction

7

1.5.3 Effector proteins secreted by TTSS

Studies suggested that Shigella TTSS delivers two sets of effectors: i) early effectors

including IpaA and IpgD, involved in the entry into the epithelial cells; ii) late effectors

including IpaH family proteins and VirA. The latter enables intracellular survival of the

bacteria, intracellular and cell-to-cell motility; and modulation of the host inflammatory

response (Shen et al., 2010). VP encoded Ipa (invasion plasmid antigen) A-D are dominant

antigens in the humoral immune response against S. flexneri infection (Hale et al., 1985; Oaks

et al., 1986). ipaA-D are present in an operon and IpaA-D proteins are essential for expression

of invasive functions of S. flexneri (Allaoui et al., 1992; Allaoui et al., 1993; Baudry et al.,

1987; Maurelli et al., 1985; Menard et al., 1993; Niebuhr et al., 2000; Sasakawa et al., 1988;

Venkatesan et al., 1988; Venkatesan et al., 1992). IpaB and IpaD colocalise at the TTSS

needle tip and act as a secretion plug (Olive et al., 2007). After secretion IpaB and IpaC form

a pore-like complex in the host cell membrane for bacterial uptake by pinocytosis and

translocation of other effectors. IpaD is essential for the invasion of the bacteria and also

associated with contact-mediated hemolysis of S. flexneri (Picking et al., 2005; Shen et al.,

2010). The other effector proteins secreted by TTSS include: IcsB, VirA, OspG, IpaH, OspF,

and OspC1. IcsB is essential for escaping autophagy (Ogawa et al., 2005), VirA enables the

movement of the bacteria through the dense and organised cytoplasmic network of the host.

Shigella mutants lacking a functional virA gene are unable to move through the cytoplasm,

and the invasiveness of these mutants is attenuated. Hence, VirA is also essential for virulence

of the bacteria (Davis et al., 2008; Yoshida et al., 2002; Yoshida et al., 2006). OspG and IpaH

modulate the inflammatory response of the host (Ashida et al., 2007; Kim et al., 2005; Okuda

et al., 2005). TTSS injects another effector protein IpgD into the epithelial cells, where IpgD

uncouples the plasma membrane from the actin cytoskeleton that allows the formation of

membrane extensions (Niebuhr et al., 2002). OspF and OspC1 have roles in the activation of

mitogen-activated protein kinase or extracellular signal-regulated kinase pathway and

transepithelial migration of PMNs, which is associated with increased inflammation and

bacterial access to the submucosa (Zurawski et al., 2006).

Introduction

8

1.5.4 IcsA protein

Initially, the intercellular motility and protrusion formation of Shigella was microscopically

observed within epithelial monolayers. However, the mechanism behind this movement was

unknown but it was observed that the movement was initiated at one pole (Ogawa et al.,

1968). The genetic basis of this movement was uncovered during transposon mutagenesis of

the VP. Tn5 insertions within an EcoRI - SalI fragment of the VP, termed virG, abolished in

vitro cell-to-cell spreading and the mutants were also avirulent in the Serény test (Makino et

al., 1986). The 1,102 amino acids protein, VirG responsible for this phenotype was identified

(Lett et al., 1989). Contemporaneously, VirG protein was identified as IcsA (intra- and

intercellular spread gene A) by Bernardini et al. (1989) and they were first to report that IcsA-

mediated motility of Shigella is dependent on polymerised host F-actin at one bacterial pole

(Bernardini et al., 1989).

IcsA is an OM protein and is polarly localised on the bacterial surface (Goldberg et al.,

1993). S. flexneri ABM has been studied in a range of model systems, including microscopy

of infected tissue culture monolayers, in vitro Xenopus cell extracts, and protein

reconstitution systems (Egile et al., 1999; Goldberg & Theriot, 1995; Loisel et al., 1999).

ABM and subsequent intercellular spreading are the key features of Shigella virulence. IcsA

mediated ABM can be measured using an in vitro plaque formation assay by measuring the

plaque sizes post S. flexneri invasion of tissue culture monolayers (Oaks et al., 1985). Mutants

lacking IcsA are non-motile (Goldberg & Theriot, 1995). The in vivo way to assess Shigella

virulence known as Serény test is the evaluation of the development of Keratoconjunctivitis

following Shigella infection of guinea pig and mouse eyes (Sereny, 1957; Wood et al., 1986).

Expression of IcsA is an absolute requirement in all these models (Goldberg & Theriot,

1995). Heterologous expression of IcsA in closely related E. coli K-12 confers the ability to

polymerise actin and exhibit ABM in vitro (Goldberg & Theriot, 1995; Kocks et al., 1995;

Monack & Theriot, 2001).

IcsA is a 120 kDa autotransporter protein and has three domains: N-terminal signal

sequence (amino acids 1-52), #-domain (amino acids 53-758), and C-terminal "-domain

(amino acids 759-1102) (Goldberg, 2001; Suzuki et al., 1995). The #-domain is the

functionally active region; C-terminal "-domain anchors the IcsA protein to the OM, exposing

Introduction

9

the N-terminal to the surface (Robbins et al., 2001). An unusually long signal sequence

directs the translocation of the IcsA polypeptide from the cytoplasm to the periplasm via Sec

pathway (Brandon et al., 2003). Formation of an intramolecular disulphide bridge occur in the

IcsA #-domain in the periplasm (Brandon & Goldberg, 2001) and when the "-domain inserts

itself into the OM via a "-barrel assembly machinery complex, the #-domain is exported to

the extracellular environment (Jain & Goldberg, 2007; Suzuki et al., 1995).

Two regions of IcsA (amino acids 1-104 and 506-620) in the #-domain have been

identified as responsible for the polar localisation of the protein (Charles et al., 2001) and

again insertion mutation at amino acids 532 and 563 of the #-domain affected IcsA polar

localisation and further supported the role of amino acids 506-620 of IcsA in polar

localisation (May & Morona, 2008). YidC a cytoplasmic chaperone protein was shown to

assist IcsA to localise at the pole (Gray et al., 2014) and FtsQ, a protein important for

bacterial cell division also facilitate IcsA polar localisation (Fixen et al., 2012). Shigella IcsP

protease releases ~95 KDa #-domain of IcsA by cleaving the protein at amino acids 758 and

759 (Egile et al., 1997; Shere et al., 1997; Steinhauer et al., 1999) and IcsP contributes to the

unipolar localisation of IcsA (Tran et al., 2013).

Intercellular movement of the bacteria is dependent on polarised actin polymerisation and

when concentrated at one pole the bacteria forms the F actin tail and resulted in unidirectional

propulsion (Cossart, 2000; Robbins et al., 2001). The #-domain stimulates actin assembly and

interacts with the host actin regulatory factor neural Wiskott-Aldrich syndrome protein (N-

WASP). IcsA has three N-WASP interacting regions: Region 1 (amino acids 185-312),

Region 2 (amino acids 330-382), and Region 3 (amino acids 508-730) (Teh et al., 2012; Teh

& Morona, 2013). Interaction of IcsA with N-WASP allows recruitment of Arp2/3 complex

leading to F-actin comet tail formation in mammalian cells (Egile et al., 1999; May &

Morona, 2008; Suzuki et al., 1995; Suzuki et al., 1996; Suzuki et al., 1998; Teh & Morona,

2013).

1.6 Lipopolysaccharide (LPS)

Gram-negative bacteria have an asymmetric OM and the inner leaflet of the OM is composed

Introduction

10

of phospholipid and the outer leaflet is comprised of lipopolysaccharide (LPS) (Dong et al.,

2014; Raetz & Whitfield, 2002; Ruiz et al., 2009). LPS is a complex lipid and forms the

protective barrier of the Gram-negative bacteria (Sperandeo et al., 2009).

1.6.1 Morphology

LPS has three domains: Lipid A, Core sugars, and Oag. The LPS structures (Fig. 1.2) of S.

flexneri can be divided into three types: Smooth LPS (S-LPS), Rough LPS (R-LPS), and

Semi-Rough LPS (SR-LPS). S-LPS is the complete LPS structure with an Oag chain length of

11 to 17 Oag tetrasaccharide repeat units (RUs). The wild type (WT) S. flexneri contains S-

LPS. The LPS structure lacking the Oag is termed R-LPS. SR-LPS contains a single Oag

tetrasaccharide RU attached to the Lipid A and core sugar (Bone, 1993; Morona et al., 1994;

Naide et al., 1965).

1.6.1.1 Lipid A

Lipid A anchors LPS in the OM through hydrophobic interactions involving fatty acids,

including laurate and myristate. It is the most highly conserved domain of the Gram-negative

bacterial LPS. Lipid A is also known as ‘endotoxin’ as it has many effects to the mammalian

system during sepsis. The innate immune response to lipid A results in cytokine production

(Bone, 1993; Muller et al., 1993; Raetz, 1993; Ranallo et al., 2010; Yethon & Whitfield,

2001). Chemically lipid A, is an ester linked di-glucosamine with both ester and amide-linked

pyrophosphates and fatty acids (Bone, 1993). S. flexneri lipid A has the beta (1-6)-linked

glucosamine disaccharide with two phosphate groups (Fig. 1.3). Both E. coli and S. flexneri

Lipid A are glucosamine disaccharide modified by six acyl chains and the fatty acid

composition and acylation are also similar. The fatty acid acylation occurs by four (R)-3-

hydroxy fatty acids at the positions O-2, O-3, O-2', and O-3' (Lindberg et al., 1991).

Introduction

11

Figure 1.2 LPS structural types S-LPS contains Lipid A, Core sugar, and Oag RUs; SR-LPS contains Lipid A, Core sugar,

and single Oag unit; and R-LPS contains Lipid A and Core sugar. n = number of Oag RUs.

Introduction

12

Figure 1.3 S. flexneri Lipid A structure S. flexneri lipid A has the beta (1-6)-linked glucosamine disaccharide with two phosphate

groups. The fatty acid acylation occurs by four (R)-3-hydroxy fatty acids at the positions O-2,

O-3, O-2', and O-3'. The numbers in the circle indicate the number of the carbon atoms

present in the fatty acids. Figure adapted from D'Hauteville et al. (2002).

Introduction

13

1.6.1.2 Core sugar

The 6' position of lipid A is glycosylated with a non-repeating oligosaccharide structure,

known as the core sugar. It is negatively charged LPS domain and forms intermolecular

cationic bridging to strengthen the rigidity of the cell wall (Knirel et al., 2011). LPS core

sugar region is divided into inner core and outer core. The inner core of most of the Gram-

negative bacteria possesses the eight-carbon sugar 3-deoxy-D-manno-oct-2-ulosonic acid

(Kdo). This Kdo unit links the core sugar region to the lipid A. In some LPS structures, L-

glycero-D-manno-heptose (Hep) residues are found to be linked to the Kdo in the inner core

region. In Enterobacteria the inner core has a common structure, either Kdo + 3Hep or Kdo +

3Hep + glucosamine (GlcN) (Kubler-Kielb et al., 2010; Raetz, 1990; Raetz & Whitfield,

2002; Yethon & Whitfield, 2001). S. flexneri has 2 residues of Kdo and 3 residues of Hep in

the inner core region (Knirel et al., 2011) (Fig. 1.4). The distal sugar region of the core is

known as outer core. The outer core is composed of glucose (Glc), galactose (Gal), and N-

acetylglucosamine (GlcNAc) residues and to which the Oag RU is attached (Kubler-Kielb et

al., 2010; Raetz, 1990; Raetz & Whitfield, 2002; Yethon & Whitfield, 2001). S. flexneri 2a

has the R3 type of LPS core (Kubler-Kielb et al., 2010) (Fig. 1.4).

1.6.1.3 O antigen (Oag)

Oag is the most variable domain of the Gram-negative bacterial LPS (Yethon & Whitfield,

2001). The presence of Oag S-LPS restricts the accessibility of colicins to their OM receptors

(van der Ley et al., 1986). Oag also acts as the attachment site of the bacteriophages. Other

than the serotype conversion, bacteriophages use Oag as a receptor for adsorption and

infection of the host bacterium (Lindberg et al., 1978). Oag consists of oligosaccharide RUs.

Differences in the sugar contents, linkage between sugar units, and number of sugar units

make this domain highly variable (Liu et al., 2008; Wang et al., 2010a). The Gram-negative

bacterial species are subdivided into various serotypes depending on the differences in the

composition of LPS-Oag (Wang et al., 2010a). So far, there are 19 known serotypes [1a, 1b,

1c (or 7a), 1d, 2a, 2b, 3a, 3b, 4a, 4av, 4b, 5a, 5b, 6, 7b, X, Xv, Y, and Yv] of S. flexneri

Introduction

14

Figure 1.4 S. flexneri 2a core The core region is composed of Kdo, Hep, Glc, Gal, and GlcNAc. The inner core is composed

of Kdo + 3Hep and the outer core is composed of Glc, Gal, and GlcNAc. Outer core is

attached to the Oag RU. Oag chain is attached to the O-4 of Glc. n = number of Oag RU.

Figure adapted from Kondakova et al. (2010).

Introduction

15

(Jakhetia et al., 2014; Sun et al., 2013a; Sun et al., 2014). Except the S. flexneri serotype 6,

the Oag of all other serotypes has the same polysaccharide backbone containing three L-

rhamnoses (Rha), and one GlcNAc (Fig. 1.5). This basic Oag structure is known as serotype

Y. In serotype Y, the Rha residues are linked by # linkage, and Rha and GlcNAc residues are

linked by " linkage [!2)-#-L-RhaIII-(1!2)-#-L-RhaII-(1!3)-#-L-RhaI-(1!3)-"-D-

GlcNAc(1!]. Addition of either glucosyl, O-acetyl, or phosphoethanolamine (PEtN) groups

by various linkages to the sugars within the tetrasaccharide repeats of serotype Y creates

different serotypes of S. flexneri (Adams et al., 2001; Allison & Verma, 2000; Jakhetia et al.,

2014; Sun et al., 2013b) (Fig. 1.5).

The genes responsible for the Oag glucosylation [gtrA, gtrB, and gtr (type)] are

arranged in a single operon (gtr cluster). Among them gtrA and gtrB are conserved, however,

gtr (type) is unique and the encoded glucosyltransferase attaches the glucosyl group to the

specific sugar in the tetrasaccharide RU (Adams et al., 2001; Adhikari et al., 1999; Allison &

Verma, 2000; Allison et al., 2002; Guan et al., 1999; Mavris et al., 1997; Stagg et al., 2009).

Presence of the oac gene for O-acetyl transferase resulted in the O-acetylation of Oag (Clark

et al., 1991; Verma et al., 1991). There is an association between bacteriophages and serotype

conversion in S. flexneri. Seven bacteriophages or prophages encode the genes known so far

for Oag modifications, among them six (SfI, SfIC, SfII, SfIV, SfV, and SfX) encode gtr gene

cluster and one (Sf6) encodes oac gene. These genes are integrated into the conserved sites of

the S. flexneri genome (Adams et al., 2001; Adhikari et al., 1999; Allison et al., 2002;

Casjens et al., 2004; Clark et al., 1991; Guan et al., 1999; Mavris et al., 1997; Stagg et al.,

2009). Glucosylation can occur on any of the residues of the basic tetrasaccharide RU but oac

mediated O-acetylation occurs at position 2 of Rha I (Jakhetia et al., 2014). Although the

glucosylation and O-acetylation are phage encoded, the PEtN modification at position 3 of

Rha II and/or Rha III is encoded by the plasmid borne opt gene (Jakhetia et al., 2014).

For S. flexneri serotypes 1b, 3a, 3b, 3c, 4b, and 7b the O-acetylation site is position 2

of Rha I (Perepelov et al., 2012). However, a new O-acetylation site have been reported:

position 3 (major) and 4 (minor) of Rha III (3/4-O-acetylation) in serotypes 1a, 1b, 2a, 5a, Y,

6 and 6a, and at position 6 of GlcNAc in serotypes 2a, 3a, and Y. The degree of 3/4-O-

Introduction

16

Figure 1.5 Chemical composition of the Oag of different S. flexneri serotypes Serotype Y has the basic Oag, consisting of repeated tetrasaccharide units of !2)-#-L-RhaIII-

(1!2)-#-L-RhaII-(1!3)-#-L-RhaI-(1!3)-"-D-GlcNAc(1!. Addition of either glucosyl, O-

acetyl, or phosphoethanolamine groups to the sugar residues within the tetrasaccharide RU via

the indicated linkages generate different serotypes. Figure adapted from (Allison & Verma,

2000; Sun et al., 2012; Sun et al., 2014).

Introduction

17

acetylation varies between the ranges 30-70% at position 3 and 15-30% at position 4 within

the strains of one serotype (Jakhetia et al., 2014; Perepelov et al., 2012). According to recent

study S. flexneri 3/4-O-acetylation is mediated by the oacB gene carried by a transposon-like

structure located upstream of the adrA gene on the chromosome (Wang et al., 2014). The

temperate bacteriophages SfII, Sf6, SfV, and SfX convert the basic serotype Y to serotypes

2a, 3b, 5a, and X, respectively (Allison & Verma, 2000). Lysogenic bacteriophage SfII adds a

glucose residue to the Rha III residue of the tetrasaccharide RU of S. flexneri Oag and confers

serotype II Oag modification (Mavris et al., 1997). Recently, a new bacteriophage Sf101 was

isolated from serotype 7a. It contains the oac gene and responsible for the O-acetyl

modification in serotype 7a (Jakhetia et al., 2014). Serotype converting temparate

bacteriophage SfV adds a glucosyl group to Rha II of the tetrasaccharide RU by an # 1,3

linkage (Allison et al., 2002). A newly emerged and most prevalent serotype in China is

serotype Xv. For serotype Xv the Oag modification occurs by the addition of PEtN group at

position 3 of one of the Rha residues (Sun et al., 2012) (Fig. 1.5). PEtN modification is also

present in 4av, a serotype 4a variant; and Yv, a serotype Y variant. In these serotypes a PEtN

residue is added mainly to Rha III and Rha II, respectively (Sun et al., 2013a).

The antigenic structure of S. flexneri is simple. S. flexneri possesses somatic Oags and

certain varieties possess K antigen. Determination of S. flexneri group is done by

agglutination with polyvalent serum and the type diagnosis is performed by a monospecific

serum obtained after absorption of the group agglutinins. Certain varieties of S. flexneri

serotype 6 are O-inagglutinable as they have K antigen (Bergey & Breed, 1957). The basic

Oag structure serotype Y is characterised by a single group 3,4 antigenic determinant. Oag

glucosylation and/or O-acetylation result in different type [I, II, III, IV, V, IC (or VIII)] and

group (3,4; 6; 7,8) antigenic determinants (Sun et al., 2012; Sun et al., 2013a) (Table 1.1).

Group 6 antigenic determinant is present in serotypes 3a, 3b, 1b, 4b, and 7b and they are

characterised by the presence of O-acetylation on Rha I. Type I, IC (or VII), II, IV, V; and

group 7,8 determinants are associated with Oag glucosylation (Sun et al., 2012; Sun et al.,

2013a). Serotypes X and Y lack type antigens but group antigens are 7,8; and 3,4;

respectively (Simmons & Romanowska, 1987). Serotype Xv can agglutinate with both MASF

IV-I and 7,8 monoclonal antibodies (Sun et al., 2012).

Introduction

18

Table 1.1 Antigenic determinants of various S. flexneri serotypes

Serotypes Type antigen Group antigen

1a I 4

1b I 6 1c (7a) - MASF 1c

1d I 7,8 2a II 3,4

2b II 7,8 3a III 6,7,8

3b III 6,3,4 4a IV 3,4

4av IV 3,4 4b IV 6

5a V 3,4 5b V 7,8

6 VI 2,4 7b - 6

X - 7,8 Xv IV 7,8

Y - 3,4 Yv IV 3,4

Introduction

19

1.6.2 LPS as a virulence factor

LPS is the immunodominant surface antigen of S. flexneri. As a surface structure it interacts

with the host and the host defense systems recognise bacteria by the elicited immune

responses to their LPS. However, inside the host LPS causes a variety of non-specific

pathological reactions, known as endotoxic reactions (Lindberg et al., 1991). The LPS

morphology of S. flexneri has a critical role in the virulence property of the bacteria (Hong &

Payne, 1997; Van den Bosch et al., 1997). An assay to measure the ability of Shigella to

invade epithelial cells, spread intercellularly, and evoke inflammatory responses is known as

the Serény test (Sereny, 1957) and previous studies showed that S. flexneri with R-LPS were

avirulent in Serény test. Strains with R-LPS could invade the tissue-culture cells but were

unable to spread intercellularly (Okamura & Nakaya, 1977; Okamura et al., 1983). S. flexenri

strains with R-LPS were also deficient in plaque formation in tissue culture monolayer, with

very small or no plaques being observed (Hong & Payne, 1997; Sandlin et al., 1995; Van den

Bosch et al., 1997; Van den Bosch & Morona, 2003).

S-LPS with very long Oag chains gives S. flexneri protection against serum killing

(Hong & Payne, 1997). LPS also plays a role in the physiological relationship between host

and the bacteria. B cells differentiate, proliferate, and secrete immunoglobulins due to the

polyclonal activation by LPS. LPS activates macrophages which enhances cytotoxicity and

phagocytosis (Lindberg et al., 1991).

A number of genes involved in the synthesis of LPS were found important for the

virulence property of Shigella spp. (Okada et al., 1991a; Okada et al., 1991b). Several studies

through mutagenesis of genes involved in the LPS biosynthesis (galU, rfe, rfb, rmlD, wecA,

wzy, and wzz) showed that the S-LPS is essential for IcsA dependent ABM and cell-to-cell

spreading (Hong & Payne, 1997; Rajakumar et al., 1994; Sandlin et al., 1995; Van den Bosch

et al., 1997; Van den Bosch & Morona, 2003). In Shigella S-LPS strains, IcsA is unipolar

however, in R-LPS strains the unipolarity is lost and IcsA can be detected over the entire

bacterial surface (Robbins et al., 2001; Van den Bosch et al., 1997). There are two proposed

hypotheses for this loss of polarity in R-LPS strains. Robbins et al. (2001) proposed that the

biophysical properties of the OM can be altered due to the mutation in the LPS biosynthesis

genes of R-LPS strains, resulting in the increased migration of IcsA from pole to the non-

Introduction

20

polar sites (Robbins et al., 2001). However, according to the other hypothesis, presence of

LPS Oags masks IcsA expression in the non-polar regions and mutants lacking Oag alter

detection of IcsA at non-polar regions in addition to the pole (Morona & Van Den Bosch,

2003a; Morona & Van Den Bosch, 2003b). Although the loss of unipolarity of IcsA is

considered the cause behind the impaired ABM but the overexpression of IcsA in the S-LPS

strains resulted in circumferential localisation of IcsA similar to the R-LPS strains and ABM

was undisturbed (Van den Bosch & Morona, 2003). Hence, LPS Oag may have other critical

roles in S. flexneri cell to cell spreading.

Lipid A is the bioactive component of LPS and the activator of the innate immune

response in host. Several previous studies suggested that the changes in lipid A acylation

effect the virulence property of the bacteria by influencing protein secretion of TTSS, cell

division, and OM function (Clements et al., 2007; Murray et al., 2001; Post et al., 2003;

Ranallo et al., 2010; Somerville et al., 1999; Watson et al., 2000). D’Hauteville et al. (2002)

investigated the effect of genetic detoxification of lipid A on the S. flexneri pathogenicity. S.

flexneri has two types of msbB genes: one chromosomal (msbB1) and another on the VP

(msbB2); both encode myristoyl transferase. They showed that in S. flexneri, lacking both the

msbB genes had reduced lipid A acylation degree, and caused reduced TNF# production and

epithelial lining inflammatory destruction of a rabbit model (D'Hauteville et al., 2002).

Ranallo et al. (2010) performed an extensive study to analyse the virulence property and

inflammatory potential of the S. flexneri 2a lacking both msbB genes and producing

underacylated lipid A. Attenuation of msbB mutants in an acute mouse pulmonary challenge

model correlated with decreases in proinflammatory cytokine production and in chemokine

release without noticeable changes in lung histopathology. After infection of mouse

macrophages with either single or double msbB mutants, production levels of IL-1", MIP-1#,

and TNF-# were also significantly reduced. The msbB double mutant showed defects in the

invasion and replication ability, and spreading within the epithelial cells. They also performed

a vaccination-challenge study in a mouse lung model and found that in msbB-immunised mice

both humoral and cellular responses were significantly strong (Ranallo et al., 2010). Paciello

et al. (2013) reported that Shigella has the ability to modify the composition of the lipid A and

core sugar domains, during proliferation within epithelial cells. They showed that the lipid A

Introduction

21

of the LPS of intracellular bacteria is hypoacylated compared to the LPS of the bacteria grown

in laboratory medium and the immunopotential of intracellular bacterial LPS is dramatically

lower than that of the LPS of the bacteria grown in laboratory medium (Paciello et al., 2013).

Rossi et al. (2014) produced generalised modules for membrane antigens (GMMA) from

Shigella. GMMA is produced from the OM and mainly contains LPS. To reduce the

reactogenicity of GMMA they modified the Lipid A by deleting the msbB genes. GMMA

with penta-acylated Lipid A from the msbB mutant strains had 600 fold reduced activity

however, GMMA with hexa-acylated Lipid A resulted by lipid A palmitoleoylation had ~10

fold higher activity to stimulate peripheral blood mononuclear cells (Rossi et al., 2014).

S. flexneri waaJ, waaD, and waaL mutant strains are unable to synthesise inner core or

are deficient in ligating Oag to the outer core, were attenuated and failed to resist killing by

antimicrobial peptides expressed in the GI tract (Cunliffe & Mahida, 2004; West et al., 2005).

The rfbA mutant deficient in converting glucose-1-phosphate to deoxythymidine diphosphate

rhamnose (dTDP-Rha) a required step for Oag biosynthesis and the cld mutant having

truncated Oag were also attenuated (West et al., 2005). gtrV gene encodes glucosyltransferase

which adds glucose to the Oag (Guan et al., 1999). West et al. (2005) reported that Shigella

strains with gtrV deletion mutation were unable to survive in the GI tract. Their work also

suggested that LPS glucosylation allows the bacteria to survive in vivo. The gtr mutants were

unable to avoid innate immune killing but the non-invasive S. flexneri with gtr mutation,

which lacks TTSS, did not show any survival disadvantage. Hence, the effect of gtr deletion

only results after invasion into the mucosa (West et al., 2005). Shigella strains with increased

Oag glucosylation has increased ability to invade epithelial cells however, the gtr mutant

strains has decreased ability of epithelial invasion compared to the WT. LPS glucosylation

affects the TTSS function which determines cell invasion (West et al., 2005).

Following Shigella WT infection and experimental challenge strong mucosal secretory

IgA anti-Oag antibody responses are observed (Levine et al., 2007). The Oag of the LPS is

the protective antigen. After S. flexneri infection the host immune response is directed against

the Oag and the immune response is serotype specific correlated with the stimulation of local

humoral and intestinal immunity against somatic antigens. The serotype specific infection

provides protection against further infection with the same serotype (Allison & Verma, 2000;

Introduction

22

Brahmbhatt et al., 1992; Formal et al., 1991; Guan & Verma, 1998; Hale & Keren, 1992;

Lindberg & Pal, 1993). In an animal experiment conducted by Formal et al. (1991), virulent S.

flexneri 2a strain was used to infect Rhesus monkeys and later the infected monkeys were

rechallenged with either S. sonnei or S. flexneri 2a strains. Monkeys rechallenged with S.

sonnei suffered disease similar to the controls, however, monkeys rechallenged with

homologous S. flexneri 2a got full protection (Formal et al., 1991). In humans, it was also

found that pre-existing serotype-specific serum antibodies against the Shigella Oag is

correlated with the resistance to homologous shigellosis (Cohen et al., 1988).

1.7 LPS biosynthesis and export

In E. coli, Shigella, and Salmonella LPS biosynthesis and export are dependent on the

products of approximately 50 genes (Schnaitman & Klena, 1993). LPS biosynthesis occurs

mainly by two separate pathways: lipid A plus core biosynthesis and Oag biosynthesis. Other

than the membrane bound enzymes for glycerophospholipids synthesis, the enzymes required

for the early stage of lipid A biosynthesis are cytoplasmic or present in the cytoplasmic

membrane (Anderson et al., 1993). In the cytoplasm after synthesis of the lipid A, the core

oligosaccharides are assembled on the lipid A. At the cytoplasmic face of the IM the Oag RUs

are assembled on Und-PP (Marino et al., 1991; Mulford & Osborn, 1983). The lipd A-core

and Oag RUs are then transported from the cytoplasmic side to the periplasmic side. The

Und-PP linked Oag RUs are polymerised into Oag chains on the periplasmic face of the

cytoplasmic membrane. The Und-P is recycled. The lipid A-core and Oag chains are ligated

(Kanegasaki & Wright, 1973; McGrath & Osborn, 1991; Mulford & Osborn, 1983;

Woodward et al., 2010). Once assembled the LPS is exported from the IM across the

periplasm, and assembled into the outer leaflet of the OM of the Gram-negative bacteria

(Sperandeo et al., 2009).

Introduction

23

1.7.1 Lipid A and core biosynthesis

Lipid A synthesis in many Gram-negative bacteria resembles the one found in E. coli (Raetz

& Whitfield, 2002). Both E. coli and S. flexneri lipid A is a glucosamine disaccharide with six

acyl chains: primary acyl chains at the 2, 3, 2’ and 3’ positions and secondary acyl chains

attached to the 2’ and 3’ primary chains (Goldman et al., 2008). In E. coli and Salmonella

typhimurium Lipid A synthesis is initiated by uridine diphosphate GlcNAc acetyltransferase

(UDP-GlcNAc acetyltransferase) LpxA. This enzyme fatty acetylates the sugar nucleotide

UDP-GlcNAc. It transfers the acyl group from R-3-hydroxymyristoyl-acyl carrier protein (R-

3-hydroxymyristoyl-ACP) to UDP-GlcNAc to form UDP-3-O-(R-3-hydroxymystristoyl)-

GlcNAc (Anderson & Raetz, 1987; Galloway & Raetz, 1990; Meier-Dieter et al., 1992;

Stevenson et al., 1994; Wyckoff et al., 1998). The following steps of Lipid A biosynthesis are

mediated by the enzymes LpxC and LpxD. LpxC deacetylates the O-acetylated UDP-GlcNAc

and LpxD performs the second deacetylation. LpxD transfers a second acyl group from R-3-

hydroxymyristoyl-ACP to O-acetylated UDP-GlcNAc and forms UDP-2,3-diacylglucosamine

(Crowell et al., 1987; Raetz et al., 2007). UDP-2,3-diacylglucosamine is the immediate

precursor of non-reducing sugar of Lipid A. Some of the UDP-2,3-diacylglucosamine is

cleaved at the phosphate bond by LpxH to generate diacylglucosamine-1-phosphate or Lipid

X (Anderson et al., 1985; Raetz et al., 2007). It is the direct precursor of the reducing sugar of

Lipid A. LpxB mediates the condensation of another molecule of UDP-2,3-diacylglucosamine

with Lipid X to form the disaccharide and releases UDP. Phosphorylation of the disaccharide

formed by LpxB generates Lipid IVA (Raetz & Whitfield, 2002). In S. flexneri two Kdo

residues are then transferred by a bifunctional transferase to form keto-doxy-octonate 2 lipid

A (D'Hauteville et al., 2002). At the final steps of S. flexneri Lipid A synthesis 12-carbon

fatty acid laurate and the 14-carbon fatty acid myristate are acyl-oxyacyl-linked to two of the

four 3-OH-myristic acids available on keto-doxy-octonate 2 lipid A. htrB encodes the

transferase (LpxL or HtrB) that catalyses the acyl-oxyacyl linkage of laurate to the 3’

hydroxymyristate that is itself linked to the 2’ position of the glucosamine and msbB encodes

the transferase MsbB (LpxM) that catalyses the acyl-oxyacyl linkage of myristate on the

hydroxy-myristate that is itself linked to the 3’ position of the glucosamine disaccharide

(D'Hauteville et al., 2002; Goldman et al., 2008).

Introduction

24

Genes of the chromosomal waa locus including waaF, waaC, waaL, waaD, waaJ,

waaY, waaI, waaP, waaG, waaQ, and waaA encodes the proteins for E. coli R3 type core

sugar synthesis (Kaniuk et al., 2004) (Fig. 1.6). The product of the waaA, WaaA catalyses the

synthesis of inner core. It transfers the first two Kdo residues of the core. (Kaniuk et al., 2004;

Sirisena et al., 1994; Vimont et al., 1997). WaaC and WaaF are the first and second

heptosyltransferases. Inner core assembly phosphate transferases are WaaP and WaaY. WaaP

trasfers phosphate to heptose I and WaaY to heptose II. WaaQ transfers heptose III to heptose

II (Kaniuk et al., 2004; Muhlradt, 1969; Sirisena et al., 1992; Yethon et al., 1998). Initially

WaaG, an UDP-glucosyl transferase, attaches the first Glc to heptose II, WaaI attaches a Gal

unit to the first Glc, and then WaaJ adds a second Glc to the Gal (Heinrichs et al., 1998;

Kaniuk et al., 2004). WaaD an #-1,2-glucosyltransferase responsible for addition of the

terminal glucose side branch (Kaniuk et al., 2004).

1.7.2 O antigen biosynthesis

The Oag synthesis genes are generally found in a single cluster, named Oag gene cluster.

Three main types of genes are found in this Oag gene cluster: nucleotide sugar synthesis,

sugar transferase, and O unit processing genes. The location of these genes within a species is

conserved (Wang et al., 2010a). Oag assembly and processing are performed by three

different pathways: Wzx/Wzy, ABC transporter, and Synthetase pathway. Among these three

pathways, the Wzx/Wzy pathway synthesises most Oags (Liu et al., 1996; Raetz & Whitfield,

2002; Wang et al., 2010a).

For all the three pathways, initially a sugar phosphate is transferred from an NDP-

sugar to Und-P at the cytoplasmic side of the IM. In the Wzx/Wzy pathway,

glycosyltransferases sequentially add sugar residues to the first sugar at the cytoplasmic side

of the IM to form the O unit. The flippase protein Wzx translocates this O unit to the

periplasmic side. At the periplasmic side the O units are polymerised at the non-reducing end

by Wzy via a block transfer mechanism to form the polymer. The chain length of the final

Oag is regulated by the protein Wzz (Wang et al., 2010a; Whitfield, 2006; Woodward et al.,

2010).

Introduction

25

Figure 1.6 Synthesis of R3 type LPS core sugar S. flexneri has R3 type LPS core sugar. The known enzymes responsible for the synthesis of

R3 core sugar are shown. Enzymes WaaA, WaaC, WaaF, WaaY, WaaQ, WaaP, and WabB

synthesise inner core region; and WaaG, WaaI, WaaJ, and WaaD are responsible for the

synthesis of outer core region (Kaniuk et al., 2004).

Introduction

26

S. flexneri follows the Wzy-dependent pathway for Oag biosynthesis. In S. flexneri

most of the Oag biosynthesis genes (except wecA) are located in the Oag biosynthesis locus

between galF and his (previously known as rfb locus) (Allison & Verma, 2000; Morona et al.,

1995) (Fig. 1.7) and the coding region for Oag biosynthesis genes is ~11 kb (Macpherson et

al., 1991). Initially, GlcNAc-phosphate (GlcNAc-1-P) is transferred from an UDP-GlcNAc to

undecaprenol phosphate (Und-P) at the cytoplasmic side of the IM by WecA. Then the

rhamnosyl transferases RfbG and RfbF add Rha residues from dTDP-Rha to the GlcNAc

(Macpherson et al., 1994; Morona et al., 1994) to form the O unit. Then rest of the proteins of

the Wzx/Wzy pathway (Wzx, Wzy, Wzz, and WaaL) complete the Oag biosynthesis (Fig.

1.8).

In the ABC transporter pathway the complete Oag chain is synthesised on the

cytoplasmic side of the IM, and the ABC transporter proteins Wzm and Wzt translocate the

Oag chain. This pathway has been described for the Oags of Klebsiella pneumoniae, Vibrio

cholerae, Yersinia enterocolitica, A-band Oag of Pseudomonas. aeruginosa, and also for the

Oags of E. coli O8, O9, O9a, O52, and O99 (Wang et al., 2010a). The Synthetase pathway is

very rare with S. enterica O54 the only known case for Oag synthesis by Synthetase pathway.

A single integral protein performs the whole Oag synthesis process and the Oag chain is

homopolymers or having two sugars (Raetz & Whitfield, 2002; Wang et al., 2010a). For all of

the three pathways, Oag ligase WaaL then ligates the Oag chains to the core-lipid A. The

complete LPS is then translocated to the OM of the bacterial cell (Whitfield & Trent, 2014).

1.7.3 LPS export

The first step of LPS export is the flipping of the LPS across the IM. The ABC transporter IM

protein MsbA translocates the R-LPS (lipid A-core) across the IM. In Oag producing strains,

the Oag is then ligated to the lipid A-core by WaaL ligase. The Lpt (LPS transport) proteins

(LptA-G) transport the LPS from the IM to the cell surface (Chng et al., 2010; Ruiz et al.,

2009; Sperandeo et al., 2009).

Introduction

27

Figure 1.7 Organisation of S. flexneri Oag biosynthesis genes S. flexneri Oag biosynthesis genes (except wecA) are located in the Oag biosynthesis locus

between galF and his. The direction of transcription is indicated by horizontal red arrows.

Figure adapted from Morona et al. (1995).

Introduction

28

Figure 1.8 S. flexneri Y serotype Oag biosynthesis S. flexneri Oag biosynthesis occurs on either side of the IM. Initially, GlcNAc-1-P is

transferred from an UDP-GlcNAc to Und-P at the cytoplasmic side of the IM by WecA. Then

the rhamnosyl transferases add Rha residues to the GlcNAc to form the O unit. Wzx

translocates this O unit to the periplasmic side. At the periplasmic side the O units are

polymerised by Wzy and the chain length of the final Oag is regulated by the protein Wzz.

WaaL then ligates the Oag chains to the core-lipid A.

Introduction

29

Recent structural studies have disclosed the molecular mechanism of LPS transport of

the Gram-negative bacteria (Bishop, 2014; Dong et al., 2014; Qiao et al., 2014). ABC

transporter complex made of LptF, LptG, and LptB separates the LPS from the external

leaflet of the IM and takes the LPS through a filament (made of LptA and LptC) which

connects IM and OM. Then the LPS is delivered to a complex made of LptD and LptE in the

OM and this complex inserts the LPS into the outer leaflet of the OM. The X-ray crystal

structure of the LptD and LPtE complex from S. flexneri and Salmonella typhimurium showed

that LptD is made of "-strands that fold into two domains: a "-jellyroll and a "-barrel. The "-

jellyroll is away from the OM and contains a greasy groove that is able to bind Lipid A

leaving the core and Oag chain exposed. A similar "-jellyroll fold was found for LptA and

LptC. These proteins interconnect and combine the greasy "-jellyroll fold to make a passage

through the space between IM and OM. LptD forms a 26 stranded "-barrel in which the LptE

forms a roll-like structure. The hydrophilic sugar chains (core and Oag) pass through the

barrel and the Lipid A inserts into the external leaflet of the OM through a lateral opening of

the LptD between "1 and "26 (Bishop, 2014; Dong et al., 2014; Qiao et al., 2014; Whitfield

& Trent, 2014).

1.8 Oag polymerisation protein Wzy

The evidence of Oag polymerisation occurring in the periplasm was detected by McGrath and

Osborn (1991) by pulse-chase experiments using doubly conditional mutants. The Wzy-

dependent pathway is one of the most widely distributed polysaccharide biosynthesis pathway

in the nature. In Wzy homologues, there are 4-5 periplasmic loops (PLs), the larger PLs are on

the periplasmic side, and the number of transmembrane segments (TMs) vary from 10-14

(Daniels et al., 1998; Islam et al., 2010; Kim et al., 2010; Marczak et al., 2013; Mazur et al.,

2003; Zhao et al., 2014). So far there is no known X-ray crystal structure for Wzy

homologues and topology models only exist for Wzy proteins of S. flexneri (Daniels et al.,

1998), Pseudomonas aeruginosa PAO1 (Islam et al., 2010); and PssT (Wzy homologue) of

Rhizobium leguminosarum bv. trifolii strain TA1 (Mazur et al., 2003).

Introduction

30

1.8.1 S. flexneri Wzy (WzySf)

The Oag polymerisation protein Wzy is encoded by the rfc/wzy gene (Macpherson et al.,

1991) (Fig. 1.7); and Morona et al. (1994) for the first time characterised S. flexneri wzy gene

(wzySf) which spans a ~2 kb region. The wzy open reading frame is located downstream of

Oag gene cluster. WT wzySf lacks detectable ribosome binding site and has four rare codons

(at positions 4, 9, 22, and 23) in the translation initiation site. The wzySf coding region has a

low G+C % and has high percentage of minor codons in the first 25 amino acids. S. flexneri

Wzy protein (WzySf) is a 43.7 kDa hydrophobic integral membrane protein (Daniels et al.,

1998; Morona et al., 1994). Daniels et al. (1998) for the first time was able to express and

visualise WzySf, and they showed by western immunoblotting using anti::PhoA serum that

WzySf::PhoA fusion protein was able to form a dimer. Based on the topological model

proposed by Daniels et al. (1998), WzySf has 12 TMs, two large PLs - PL3 and PL5; and the

amino and carboxy terminal ends are located on the cytoplasmic side of the IM (Fig. 1.9).

wzySf mutants produce SR-LPS (Morona et al., 1994). They are avirulent in Serény test and

unable to produce plaque on tissue culture cells, and have IcsA distributed over the entire cell

surface (Sandlin et al., 1996).

1.8.2 Wzy proteins of different bacterial species

Wzy proteins have extremely low detectability in cells (Abeyrathne & Lam, 2007; Collins &

Hackett, 1991). Optimising conditions for overexpression and purification of Wzy is a

laborious and time-consuming process due to the hydrophobic and transmembrane nature of

the protein, presence of rare codons in the 5’ regions, and weak ribosomal binding site (Kim

et al., 2010; Wong et al., 1999). So far, limited work has been conducted on biochemical and

functional characterisation of Wzy proteins of different bacterial species. Kim et al. (2010)

performed site-directed mutagenesis on the Francisella tularensis LVS wzy (wzyFt) and found

that modifications of the residues G176, D177, G323, and Y324 resulted in loss of Oag

Introduction

31

Figure 1.9 S. flexneri Wzy (WzySf)

WzySf is an integral membrane protein. It has 12 TMs and two large PLs (PL3 and PL5). The

positions of the PLs (PL1-6) and TMs (TM1-12), and cytoplasmic loops (CL) (CL1-5) are

indicated. WzySf topology map is adapted from Daniels et al. (1998).

Introduction

32

polymerisation by F. tularensis Wzy (WzyFt). Comparing amino acid sequences and predicted

theoretical topology models by TMHMM server of the Oag polymerase of different bacterial

species, and the previously determined Shigella flexneri topology model by Daniels et al.

(1998), they found that these amino acids are in close proximity on the bacterial surface (Kim

et al., 2010). Woodward et al. (2010) for the first time were able to purify a Wzy protein,

from E. coli O86 (WzyEc), and suggested that WzyEc may form dimers. They also established

an in vitro Oag polymerisation system using the purified WzyEc (Woodward et al., 2010).

Extensive work on Pseudomonas aeruginosa PAO1 Wzy (WzyPa) was performed by

Islam et al. (Islam et al., 2010; Islam et al., 2011; Islam et al., 2013). They found that PL3

and PL5 of WzyPa contain RX10G motifs and also identified sequence conservation between

PL3 and PL5 of WzyPa. Site-directed mutagenesis on the Arg residues within these two

RX10G motifs showed that these Arg residues are important for WzyPa function. They found

that at physiological pH PL3 possesses a net positive charge (pI 8.59) and PL5 possesses a net

negative charge (pI 5.49). From these findings they proposed a “catch-and-release”

mechanism of Oag polymerisation by Wzy. According to this model PL3 acts as a “capture

arm” and catches incoming negatively charged Oag subunit for subsequent transfer to PL5,

which acts as a “retention arm” and involves relatively transient interaction with the Oag. PL5

is associated with constant binding and release of the growing Oag (Islam et al., 2011). They

claimed a widespread presence of the “catch-and-release” mechanism by finding homologues

Wzy proteins to WzyPa in other bacteria using a “jackhammer” search. Interestingly, their

search was unable to find any Wzy homologues to WzyPa in Enterobacteriaceae (Islam et al.,

2013).

Zhao et al. (2014) showed that WzyEc works in a distributive manner where the

polymerisation product leaves the active site of WzyEc after each round of reaction. They

found that at physiological pH the major PLs (PL3 and PL4) of WzyEc are positively and

negatively charged (pI values 9.08 and 4.29, respectively). Hence, they proposed that WzyEc

also follows the catch-and-release mechanism. However, the number of TMs and amino acid

sequence of WzyEc are different from WzyPa (Zhao et al., 2014).

PssT protein in R. leguminosarum is the counterpart of the Gram-negative bacterial

Wzy. Mazur et al. (2003) predicted the topology model of PssT having 12 TM and a large PL

Introduction

33

between TM9 and 10. Initially, a RX10G motif was found in PssT between amino acids 350-

361 and then bioinformatics analysis identified two new RX10G motifs between amino acids

159-170 and 229-240. According to the previously predicted topology model these new

RX10G motifs are present in cytoplasmic loops (CL) 2 and 3 but studies of PssT-PhoA fusion

protein Ala-158 suggested translocation of these segment to the periplasm (Marczak et al.,

2013). Hence, Marczak et al. (2013) proposed that these motifs may have important role in

PssT polymerisation activity similar to WzyPa.

There is little knowledge about the substrate specificity and cross complementation of

different Wzy proteins. Studies showed that in Salmonella enterica A, B1, and D1 Wzy

proteins from different serogroups cross-complemented and produced relevant Oags (Makela,

1965; Nurminen et al., 1971; Valtonen et al., 1975) but were unable to produce Oag of

serogroup C2 (Naide et al., 1965). WzySf was also unable to replace WzyFt and synthesise

relevant Oag (Kim et al., 2012).

1.8.3 Comparison of WzySf with other Wzy proteins

There is very little sequence identity between wzy gene and Wzy proteins of different

bacterial species (Kim et al., 2010). The number of TMs in different Wzy proteins also varies:

WzyFt has 11 TMs (WzyFt topology model generated using TMHMM-2.0 and S. flexneri

topology model), WzyPa has 14 TMs, WzyEc has 10 TMs; however, WzySf and PssT have 12

TMs (Daniels et al., 1998; Islam et al., 2011; Kim et al., 2010; Mazur et al., 2003; Zhao et

al., 2014). WzyPa, WzyFt, and WzySf have two major PLs - PL3 and PL5 (Daniels et al., 1998;

Islam et al., 2011; Kim et al., 2010), WzyEc has two major PLs - PL3 and PL4 (Zhao et al.,

2014); however PssT has only one major PL between TM9 and TM10 (Mazur et al., 2003).

There is also a difference in the charged property of substrate of different Wzy proteins such

as, the Oag of P. aeruginosa is negatively charged (Knirel et al., 2006) but the Oag of WzySf

is neutral. WzySf is also very flexible about its substrate recruitment compared to other Wzy

proteins as polymerisation of Oags of all the serotypes of S. flexneri are performed by a single

type of WzySf.

Introduction

34

1.9 Oag chain length regulator Wzz

Bacterial cell surface polysaccharides are regulated by the members of a large family of

proteins known as polysaccharide co-polymerase (PCP) family, anchored in the IM. PCPs are

subdivided into three classes (PCP1, PCP2, and PCP3) based on different characteristics such

as their association with a Wzy-dependent or ATP-binding cassette (ABC) transporter-

dependent pathway, and by the presence or absence of an additional cytoplasmic domain

(Morona et al., 2000b; Morona et al., 2009; Purins et al., 2008; Tocilj et al., 2008). All the

PCPs share some common structural features: N-terminal and C-terminal transmembrane

helices (transmembrane helices 1 and 2) separated by a polypeptide segment (~130 to 400

amino acid residues) with a coiled-coil region located externally or in the periplasm, and a

proline or glycine rich motif adjacent to the C-terminal transmembrane helix (Tocilj et al.,

2008). PCP1 and PCP2 proteins are involved in the Wzy-dependent pathway; and PCP3

proteins are involved in ABC dependent capsular polysaccharide synthesis (Morona et al.,

2009).

1.9.1 S. flexneri Wzz

The modal length of the Oag chain is regulated by Wzz proteins, members of the PCP1 family

(Morona et al., 2000a). Initially, wzz was named as regulator of O-chain length (rol) or chain

length determinant (cld) (Bastin et al., 1993; Batchelor et al., 1991). S. flexneri 2a has S-LPS

with two types of modal chain length (Fig. 1.10), short (S) type (11-17 Oag RUs) and very

long (VL) type (>90 Oag RUs), and the S-type and VL-type Oag chain lengths are determined

by WzzSf and WzzpHS2, respectively (Morona et al., 2003; Van den Bosch & Morona, 2003).

Both WzzSf and WzzpHS2 are members of the PCP1 subclass (Morona et al., 2009). Wzz is

characterised by two transmembrane helices and a large PL that comprises more than 85% of

the protein. Special feature of the PL of Wzz is the coiled coil formed by three regions

Introduction

35

Figure 1.10 S. flexneri modal chain length LPS from 108 bacteria of S. flexneri 2a strain 2457T was electrophoresed on a 15% (w/v)

polyacrylamide gel and silver stained. The VL-type LPS and the S-type LPS are shown.

Figure adapted from May (2007).

Introduction

36

(Marolda et al., 2008; Morona et al., 2000b). The coiled coil feature is a bundle of #-helices

that wind to form a superhelix (Lupas, 1996; Marolda et al., 2008).

Daniels and Morona (1999) performed site-directed mutagenesis to understand the

functional importance of the conserved residues in the carboxy and amino terminal ends of

WzzSf and found that not only the carboxy and amino terminal ends, but the amino acid

residues of the entire length of WzzSf are important for the Oag modal chain length control by

WzzSf. They also showed that WzzSf forms a dimer in vivo and may oligomerise up to a

hexamer (Daniels & Morona, 1999). Marolda et al. (2008) performed mutagenesis in the

coiled coil region of WzzSf and found that mutagenesis in region I (amino acid 108-130)

resulted in partial defects on the Oag modal chain length, region II (amino acids 153-173)

resulted in elimination of WzzSf function, and region III (amino acids 209-233) had no effect

on the LPS Oag modal chain length. Mutations in the coiled coil region of WzzpHS2 also

resulted in loss of Oag modal chain length control (Purins et al., 2008). Papadopoulas and

Morona (2010) performed random in-frame linker mutagenesis on wzzSf and created five

different classes (I-V) of mutants with varied Oag modal chain length [inactive (I), shorter (II

and III), nearly wild type (IV), and increased (V)]. In vivo chemical cross-linking showed

that class V mutants formed high-molecular weight oligomers but classes II and III were

unable to cross-link. Mapping of the class V mutations on the three dimensional structures

located the mutations within the inner cavity of PCP oligomer. Hence, they concluded that

stable dimer formation may be important for Oag modal chain length control by WzzSf

(Papadopoulos & Morona, 2010). Tran and Morona (2013) performed single amino acid

substitution on E. coli FepE which is a PCP1a protein and confers very long Oag modal chain

length (>80 Oag RUs). Analysis of the LPS profiles conferred by FepE mutants and chemical

cross-linking properties of the mutant FepE oligomer suggested that FepE residues located

within the internal cavity of the FepE oligomer contribute to Oag modal chain length but not

the oligomeric state of the protein (Tran & Morona, 2013). However, study of chimeric

molecules by Kalynych et al. (2012b) suggested that differences in the modal chain length

depends on the surface exposed amino acid residues in specific regions of WzzSf rather than

the oligomeric state. Cross complementation and chimeric studies using wzz from

phylogenetically similar species: E. coli, Salmonella enterica, and S. flexneri showed that

inherent variability of different Wzz lies in the PLs (Daniels & Morona, 1999; Kalynych et

Introduction

37

al., 2011; Klee et al., 1997). Hence, the difference in chain length regulation by Wzz proteins

is dependent on the PLs. However, as mentioned before Daniels and Morona (1999) showed

that the entire WzzSf protein has role in Oag modal chain length regulation and recent studies

also showed that the TMs of Wzz are also important for modal chain length regulation (Islam

et al., 2013; Taylor et al., 2013).

Tran et al. (2014) analysed the colicin E2 (ColE2) sensitivity of all the classes of

WzzSf mutants generated by Papadopoulos and Morona (2010) and found that mutants with S-

type or long type (16-28 Oag RUs) modal chain lengths were more resistant to ColE2

compared to the mutants with intermediate S-type (8-14 Oag RUs), very S-type (2-8 Oag

RUs), and VL-type (>80 Oag RUs) Oag modal chain lengths. From this data they concluded

that the LPS Oag modal chain length control by WzzSf may be evolved due to selection

pressure from colicin in the environment (Tran et al., 2014).

1.9.2 Chain length and virulence

Oag is one of the virulence determinants of S. flexneri, however presence of Oag is not the

sole determinant of LPS for providing virulence but its modal chain length also plays a

critical role in the pathogenesis of the bacteria. S. flexneri wzz mutants are unable to either

form plaques on HeLa cell monolayer or form F-actin comet tails. Hence, WT Oag modality

is important for cell-to-cell spreading of S. flexneri (Morona et al., 2003). Studies showed that

S-type Oag modal chain is essential for the unipolar localisation of IcsA and efficient ABM

(Morona & Van Den Bosch, 2003a; Robbins et al., 2001; Sandlin et al., 1995). S. flexneri

strains with WzzpHS2 as the sole determinant of the Oag modal chain length and hence

producing VL-type Oag chain, are unable to form plaques on HeLa cell monolayer, having

reduced level of IcsA on cell surface, reduced virulence in Serény test, and reduced ability to

form F-actin comet tails (Van den Bosch et al., 1997). WzzSf and WzzpHS2 compete for the

available WzySf (Carter et al., 2009). Hong and Payne (1997) showed that WzzpHS2 is required

for resistance to serum killing however, WzzSf is required for invasion and intercellular spread

(Hong & Payne, 1997). Both WzzSf and WzzpHS2 are required for the optimal virulence

property of the bacteria.

Introduction

38

1.10 Association of the proteins of the Oag biosynthesis pathway

Understanding the association of the participant proteins of the Wzy-dependent Oag

biosynthesis pathway is important to understand the actual mechanism behind this

biosynthesis. For a long time several research group suggested the multi-protein complex

formation in the Wzy-dependent pathway (Marolda et al., 2006; Whitfield, 2006; Whitfield,

2010). There are several proposed mechanisms about these associations such as molecular-

clock model (Bastin et al., 1993) and molecular chaperone model (Morona et al., 1995).

According to the proposed molecular-clock model, Wzz acts as a molecular clock and

regulates Wzy activity between two states: the E, or extension, state favours polymerisation,

and the T, or transfer, state favours the ligation reaction (Bastin et al., 1993). The molecular-

chaperone model describes Wzz as a typical molecular chaperone that regulates the overall

ratio of Wzy and WaaL in a complex and controls the enzyme kinetics of the ligation reaction

to define the modality (Morona et al., 1995). Tocilj et al. (2008) suggested that Wzz may

form a scaffold that recruits Wzy.

Kintz and Goldberg (2011) proposed a ruler model, which suggested that Wzz periplasmic

barrel, acts as a ruler and the Oag chain length is determined by the direct physical interaction

of the Oag polymer with the barrel of the Wzz oligomer. A more compact barrel generates

shorter Oag chain length as the area of interaction between Wzz and Oag polymer is reduced.

According to them the amount of Wzy has no correlation with the chain length of Oag (Kintz

& Goldberg, 2011). According to the model of Kalynych et al. (2012a) the growing Oag chain

adopt a higher order structure and may interfere with the proper positioning within the Wzy-

binding site. Wzz binds with the growing Oag and allows further polymerisation. After

achieving certain length the Oag can no longer be bound with the Wzz and dissociates

(Kalynych et al., 2012a). However, both of these models suggested the direct interaction of

the Oag polymer with Wzz or Wzz and Wzy but did not suggest any interaction between Wzz

and Wzy.

Islam and Lam (2014) proposed a hybrid model based on the Kintz and Goldberg (2011)

and Kalynych et al. (2012a) models. According to the hybrid model the Wzy dimer binds

with the TMs of the Wzz protomer within the PCP conserved bell-shaped quaternary

structure. The Oag chain is elongated due to the polymerisation by Wzy and the higher order

Introduction

39

Oag structure destabilises the interaction of the polymer with Wzz. As the tip of the growing

Oag chain reaches the apex of the Wzz bell, the mechanical feedback of the interaction of Oag

polymer and Wzz transmits through the Oag chain to the basal Oag RU in the Wzy active site

and the Oag chain dissociates from Wzy (Islam & Lam, 2014). However, there is no direct

evidence to date on how these proteins are associated except the study of Marolda et al.

(2006) that provided the genetic data about the interaction of Wzx, Wzz, and Wzy. The work

of Carter et al. (2009) contradicts their data and suggested that may be there is no direct

physical interaction between WzzSf and WzySf.

Woodward et al. (2010) provided evidence supporting the association of Wzz and Wzy

through their in vitro polymerisation assay and suggested that Wzz and Wzy are enough to

shape the Oag chain. Islam et al. (2013) suggested that the chain length of the Oag is

determined by the interaction of Wzz and Wzy. Taylor et al. (2013) showed that the inhibitor

of # polymerase (Iap) peptide inhibits Wzy# (Oag polymerase of P. aeruginosa PAO1

serotype O5) by mimicking the Wzz TM segment and provided the evidence of direct

interaction between Wzz and Wzy. Bacterial two-hybrid system analysis in R. leguminosarum

showed that PssP protein, which is a PCP, interacts with PssL and PssT, which are Wzx and

Wzy, respectively (Marczak et al., 2013; Marczak et al., 2014). However, to date there is a

lack of evidence on the interaction of Wzy with the other proteins of the Wzy-dependent Oag

biosynthesis pathway using more direct approaches. Several studies pointed towards the

complex formation of the IM proteins during the Wzy-dependent pathway. Hence a direct

evidence of the complex formation is required to confirm these models and to know the actual

mechanism behind this pathway.

1.11 Aims and hypotheses

WzySf is an important protein in synthesising Oag, the key virulence determinant of S.

flexneri. WzySf was identified more than 20 years ago (Morona et al., 1994) but there is lack

of knowledge about its functional amino acid residues and its association with the other

proteins of the Oag biosynthesis pathway. In this thesis, I have investigated the biochemical

Introduction

40

and functional characteristics of WzySf. Based on the earlier studies following hypotheses

were generated:

1. WzySf has variety of regions that are important for Oag polymerisation and chain

length control.

2. WzySf contains Arg residues in PL3 and PL5 which are essential for Oag

polymerisation.

3. WzySf interacts with WzzSf .

4. WzySf interacts physically with the other proteins of the Wzy-dependent pathway.

The following aims investigated the hypotheses:

1. To construct a suitable wzySf expression system for performing experiments to

understand the biochemical and functional characteristcs of WzySf.

2. To perform random mutagenesis on wzySf to identify the amino acid residues important

for WzySf polymerisation function.

3. To perform site-directed mutagenesis on the Arg residues on PL3 and PL5 of WzySf

and characterising the mutants based on their polymerisation activity and association

with WzzSf.

4. To optimise a purification method of S. flexneri WzySf.

5. To perform in vivo cross-linking followed by purification of WzySf to monitor the

direct physical association of the proteins of the Wzy-dependent pathway.

1.12 Thesis Organisation

The dissertation is organised as follow (Table 1.2).

Introduction

41

Table 1.2 Overview of the chapters in this PhD thesis

Chapter Purpose and investigated research aims

Chapter 1 (Introduction) Introduces the research domain and provides context of the research

Chapter 2 (Materials and Methods)

Describes the experimental methods and the materials used to perform the studies

Chapter 3 Construction of a wzySf expression system, identifying functional amino acid residues of WzySf, understanding the role of WzzSf in the Oag polymerisation (Aims 1 and 2)

Chapter 4 Identifying the importance of the Arg residues in PL3 and PL5 of WzySf and characterising the WzySf mutants based on their polymerisation function and association with WzzSf (Aim 3)

Chapter 5 Purifying WzySf, investigating dimer formation of WzySf and relation of the dimer formation with the functioning of the protein, monitoring the direct physical interaction of the proteins of the Wzy-dependent pathway (Aims 4 and 5)

Chapter 6 (Conclusions) Concludes the thesis by discussing key contributions and direction for future research

42

43

Chapter 2

Materials and Methods


44

Chapter 2: Materials and Methods

2.1 Bacterial strains and plasmids

A listing of Shigella flexneri and Escherichia coli host strains used in this work and a

complete list of strains constructed during this work is summarised in Table 2.1. Cloning

vectors and plasmids used in these studies are listed in Table 2.2.

2.2 Bacterial growth media and growth condition

2.2.1 Liquid growth media

All E.coli and S. flexneri strains were grown at 37°C in lysogeny broth (LB) (10 g/litre

tryptone, 5 g/litre yeast extract, 5 g/litre NaCl) and LB agar (LB broth, 15 g/litre Bacto agar).

Under induction conditions, 0.4 mM isopropyl-"-D-thiogalactopyranoside (IPTG) or 0.2%

(w/v) L-arabinose was added to the cultures, and they were grown for 20 h at 20°C.

Antibiotics were added as required to the media at the following final concentrations:

kanamycin (Km), 50 µg/ml, and chloramphenicol (Cm), 25 µg/ml, Ampicillin (Amp), 100

µg/ml. Liquid growth media were prepared in distilled water and sterilised by autoclaving.

2.2.2 Solid growth media

Bacteria were stored in a suspension of glycerol [30% (w/v), Invitrogen] and peptone [1%

(w/v), Difco] in Wheaton vials at -80°C. From these glycerol stocks fresh cultures were

prepared by streaking a loopful of the suspension onto LB agar with appropriate antibiotics,

followed by incubation for 16-18 h at 37°C to achieve adequate growth.

When blue/white colony screening was required, plates were supplemented with 40

µg/ml 5-bromo-4-chloro-3-indolyl-"-D-galactoside (X-Gal) and 0.5 mM IPTG that allowed

identification of recombinant plasmids in suitable E. coli via disruption of the LacZ# peptide

in pGEMT-Easy (Promega) cloning plasmids (Kovach et al., 1994).


45

Table. 2.1 Bacterial strains used in this study

Strain Relevant characteristics # Source E. coli DH5# F- endA1 glnV44 thi-1 recA1 relA1 gyrA96

deoR nupG #80dlacZ!M15 !(lacZYA-argF)U169 hsdR17(rK- mK+)

Lab stock

XL10-Gold Tet r!(mcrA)183 !(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F´ proAB lacIqZ!M15Tn10 (Tetr) Camr]

Stratagene

Lemo21(DE3)

fhuA2 (lon) ompT gal ($ DE3) (dcm) "hsdS/ pLemo(Cmr)

New England Biolabs

PNRM1 DH5# (pGEMT-Easy-wzySf) This study PNRM3 DH5# (pWaldo-TEV-GFP) This study PNRM4 DH5# (pRMPN1) This study PNRM15 Lemo21(DE3) (pRMPN1) This study PNRM22 XL10-Gold (pRMPN2) This study PNRM24 XL10-Gold (pRMPN3) This study PNRM26 XL10-Gold (pRMPN4) This study PNRM27 XL10-Gold (pRMPN5) This study PNRM 30 XL10-Gold (pRMPN6) This study PNRM53 XL10-Gold (pRMPN7) This study PNRM56 XL10-Gold (pRMPN8) This study PNRM57 XL10-Gold (pRMPN9) This study PNRM59 XL10-Gold (pRMPN10) This study PNRM61 XL10-Gold (pRMPN11) This study PNRM64 XL10-Gold (pRMPN12) This study PNRM66 XL10-Gold (pRMPN13) This study PNRM67 XL10-Gold (pRMPN14) This study PNRM69 XL10-Gold (pRMPN15) This study PNRM71 XL10-Gold (pRMPN16) This study PNRM73 XL10-Gold (pRMPN17) This study PNRM95 XL10-Gold (pRMPN19) This study PNRM98 XL10-Gold (pRMPN21) This study PNRM100 XL10-Gold (pRMPN22) This study PNRM103 XL10-Gold (pRMPN23) This study PNRM114 XL10-Gold (pRMPN24) This study


46

Table 2.1 continued Strain Relevant characteristics # Source PNRM116 XL10-Gold (pRMPN25) This study PNRM179 XL10-Gold (pRMPN27) This study PNRM182 XL10-Gold (pRMPN28) This study PNRM184 XL10-Gold (pRMPN29) This study PNRM185 XL10-Gold (pRMPN30) This study PNRM187 XL10-Gold (pRMPN31) This study PNRM209 XL10-Gold (pRMPN32) This study PNRM210 XL10-Gold (pRMPN33) This study PNRM211 XL10-Gold (pRMPN34) This study PNRM212 XL10-Gold (pRMPN36) This study PNRM230 XL10-Gold (pRMPN35) This study S. flexneri PE638 S. flexneri Y rpoB (Rifr) (Morona et al., 1995) RMM109 PE638 !wzy (Rifr) (Morona et al., 1994) RMA4337 RMM109 !wzz (Rifr Tetr) This study PNRM5 RMM109 (pWaldo-TEV-GFP) This study PNRM6 RMM109 (pAC/pBADT7-1) This study PNRM7 PE638 (pWaldo-TEV-GFP) This study PNRM8 PE638 (pAC/pBADT7-1) This study PNRM9 RMM109 (pRMPN1) This study PNRM10 PE638 (pRMPN1) This study PNRM11 PNRM6 (pWaldo-TEV-GFP) This study PNRM12 PNRM8 (pAC/pBADT7-1) This study PNRM13 PNRM6 (pRMPN1) This study PNRM14 PNRM10 (pAC/pBADT7-1) This study PNRM16 PNRM6 (pRMPN2) This study PNRM17 PNRM6 (pRMPN3) This study PNRM18 PNRM6 (pRMPN4) This study PNRM19 PNRM6 (pRMPN5) This study PNRM20 PNRM6 (pRMPN6) This study PNRM75 PNRM6 (pRMPN7) This study PMRM76 PNRM6 (pRMPN8) This study PNRM77 PNRM6 (pRMPN9) This study PMRM78 PNRM6 (pRMPN10) This study


47

Table 2.1 continued Strain Relevant characteristics # Source PMRM79 PNRM6 (pRMPN11) This study PMRM80 PNRM6 (pRMPN12) This study PMRM81 PNRM6 (pRMPN13) This study PMRM82 PNRM6 (pRMPN14) This study PMRM83 PNRM6 (pRMPN15) This study PMRM84 PNRM6 (pRMPN16) This study PMRM85

PNRM6 (pRMPN17) This study PMRM87 PNRM7 (pRMPN2) This study PMRM88 PNRM7 (pRMPN3) This study PMRM89 PNRM7 (pRMPN5) This study PMRM90 PNRM7 (pRMPN6) This study PMRM91 PNRM7 (pRMPN7) This study PMRM92 PNRM7 (pRMPN8) This study PMRM93 PNRM7 (pRMPN15) This study PMRM94 PNRM7 (pRMPN16) This study PMRM119 PNRM6 (pRMPN19) This study PMRM120 PNRM6 (pRMPN20) This study PMRM121 PNRM6 (pRMPN22) This study PMRM122 PNRM6 (pRMPN23) This study PMRM123 PNRM6 (pRMPN24) This study PMRM124 PNRM6 (pRMPN25) This study PNRM126 RMA4337 (pAC/pBADT7-1) This study PMRM127 PNRM126 (pRMPN2) This study PMRM128 PNRM126 (pRMPN3) This study PMRM129 PNRM126 (pRMPN5) This study PMRM130 PNRM126 (pRMPN6) This study PMRM131 PNRM126 (pRMPN7) This study PMRM132 PNRM126 (pRMPN15) This study PMRM133 PNRM126 (pRMPN16) This study PMRM134 PNRM126 (pRMPN1) This study PMRM135 PNRM126 (pWaldo-TEV-GFP) This study PMRM136 PNRM126 (pRMPN8) This study PMRM137 PNRM126 (pRMPN10) This study


48

Table 2.1 continued Strain Relevant characteristics # Source PNRM140 PNRM126 (pRMPN13) This study PNRM141 PNRM126 (pRMPN14) This study PMRM142 PNRM126 (pRMPN9) This study PMRM143 PNRM126 (pRMPN11) This study PMRM144 PNRM126 (pRMPN19) This study PMRM145 PNRM126 (pRMPN24) This study PMRM146 PNRM126 (pRMPN25) This study PMRM147 PNRM126 (pRMPN23) This study PMRM148 PNRM126 (pRMPN21) This study PMRM149 PNRM126 (pRMPN22) This study PMRM150 PNRM126 (pRMPN12) This study PMRM151 PNRM126 (pRMPN17) This study PNRM153 PNRM126 (pRMPN4) This study PNRM159 PNRM13 (pWSK29-wzzSf) This study PNRM161 PNRM13 (pWSK29) This study PNRM190 PNRM6 (pRMPN27) This study PNRM192 PNRM6 (pRMPN28) This study PNRM194 PNRM6 (pRMPN29) This study PNRM196 PNRM6 (pRMPN30) This study PNRM198 PNRM6 (pRMPN31) This study PNRM216 PNRM6 (pRMPN32) This study PNRM218 PNRM6 (pRMPN33) This study PNRM220 PNRM6 (pRMPN34) This study PNRM222 PNRM6 (pRMPN36) This study PNRM232 PNRM6 (pRMPN35) This study PNRM246 PNRM126 (pRMPN27) This study PNRM248 PNRM126 (pRMPN28) This study PNRM250 PNRM126 (pRMPN29) This study PNRM252 PNRM126 (pRMPN30) This study PNRM252 PNRM126 (pRMPN30) This study PNRM254 PNRM126 (pRMPN31) This study PNRM256 PNRM126 (pRMPN32) This study PNRM258 PNRM126 (pRMPN33) This study


49

Table 2.1 continued Strain Relevant characteristics # Source PNRM260 PNRM126 (pRMPN34) This study PNRM262 PNRM126 (pRMPN35) This study PNRM264 PNRM126 (pRMPN36) This study PNRM271 RMM109 (pWSK29-wzzSf) This study PNRM289 PNRM16 (pWSK29-wzzSf) This study PNRM293 PNRM122 (pWSK29-wzzSf) This study PNRM299 PNRM85 (pWSK29-wzzSf) This study PNRM301 PNRM192 (pWSK29-wzzSf) This study

# Rifr, rifampicin resistant; Kmr, kanamycin resistant; Cmr, chloramphenicol resistant; Tetr,

tetracycline resistant. $DE3 is $ sBamHIo !EcoRI-B int::(lacI::PlacUV5::T7 gene1)i21

!nin5. pLemo is pACYC184-PrhaBAD-lysY.


50

Table 2.2 Plasmids used in this study

Plasmid Description # Source pGEMT-Easy Cloning vector Promega pRMCD6 Source of wzySf (modified codons at

positions 4, 9, and 23) (Daniels et al., 1998)

pAC/pBADT7-1 Source of T7 RNA polymerase; Cmr (McKinney et al., 2002) pWaldo-TEV-GFP Cloning vector with GFP tag; Kmr (Waldo et al., 1999) pGEMT-Easy-wzySf wzySf with BamHI and KpnI sites This Study pRMPN1 pWaldo-wzySf-TEV-GFP This Study pCACTUS Suicide vector containing sacB, Cmr,

and orits (Morona et al., 1995)

pRMA577 Suicide vector contatining SphI-SphI fragment with the rol gene

(Morona et al., 1995)

pCACTUS-wzzSf::Tcr Suicide mutagenesis construct to construct the strain RMA4337

This Study

pWSK29 Cloning vector; Ampr (Wang & Kushner, 1991) pWSK29-wzzSf pWSK29 with S. flexneri 2a wzzSf (Murray et al., 2006)

pRMPN2 pRMPN1 with R164A point mutation in the wzySf

This Study


This Study


This Study


This Study


This Study

pRMPN7 pRMPN1 with G130V point mutation in the wzySf

This Study

pRMPN8 pRMPN1 with L11I point mutation in the wzySf

This Study

pRMPN9 pRMPN1 with N86K point mutation in the wzySf

This Study

pRMPN10 pRMPN1 with L28V point mutation in the wzySf

This Study

pRMPN11 pRMPN1 with P165S point mutation in the wzySf

This Study


51

Table 2.2 continued Plasmid Description # Source

pRMPN12 pRMPN1 with G82C point mutation in the wzySf

This Study

pRMPN13 pRMPN1 with N147K point mutation in the wzySf

This Study

pRMPN14 pRMPN1 with L191F point mutation in the wzySf

This Study

pRMPN15 pRMPN1 with L214I point mutation in the wzySf

This Study

pRMPN16 pRMPN1 with P352H point mutation in the wzySf

This Study

pRMPN17 pRMPN1 with V92M point mutation in the wzySf

This Study

pRMPN19 pRMPN1 with F52Y point mutation in the wzySf

This Study

pRMPN21 pRMPN1 with F52C/I242T point mutation in the wzySf

This Study

pRMPN22 pRMPN1 with C60F point mutation in the wzySf

This Study

pRMPN23 pRMPN1 with Y137H point mutation in the wzySf

This Study

pRMPN24 pRMPN1 with L49F/T328A point mutation in the wzySf

This Study

pRMPN25 pRMPN1 with F54C point mutation in the wzySf

This Study

pRMPN27 pRMPN1 with R164K point mutation in the wzySf

This Study


This Study


This Study


This Study


This Study

pRMPN32 pRMPN1 with R164E point mutation in the wzySf

This Study


This Study


This Study


52

Table 2.2 continued Plasmid Description # Source


This Study


This Study

# Kmr, kanamycin resistant; Cmr, chloramphenicol resistant; Tcr or Tetr, tetracycline resistant.


53

2.3 Chemicals and reagents

Unless otherwise stated chemicals and reagents were sourced from the following suppliers:

Promega, Invitrogen, Sigma-Aldrich, Roche, BD, Qiagen, New England Biolabs, and

Anatrace. Key chemicals used in this study were L-arabinose (Sigma, Catalogue number

A3256), IPTG (Biovectra, Catalogue number 1882), Sodium dodecanoyl sarcosine (Anatrace,

Catalogue number S300), n-Dodecyl-"-D-maltopyranoside (DDM, Sigma Catalogue number

D4641), 40% Bis-Acrylamide solution (BioRad, Catalogue number 161-0141), Ammonium

persulphate (BioRad, Catalogue number 161-0700), N,N,N',N'-Tetramethyl-ethylenediamine

(TEMED, Sigma Catalogue number T22500), Formaldehyde (Sigma, Catalogue number

F8775), Proteinase K (Invitrogen, Catalogue number 25530-015), and Dithiobis(succinimidyl

propionate) (DSP) (Thermo Fischer Scientific, Catalogue Number 22585).

2.4 Antibodies and antisera

Affinity purified rabbit polyclonal anti-WzzSf was made by Daniels and Morona (1999). The

WzzSf antibody was used at 1:750 for Western immunoblotting. Mouse monoclonal His6

antibody (Genscript) was used at 1:50000 for Western immunoblotting. For Western

immunoblotting goat anti-rabbit horseradish-peroxidase(HRP)-conjugate or a goat anti-mouse

HRP-conjugate (KPL) was used as secondary antibody at 1:30,000.

2.5 Nucleic acid methods

2.5.1 Isolation of plasmid DNA and DNA preparation

Plasmid DNA was isolated by using the QIAprep spin miniprep kit (Qiagen) following the

manufacturer’s instructions. PCR amplified DNA and restriction enzymes digests were

purified using QIAquick PCR purification kit (Qiagen). Gel extracted DNA was purified

using PureLink gel extraction kit (Invitrogen).


54

2.5.2 Quantitation of DNA

Concentration of DNA was determined by the absorption measurement at 260 nm assuming

that an optical density (OD) of 1.0 is equal to 50 µg/ml double stranded DNA. Purity of DNA

was evaluated by the ratio of A260nm to A280nm and the DNA was considered pure and used for

further manipulation if the ratio is ~2.0.

2.5.3 Restriction enzyme digestion

Restriction digestion of plasmid DNA and PCR amplicons were prepared as per the

manufacturer’s (New England Biolabs) instructions. Where required the restriction enzymes

were heat inactivated prior to further manipulation according to the manufacturer’s

instructions.

2.5.4 Agarose gel electrophoresis

DNA samples for electrophoresis were mixed with one-tenth volume of tracking dye [0.1 %

(w/v) bromophenol blue, 20% (v/v) glycerol, 0.1 mg/ml RNase; heated at 100°C for 30 min]

and separated on 1% (w/v) agarose gels using 1x TBE buffer (67 mM Tris, 22 mM boric acid,

1 mM EDTA) at 120 V for 45 min. EcoRI-restricted bacteriophage SPP1 known standards

were used to determine the sizes of the DNA. The SPP1 sizes (kb) were: 8.51, 7.35, 6.11,

4.84, 3.59, 2.81, 1.95, 1.86, 1.51, 1.39, 1.16, 0.98, 0.72, 0.48, 0.36, and 0.09. The EcoRI

digested SPP1 molecular weight standards were prepared as described previously (Ratcliff et

al., 1979). Gels were stained with 3x GelRed (Biotium) for 15-30 min. DNA bands were

visualised using GelDoc XR system (Biorad).

2.5.5 DNA sequencing

2.5.5.1 Sample preparation

A mixture of 600-1200 ng plasmid DNA and 1 µl primer was adjusted to 12 µl with Milli Q

water in a 1.5 ml reaction tube. The samples were sent to the Australian Genome Research


55

Facility (AGRF), Plant Genomic Centre, University of Adelaide, PMB1 Glen Osmond,

Urrbrae, SA 5042.

2.5.5.2 Capillary separation DNA sequencing

DNA sequencing of the plasmid DNA was carried out at AGRF using the ABI Prism Big Dye

Terminator Version 3.1 in an Eppendorf Mastercycler. The 12 µl of the total reaction volume

contained 1 µl of template DNA, 1 µl of primer, and 4 µl of BIG DYE reaction mix. The

sequencing reaction consisted of 25 cycles of DNA denaturation (95°C, 30 sec), primer

annealing (50°C, 15 sec), and extension (60°C, 4 min). The reaction mix was then precipitated

using the ethanol/sodium acetate method. Briefly, 8 µl Milli Q water was added to the

reaction mix to adjust the volume to 20 µl. Then 3 µl 3 M sodium acetate (pH 4.6), 62.5 µl

non-denatured 95% ethanol, and 14.5 µl Milli Q water were added to 20 µl reaction mix and

incubated at room temperature (RT) for 1 h. DNA was collected from the reaction mix as

pellet by centrifugation (13,200 x g, 20 min, RT, Eppendorf Centrifuge 5415R), DNA pellet

was washed in 250 µl 70% (v/v) ethanol, and was dried for 1 h at RT. Sequencing reactions

were analysed by AGRF.

2.5.5.3 Sequencing analysis

DNA sequencing data obtained from AGRF was analysed and aligned with the native DNA

sequence using DNAMAN (Version 4.22).

2.6 Polymerase chain reaction (PCR)

Oligonucleotides used in this study are purchased from GeneWorks (Adelaide) and Integrated

DNA Technologies (USA). The oligonucleotides are listed in Table 2.3. General PCR was

carried out in a 20 µl reaction mix containing 1x ThermoPol Reaction Buffer (NEB), 200 µM

dNTPs (Sigma), 50 µM of each primer, 100 ng DNA template (either plasmids or genomic

DNA), and 0.25 Unit Taq DNA polymerase (NEB). PCR reactions were performed in an


56

Table 2.3 Differenet primers made in this study

Primer* Sequence (5’-3’) # Purpose PN1_wzySfKpnF GGCGGTACCATGAATAATATTAATAAAATT

TTTATTACA amplification Of wzySf

PN2_wzySfBamHR GCGGGATCC TTTTGCTCCAGAAGT GAGGTTA

amplification of wzySf

ET35_F AGAGTAGAAAATAATAATGTTTCT

Construction of RMA4337

ET35_R GGCAAGCTTTTACTTCGCGTTGTAATTACG Construction of RMA4337

PN5_R164A_F AGCGAGTTCTTTTTTGCCCCCGATGGGGC R164A mutation PN6_R164A_R GCCCCATCGGGGGCAAAAAAGAACTCGCT R164A mutation PN9_R250A_F ATGCTTTACATGGTCGGATCAGCCAGTGAA

GATTCTGAC R250A mutation

PN10_R250A_R GTCAGAATCTTCACTGGCTGATCCGACCATGTAAAGCAT

R250A mutation

PN11_R258A_F GTGAAGATTCTGACTCTGTTGCCTTTAATGATTTATATTTTTATTATAAAAATGTTG

R258A mutation

PN12_R258A_R CAACATTTTTATAATAAAAATATAAATCATTAAAGGCAACAGAGTCAGAATCTTCAC

R258A mutation

PN13_R278A_F GCGACGTTCTTGTTTGGAGCCGGATTTGGTTCATTTATATTAG

R278A mutation

PN14_R278A_R CTAATATAAATGAACCAAATCCGGCTCCAAACAAGAACGTCGC

R278A mutation

PN15_R289A_F TCATTTATATTAGATCGATTAGCCATTGAAATAGTACCTCTTGAG

R289A mutation

PN16_R289A_R CTCAAGAGGTACTATTTCAATGGCTAATCGATCTAATATAAATGA

R289A mutation

PN27_R164K_F GACTAGCGAGTTCTTTTTTAAACCCGATGGGGC

R164K mutation

PN28_R164K_R GCCCCATCGGGTTTAAAAAAGAACTCGCTAGTC

R164K mutation

PN29_R250K_F ATGCTTTACATGGTCGGATCAAAAAGTGAAGATTCTGAC

R250K mutation

PN30_R250K_R GTCAGAATCTTCACTTTTTGATCCGACCATGTAAAGCAT

R250K mutation

PN31_R258K_F CGGATCACGCAGTGAAGATTCTGACTCTGTTAAATTTAATGATT

R258K mutation

PN32_R258K_R AATCATTAAATTTAACAGAGTCAGAATCTTCACTGCGTGATCCG

R258K mutation


57

Table 2.3 continued Primer* Sequence (5’-3’) # Purpose PN33_R278K_F GCGACGTTCTTGTTTGGAAAAGGATTTGGTT

CATTTATATTAG R278K mutation

PN34_R278K_R CTAATATAAATGAACCAAATCCTTTTCCAAACAAGAACGTCGC

R278K mutation

PN35_R289K_F GGTTCATTTATATTAGATCGATTAAAAATTGAAATAGTACCTCTTGAG

R289K mutation

PN36_R289K_R CTCAAGAGGTACTATTTCAATTTTTAATCGATCTAATATAAATGAACC

R289K mutation

PN41_R164E_F GACTAGCGAGTTCTTTTTTGAGCCCGATGGGGC

R164E mutation

PN42_R164E_R GCCCCATCGGGCTCAAAAAAGAACTCGCTAGTC

R164E mutation

PN43_R250E_F ATGCTTTACATGGTCGGATCAGAGAGTGAAGATTCTGAC

R250E mutation

PN44_R250E_R GTCAGAATCTTCACTCTCTGATCCGACCATGTAAAGCAT

R250E mutation

PN45_R258E_F CGGATCACGCAGTGAAGATTCTGACTCTGTTGAGTTTAATGATT

R258E mutation

PN46_R258E_R AATCATTAAACTCAACAGAGTCAGAATCTTCACTGCGTGATCCG

R258E mutation

PN47_R278E_F GCGACGTTCTTGTTTGGAGAGGGATTTGGTTCATTTATATTAG

R278E mutation

PN48_R278E_R CTAATATAAATGAACCAAATCCCTCTCCAAACAAGAACGTCGC

R278E mutation

PN49_R289E_F GGTTCATTTATATTAGATCGATTAGAGATTGAAATAGTACCTCTTGAG

R289E mutation

PN50_R289E_R CTCAAGAGGTACTATTTCAATCTCTAATCGATCTAATATAAATGAACC

R289E mutation

*F, Forward; R, Reverse; # Bold and underlined - restriction enzyme coding region; Bold -

changed codon.


58

Eppendorf Mastercycler Gradient PCR thermocycler with 25-35 amplification cycles adjusted

according to the DNA being amplified. Each standard cycle involved denaturation of the

template at 95ºC for 30 sec, annealing of primers to the DNA at a temperature ranging from

55-70ºC for 30 sec, and extension at 72ºC for 1 min/kb of DNA. Amplification of specific

gene sequences for cloning was performed in a 50 µl reaction volume in an Eppendorf

Mastercycler Gradient PCR thermocycler using Phusion high-fidelity DNA polymerase

(Finnzymes) following manufacturer’s protocol.

2.7 DNA purification

2.7.1 DNA gel extraction

10 µl tracking dye (See Section 2.5.4) was added to 50 µl overnight restriction enzyme

digested DNA samples and were electrophoresed in 1% agarose in TBE at 120 V for 45 min.

50 µl DNA sample was loaded into the center well and 4 µl of DNA samples were loaded into

the flanking wells. The flanking lanes were cut from the rest of the gel and stained with 3X

Gel Red. Flanking lanes were exposed to UV light and the desired DNA band was marked.

The central lane was then aligned with the marked flanking lanes and the DNA band of

interest was excised from the central lane. The extracted DNA was purified using the

PureLink Quick Gel Extraction kit (Invitrogen) following the manufacturer’s protocol. The

purified DNA was used in the ligation reactions.

2.7.2 Purification of PCR products

PCR products were purified using the QIAquick PCR purification Kit (Qiagen) to remove the

enzymes, oligonucleotides, and salts prior to use in the ligation reactions.

2.8 Ligation of DNA fragments into cloning vectors

Ligation reactions were performed following the manufacturer’s (NEB) protocol where PCR

products and plasmids for ligations were mixed in a molar ratio of 3:1 (insert:vector).


59

Ligation with the pGEMT-Easy vector was performed as instructed by the manufacturer

(Promega).

2.9 Transformation

2.9.1 Preparation of chemically competent cells

Overnight cultures were diluted 1:20 in 10 ml LB broth and incubated at 37ºC to an OD600 of

~0.4-0.6. Cultures were then centrifuged (2,200 x g, 10 min, 4ºC) to get the bacterial cell

pellet. The cell pellet was resuspended in ice-cold 0.1 M MgCl2, and re-centrifuged as before.

Then the cell pellet was resuspended in 1 ml ice-cold 0.1 M CaCl2 and incubated on ice for 1

h, followed by similar centrifugation as above. Finally, the bacterial cell pellet was

resuspended in 0.5 ml ice-cold 0.1 M CaCl2 containing 15% (v/v) glycerol. The chemically

competent bacterial cells were stored at -80ºC or used fresh.

2.9.2 Heat shock transformation

Chemically competent cells were thawed on ice for 10 min and mixed with 6-10 µl of ligation

product. The mix was then left on the ice for 30 min prior to 3 min heat shock in a 37°C water

bath followed by 5 min incubation on ice. 900 µl of SOC medium [20 g/litre tryptone (BD), 5

g/litre yeast extract, 2.5 mM KCl, 20 mM MgSO4, 8.6 mM NaCl] with 0.2% (w/v) glucose

was added to the mix and the mix was incubated at 37°C for 1-3 h to allow the expression of

the antibiotic resistance genes in the plasmids. Cells were plated onto LB agar plates with

appropriate antibiotics and incubated at 37°C for 16-18 h.

2.9.3 Preparation of electrocompetent cells

Overnight cultures were sub-cultured 1:20 in 10 ml LB broth and incubated at 37ºC to an

OD600 of ~0.4-0.6 and centrifuged (2,200 x g, 10 min, 4ºC) to get the cell pellet. The cell

pellet was resuspended in 10 ml ice-cold Milli Q water and centrifuged as before. The cell


60

pellet was again resuspended in 5 ml ice-cold Milli Q water and re-centrifuged as before. The

bacterial cell pellet was resuspended in 200 µl ice-cold 10% (v/v) glycerol. 100 µl aliquots of

the suspension were stored at -80°C or used fresh.

2.9.4 Electroporation

A 100 µl aliquot of the electrocompetent cells was thawed on ice, 2-5 µl of the plasmid DNA

was added to the aliquot, and then the cells and plasmid mix was added to a pre-chilled

electroporation cuvette (0.2 cm gap, Bio-Rad). The mix was then electroporated using an

electroporation device (Bio-Rad Gene Pulser, 2.5 kV, 25 µF, Pulse Controller 200 %). 900 ml

of SOC medium (Section 2.9.2) with 0.2% (w/v) glucose was added to the mix and incubated

for 1-3 h at 37°C. The electroporated cells were plated on LB agar with appropriate antibiotics

and incubated for 16-18 h at 37°C.

2.10 Mutagenesis

2.10.1 Random mutagenesis

Random mutagenesis was performed using the GeneMorph II EZ-Clone Domain Mutagenesis

Kit (Catalogue number 200552; Stratagene) according to the manufacturer’s instructions with

the primers PN1_wzySfKpnF and PN2_wzySfBamHR (Table 2.3). S. flexneri wzy (wzySf) coding

region in plasmid pRMPN1 (Table 2.2) was mutagenised with an error-prone DNA

polymerase. The mutagenised plasmids were transformed into competent XL10-Gold cells

(Agilent Technologies). Plasmid DNA was then isolated from randomly chosen transformed,

mutated colonies and transformed into strain PNRM6 (Table 2.1). Colicin swab assays were

performed to screen the mutants (see below). Plasmid DNA was isolated from putative

mutants, transformed into XL10-Gold cells, and subjected to DNA sequencing (AGRF,

Adelaide, Australia).


61

2.10.2 Site-directed mutagenesis

QuickChange Lightning Site-Directed Mutagenesis Kit (Catalogue number 210518,

Stratagene) was used to perform site-directed mutagenesis on wzySf in pRMPN1 plasmid

following the manufacturer’s instructions. Mutagenised plasmids were transformed into

XL10-Gold. Plasmids were isolated from the mutated strains and the precise position of the

mutation within the coding region was determined by DNA sequencing (AGRF, Adelaide,

Australia). Finally, the mutated plasmids were transformed into PNRM6. The oligonucleotide

primers used for site-directed mutation are listed in Table 2.3.

2.11 Characterisation of mutants

2.11.1 Colicin sensitivity assay

For the colicin sensitivity assay His6-colicin E2 (ColE2) with an initial concentration of 1

mg/ml was purified from E. coli BL21(DE3) carrying pET41b expressing C-terminal His-

tagged colicin E2 (Sharma et al., 2009; Tran et al., 2014).

2.11.1.1 ColE2 swab assay

A 2-fold serial dilution of 1 µg/ml ColE2 was swabbed onto antibiotic-selective LB agar

plates with a cotton swab. The plates were left to dry for 1 h at RT. Overnight cultures were

diluted 1:20 in LB with antibiotics and incubated at 37ºC to an OD600 of ~0.4-0.6 and

centrifuged. Cultures were induced where required. Individual cultures were then swabbed

perpendicular to the ColE2 streak, and the plates were left to dry for another 1 h at RT. The

plates were then incubated for 16-18 h at 37°C. The ColE2 susceptibilities of the test strains

were recorded and images were taken using a Canon scanner (CanoScan 9000F) against a

dark background.

2.11.1.2 ColE2 spot assay

A spot assay was performed using the serial dilutions of ColE2. Overnight cultures were


62

diluted 1:20 in LB with antibiotics, incubated at 37ºC to an OD600 of ~0.4-0.6 and centrifuged.

Cultures were induced where required. One hundred microlitres of the individual cultures

were spread onto LB agar plates with appropriate antibiotics. The plates were left to dry for 2

h at RT. A 2-fold serial dilution of 1 µg/ml ColE2 [denoted neat (N)] was spotted on the dried

plates, and the plates were left to dry for another 3 h at RT. The plates were then incubated for

16-18 h at 37°C. The endpoints of the killing zones of test strains were recorded. Images were

taken as described above.

2.11.2 Bacteriophage sensitivity assay

The procedures of phage propagation and phage stock preparation have been described

previously (Mavris et al., 1997; Morona et al., 1994). The concentration of the bacteriophage

Sf6c stock used was 8.6 x 107 p.f.u./ml. Overnight cultures were diluted 1:20 in LB with

antibiotics and incubated at 37ºC to an OD600 of ~0.4-0.6 and centrifuged. Cultures were

induced where required. 100 µl of the individual cultures was spread onto LB agar plates with

appropriate antibiotics. The plates were left to dry for 2 h at RT. Serial dilutions of the

bacteriophage Sf6c stock were spotted on the dried plates, and the plates were dried for a

further 3 h at RT. The plates were incubated for 18 h at 37°C. The phage sensitivities of the

test strains were recorded. Images were taken as described above.

2.12 Protein Techniques

2.12.1 Preparation of whole cell lysate

Overnight cultures were diluted 1:20 in LB with antibiotics and incubated at 37ºC to an OD600

of ~0.4-0.6 and centrifuged. Cultures were induced where required. Cultures equivalent to

5x108 bacteria were harvested by centrifugation (16,000 x g, 1 min, 4ºC, Eppendorf

Centrifuge 5415R). For Western immunoblotting the pellet was resuspended in 100 µl 1x

sample buffer [0.0625 M Tris- HCl, 10% (v/v) glycerol, 2% (w/v) SDS, 0.02% (v/v)

bromophenol blue, and 5% (v/v) "-mercaptoethanol ("-Me) (Sigma)]. For in-gel fluorescence

the pellet was resuspended in 20 µl PBS, and 20 µl buffer A [200 mM Tris-HCl (pH 8.8),


63

20% (v/v) glycerol, 5 mM EDTA (pH 8.0), 0.02% (w/v) bromophenol blue, 4% (w/v) SDS,

and 0.05 M dithiothreitol (DTT)] was then added to the cell suspension. Solubilised cell

suspensions were incubated at 37ºC for 5 min unless otherwise stated.

2.12.2 Preparation of whole membrane fraction

Overnight cultures were diluted 1:20 in LB with antibiotics and incubated at 37ºC to an OD600

of ~0.4-0.6 and centrifuged. Cultures were induced where required. Cells were harvested from

the 50 ml culture by centrifugation (9,800 x g, Beckman J2-21M induction drive centrifuge,

10 min, 4°C), and the cell pellet was resuspended in 4 ml sonication buffer (20 mM Tris-HCl,

150 mM NaCl, pH 7.5). The mixture was then lysed by sonication, followed by centrifugation

(2,200 x g, Sigma 3K15 tabletop centrifuge, 10min, 4°C) to remove debris.

Ultracentrifugation was performed in a Beckman Coulter Optima MAX-XP tabletop

ultracentrifuge (126,000 x g, 1 h, 4°C) to isolate the whole-membrane (WM) fraction. The

WM fraction was resuspended in PBS and then solubilised in buffer A (Section 2.12.1) and

was incubated at 37ºC for 5 min.

2.12.3 SDS-PAGE

Samples for SDS-PAGE were heated at 37ºC for 5 min prior to loading, unless otherwise

stated. Samples were electrophoresed on 15% (w/v) acrylamide gels in PAGE running buffer

[0.025 M Tris-HCl, 0.2 M glycine, 0.1% (w/v) SDS] at 200 V for 1-2 h using either the Bio-

Rad Mini-Protean System III or the Sigma vertical gel electrophoresis unit. BenchMark Pre-

stained Protein standard (Invitrogen; 190 kDa, 120 kDa, 85kDa, 60 kDa, 50 kDa, 40 kDa, 25

kDa, 20 kDa, 15 kDa, 10 kDa) and BenchMark Fluorescent Protein standard (Invitrogen; 155

kDa, 100 kDa, 65kDa, 41 kDa, 33 kDa, 23 kDa, 12 kDa) were used as guides to estimate the

protein molecular mass.


64

2.12.4 In-gel fluorescence

In-gel fluorescence was performed as described before (Drew et al., 2006) with some

modifications. Purified protein samples were mixed 1:1 with buffer A (Section 2.12.1) and

heated at 37ºC for 5 min. The whole cell (Section 2.12.1) and WM (Section 2.12.2) samples;

and purified protein samples were separated on 15% (w/v) SDS polyacrylamide gels and

BenchMark Fluorescent Protein Standard (Section 2.12.3) was used as a molecular mass

standard. Gels were rinsed with distilled water and fluorescent imaging of the gel was

performed to detect the protein expression with a Bio-Rad Gel Doc XR + System using Image

Lab software (excitation at 485 nm and emission at 512 nm).

2.12.5 Coomassie blue staining

Protein samples separated on SDS-PAGE (Section 2.12.2) were stained with Coomassie Blue

staining solution [45% (v/v) methanol, 10 % (v/v) glacial acetic acid, and 0.3 % (w/v)

Coomassie Brilliant Blue R-250 (Thermo Scientific)] for overnight at RT with agitation, then

destained with destaining solution [40% (v/v) methanol, 10 % (v/v) glacial acetic acid] until

the background stain was removed.

2.12.6 Western immunoblotting

Proteins separated on SDS-PAGE (Section 2.12.3) were transferred to a 0.45 µm

nitrocellulose membrane (Bio-Rad) for 1-2 h at 400 mA in transfer buffer [3.06 g/litre Tris,

0.2 M glycine, 5% (v/v) methanol]. The membrane was blocked for 1 h at RT with 5% (w/v)

skim milk in TTBS [2.4 g/litre Tris, 0.12 M NaCl, 0.05% (v/v) Tween 20 (Sigma)] and then

incubated with the appropriate primary antibody (Section 2.4) in the same TTBS buffer for

overnight at RT. The membrane was then washed 3 times with TTBS (10 min each) followed

by incubation in appropriate secondary antibody in TTBS buffer for 2 h at RT. Then the

membrane was washed 3 times with TTBS (5 min each) and 3 times with TBS [2.4 g/litre

Tris, 0.12 M NaCl] (5 min each). Chemiluminescent Peroxidase Substrate-3 (Sigma) was

used following the manufacturer’s protocol for detection. The membrane was then exposed to


65

an X-ray film. For visualisation of bands of interest the film was developed using a Curix 60

automatic X-ray film processor (AGFA).

2.12.7 Over-expression of WzySf-GFP-His8

Bacterial strains harbouring pRMPN1 plasmid (pWaldo-TEV-wzySf-GFP-His8) (Table 2.2)

were grown for 16-18 h at 37°C in LB broth with appropriate antibiotics and then diluted 1/20

into fresh LB broth and grown to mid-exponential phase (OD600 of ~ 0.4 to 0.6). Where

required, the growth medium was supplemented with 0.2% (w/v) glucose to suppress protein

expression. Before induction, cells were centrifuged (2,200 x g, Sigma 3K15 tabletop

centrifuge, 10 min, 4°C) and washed twice with LB broth to remove glucose. Under induction

conditions, 0.4 mM IPTG or 0.2% (w/v) L-arabinose was added to the cultures, and they were

grown for 20 h at 20°C. Antibiotics were added as required to the media. In S. flexneri strains,

the plasmid pAC/pBADT7-1 (Table 2.2) encodes T7 RNA polymerase which drives the

expression of the wzySf-gfp gene in pRMPN1 plasmid.

2.12.8 Purification of His-tagged membrane protein

WzySf-GFP-His8 was purified following the method of Woodward et al. (2010) with some

modifications. PNRM15 [Lemo21(DE3) (pRMPN1)] strain were grown for 16-18 h at 37 °C

in LB broth with appropriate antibiotics and then diluted 1/20 into fresh LB broth and grown

to mid-exponential phase (OD600 of ~ 0.4-0.6). Then 0.4 mM IPTG was added to the cultures,

and they were grown for 20 h at 20°C. Cells were harvested from 200 ml induced culture by

centrifugation (9600 x g, Beckman Coulter Avanti J-26XP centrifuge, 8 min, 4°C) and the

cell pellet was washed with sonication buffer (20 mM Tris-HCl, 150 mM NaCl, pH7.5)

followed by disruption of the cell by sonication. Cell debris was removed by centrifugation

(2200 x g, SIGMA 3K15 table top centrifuge, 10 min, 4°C). Then the WM fraction was

isolated by ultracentrifugation (Beckman Coulter Optima L-100 XP ultracentrifuge, 250,000 x

g, 1 h, 4°C) and solubilised in 500 µl Milli Q water and 500 µl 2x solubilisation buffer [40

mM Tris-HCl, 300 mM NaCl, 10% (w/v) sodium dodecanoyl sarcosine (SDDS) (Anatrace),

pH 7.5] at 4°C for overnight. Unsolubilised material was removed by ultracentrifugation


66

(Beckman Coulter Optima Max-XP tabletop ultracentrifuge, 160,000 x g, 1 h, 4°C) and the

solubilised supernatant was incubated with 100 µl IMAC Ni-Charged Resin (Bio-Rad) pre-

equilibrated with equilibration buffer [20 mM Tris-HCl, 150 mM NaCl, 5 mM imidazole,

0.1% (w/v) n-dodecyl-"-Dmaltopyranoside (DDM) (Sigma), 10% (v/v) glycerol, pH 7.5] for

1 h at RT. Incubated Ni-NTA beads were washed with wash buffers [20 mM Tris-HCl, 150

mM NaCl, 0.1% (w/v) DDM, 10% (v/v) glycerol, pH 7.5] of different imidazole

concentrations (10 mM, 20 mM, and 50 mM). Finally, WzySf-GFP-His8 was eluted in 200 µl

elution buffer [20 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, 0.1% (w/v) DDM, 10%

(v/v) glycerol, pH 7.5].

2.12.9 In vivo chemical crosslinking

In vivo crosslinking with DSP was performed as described before (Daniels & Morona, 1999)

with some modifications. Overnight cultures were diluted 1:20 in LB with antibiotics and

incubated at 37ºC to an OD600 of ~0.4-0.6 and centrifuged. Cultures were induced where

required. 200 ml cultures were centrifuged (9600 x g, Beckman Coulter Avanti J-26XP

centrifuge, 8 min, 4°C) and the pellets were washed with DSP cross-linking buffer [150 mM

NaCl, 20 mM sodium phosphate buffer (Na2PO4/NaH2PO4), pH 7.2]. Then the pellets were

resuspended in 200 ml of DSP cross-linking buffer followed by cross-linking with 0.2 mM

DSP for 45 min at 37°C. Excess DSP was quenched with 20 mM Tris-HCl, pH 7.5 and the

cells were washed again with DSP cross-linking buffer. Harvested cells were then either

stored at -20°C or immediately disrupted by sonication for protein purification.

2.12.10 Co-purification or pull down

Cells (from Section 2.12.9) were washed with sonication buffer (20 mM Tris-HCl, 150 mM

NaCl, pH 7.5) and were disrupted by sonication (Branson B15) followed by centrifugation

(2200 x g, SIGMA 3K15 table top centrifuge, 10 min, 4°C) to remove cell debris. Then the

WM fractions were isolated by ultracentrifugation (Beckman Coulter Optima L-100 XP

ultracentrifuge, 250,000 x g, 1 h, 4°C) and were solubilised in 1 ml solubilisation buffer [20

mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 4% (w/v) DDM, 10% (v/v) glycerol, pH 7.5]


67

overnight at 4°C. Unsolubilised material was removed by ultracentrifugation (Beckman

Coulter Optima Max-XP tabletop ultracentrifuge, 160,000 x g, 1 h, 4 °C). Then the soluble

material was incubated with 200 µl of Ni-NTA resin (QIAGEN) pre-equilibrated with

equilibration buffer [20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 0.1% (w/v) DDM,

10% (v/v) glycerol, pH 7.5] for 1 h at RT. The Ni-NTA resin was washed with wash buffers

[20 mM Tris-HCl, 500 mM NaCl, 0.1% (w/v) DDM, 10% (v/v) glycerol, pH 7.5] of different

imidazole concentrations (20 mM, 30 mM, 50 mM, and 80 mM). Finally, the proteins were

eluted from the resin with 200 µl of elution buffer [20 mM Tris-HCl, 500 mM NaCl, 250 mM

imidazole, 0.1% (w/v) DDM, 10% (v/v) glycerol, pH 7.5].

2.12.11 Proteomics analysis

Liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS)

was performed at the Adelaide Proteomics Centre. Protein samples were electrophoresed on

4-12% SDS-PAGE gels (Catalogue number NPO322BOX, Invitrogen) followed by staining

with Brilliant Blue G (Sigma). Novex sharp unstained protein standard (Thermo Fisher

Scientific) was used as molecular mass standard. Regions and bands of interest were excised

and destained with 100 mM ammonium bicarbonate (NH4HCO3) in 30% (v/v) acetonitrile

(ACN). Samples were then washed with 50 mM NH4HCO3, reduced with 0.5 µmol DDT in

50 mM NH4HCO3, and alkylated with 2.75 µmol iodoacetamide in 100 mM NH4HCO3

followed by digestion with 100 ng trypsin (Promega) in 5 mM NH4HCO3 in 10% (v/v) ACN.

Resulting peptides were extracted using three washes of 1% (v/v) formic acid (FA) in water,

1% (v/v) FA in 50% (v/v) ACN, and 100% (v/v) ACN, respectively. The volumes of the

resulting peptide extracts were reduced by vacuum centrifugation to approximately 1 µl and

resuspended with 0.1% (v/v) formic acid in 2% (v/v) ACN to a totalvolume of 10 µl prior to

LC-ESI-MS/MS analysis. LC-ESI-MS/MS was performed using an Ultimate 3000 nano-flow

system (Dionex) coupled to an Impact II QTOFmass spectrometer (Bruker Daltonics) via an

Advance CaptiveSpray source (Bruker Daltonics). Post acquisition, acquired spectra were

subjected to peak detection and deconvolution using Compass Data Analysis for OTOF


68

(Version 1.7, Bruker Daltonics). Processed MS/MS spectra were then exported to Mascot

generic format and submitted to Mascot (Version 2.3.02) for identification.

2.13 Lipopolysaccharide (LPS) Techniques

2.13.1 Preparation of LPS samples

LPS samples were prepared as described previously (Hitchcock & Brown, 1983). Overnight

culture of bacteria was diluted 1:20 and at 37ºC with aeration to OD600 of ~0.6. If required the

culture was induced. The equivalent of 109 bacteria were harvested by centrifugation (16,000

x g, 1 min, 4ºC, Eppendorf Centrifuge 5415R) and resuspended in 50 µl lysing buffer [10%

(w/v) glycerol, 2% (w/v) SDS, 4% (w/v) "-ME, 0.1% (w/v) bromophenol blue, 1 M Tris-HCl,

pH 7.6]. Samples were heated for 5-10 min at 100ºC, then 10 µl of 2.5 mg/ml proteinase K

(Invitrogen) was added followed by incubation at 56ºC for ~16 h. LPS samples were stored at

-20ºC or used immediately.

2.13.2 Analysis of LPS by silver-stained SDS-PAGE

Silver-staining was performed as described previously (Tsai & Frasch, 1982) with minor

changes. LPS samples (Section 2.13.1) were heated at 100ºC for 5-10 min. Then 5-10 µl of

the heated samples were loaded on an SDS 15% (w/v) polyacrylamide gel and eletrophoresed

using the Sigma vertical gel electrophoresis unit (gel dimension: 16.5 cm x 22 cm) at 12 mA

for 16-18 h. The gel was fixed for 2 h in fixing solution [40% (v/v) ethanol, 5% (v/v) glacial

acetic acid in Milli Q water] with gentle agitation and then oxidised in oxidising solution

[40% (v/v) ethanol, 5% (v/v) glacial acetic acid, 0.7% periodic acid in Milli Q water] for 5

min. After 1.5-2 h washing in Milli Q water (changed water at 15 min intervals), the gel was

stained for 10 min in staining solution [2 ml NH4OH, 0.12 g NaOH, 1 g solid silver nitrate in

150 ml Milli Q water]. The gel was washed again in Milli Q water for 1 h (changed water at

10 min intervals) and developed with pre-warmed (42ºC) developing solution [50 mg/ml

citric acid in 1 litre Milli Q water (warmed to 42ºC) with 500 µl 37% (v/v) formaldehyde


69

solution (added just prior to developing)] and stopped by addition of the stopping solution

[4% (v/v) glacial acetic acid in Milli Q water].

!

70

!

71

Chapter 3

Mutational analysis of the Shigella flexneri O antigen

polymerase Wzy; identification of Wzz-dependent Wzy mutants

Mutational analysis of the Shigella flexneri O antigen polymerase Wzy; identification of Wzz-

dependent Wzy mutants

72

Mutational analysis of the Shigella flexneri O antigen

polymerase Wzy; identification of Wzz-dependent Wzy

mutants

Pratiti Nath, Elizabeth Ngoc Hoa Tran, and Renato Morona

Discipline of Microbiology and Immunology, School of Molecular and

Biomedical Science, University of Adelaide, Adelaide 5005, Australia

J Bacteriol. 2015 Jan 1;197(1):108-19. doi: 10.1128/JB.01885-14. Epub 2014 Oct 13.



73

Statement of Authorship

Title of Paper Mutational analysis of the Shigella flexneri O antigen polymerase Wzy; identification of Wzz-dependent Wzy mutants

Publication Status Published Publication Details J Bacteriol. 2015 Jan 1;197(1):108-19. doi: 10.1128/JB.01885-14. Epub 2014 Oct

13.

Author Contributions By signing the Statement of Authorship, each author certifies that their stated contribution to the publication is accurate and that permission is granted for the publication to be included in the candidate’s thesis. Name of Principal Author (Candidate)

Pratiti Nath

Contribution to the Paper Performed all experiments, performed analysis on all samples, interpreted data, and wrote manuscript.

Signature

Date 20.5.15

Name of Co-Author Elizabeth Ngoc Hoa Tran Contribution to the Paper Purified His6-ColE2 and constructed the strain RMA4337.

Signature

Date

Name of Co-Author Renato Morona Contribution to the Paper Supervised development of work, helped in data

interpretation, manuscript evaluation, and editing.

Signature

Date



74

Chapter 3: Third paper

3.1 Abstract

The O antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant

of Shigella flexneri and is synthesied by the Oag polymerase, WzySf. Oag chain length is

regulated by chromosomally encoded WzzSf and pHS-2 plasmid encoded WzzpHS2. To

identify functionally important amino acid residues in WzySf, random mutagenesis was

performed on the wzySf in a pWaldo-TEV-GFP plasmid, followed by screening with colicin

E2. Analysis of the LPS conferred by mutated WzySf proteins in the wzySf deficient ("wzy)

strain identified 4 different mutant classes, with mutations found in the Periplasmic Loops

(PL) - 1, 2, 3, and 6; Trans-membrane (TM) regions - 2, 4, 5, 7, 8, and 9; and Cytoplasmic

Loops (CL) - 1 and 5. The association of WzySf and WzzSf was investigated by transforming

these mutated wzySf plasmids into a wzySf and wzzSf deficient ("wzy "wzz) strain. Comparison

of the LPS profiles in the "wzy and "wzy "wzz backgrounds identified WzySf mutants whose

polymerisation activity was WzzSf-dependent. Colicin E2 and bacteriophage Sf6c sensitivities

were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP

mutants in the "wzy and "wzy "wzz backgrounds identified a role for WzzSf in WzySf

stability. Hence, in addition to its role in regulating Oag modal chain length, WzzSf also

affects WzySf activity and stability.

3.2 Introduction

Shigella flexneri lipopolysaccharide (LPS) is crucial for pathogenesis (Sperandeo et al.,

2009). LPS is exclusively located in the outer leaflet of the outer membrane (OM) and has

three domains: 1) Lipid A - a hydrophobic domain which anchors LPS to the OM, 2) the core

oligosaccharides - a non-repeating oligosaccharide domain, and 3) the O-antigen (Oag)

polysaccharide - an oligosaccharide repeat domain (Sperandeo et al., 2009) (Raetz &

Whitfield, 2002). The complete LPS structure with Oag chains is termed smooth LPS (S-

LPS). However, the LPS structure lacking the Oag is termed rough LPS (R-LPS), and LPS



75

with a single Oag tetrasaccharide repeat unit (RU) attached to the Lipid A and core sugar is

termed semi-rough LPS (SR-LPS) (Morona et al., 1994). S. flexneri is subdivided into various

serotypes depending on the differences in the composition of LPS Oag. So far there are 17

known serotypes of S. flexneri (Sun et al., 2013b). Except for S. flexneri serotype 6, the Oag

of all other serotypes has the same polysaccharide backbone containing three L-rhamnose

residues (Rha), and one N-acetylglucosamine (GlcNAc). This basic Oag structure is known as

serotype Y. Addition of either glucosyl, O-acetyl, or phosphoethanolamine (PEtN) groups by

various linkages to the sugars of the Y serotype tetrasaccharide repeat creates different S.

flexneri serotypes (Allison & Verma, 2000; Sun et al., 2012; Wang et al., 2010b). Oag is the

protective antigen as immunity against the S. flexneri infection is serotype specific (Jennison

& Verma, 2004; Stagg et al., 2009). S-LPS confers resistance to complement (Hong & Payne,

1997) and colicins (Tran et al., 2014; van der Ley et al., 1986), and Y serotype Oag acts as a

receptor to bacteriophage Sf6 (Lindberg et al., 1978).

S. flexneri Oag biosynthesis occurs by the Wzy-dependent pathway. Most of the Oag

biosynthesis genes (except wecA) of S. flexneri are located in the Oag biosynthesis locus

between galF and his (Allison & Verma, 2000; Morona et al., 1995). S. flexneri Oag

biosynthesis occurs on either side of the inner membrane (IM). Initially N-acetylglucosamine

phosphate (GlcNAc-1-P) is transferred from uridine diphosphate-GlcNAc (UDP-GlcNAc) by

WecA to undecaprenol phosphate (Und-P) at the cytoplasmic side of the IM (Guo et al.,

2008; Liu et al., 1996; Wang et al., 2010b). RfbG and RfbF then add Rhamnose (Rha)

residues from thymidine diphospho-rhamnose (dTDP-Rha) to the GlcNAc (Macpherson et al.,

1995; Morona et al., 1994) to form the O unit. In the Wzy-dependent model of LPS assembly,

the flippase protein Wzx translocates this O unit to the periplasmic side. At the periplasmic

side, the O units are polymerised at the non-reducing end by the Oag polymerisation protein

Wzy via a block transfer mechanism to form the polymer. The chain length of the final Oag is

regulated by the protein Wzz (Daniels et al., 1998; Morona et al., 1994). Finally, the Oag

ligase WaaL ligates the Oag chains to the previously synthesied core-lipid A. The Lpt

proteins (Lpt A-G) then transport the LPS from the IM to the OM (Ruiz et al., 2008;



76

Sperandeo et al., 2009).

The Oag polymerisation protein WzySf is encoded by the rfc/wzy gene. WzySf is a 43.7

kDa hydrophobic integral membrane protein. It has 12 transmembrane (TM) segments and

two large periplasmic (PL) domains (Daniels et al., 1998; Morona et al., 1994). Based on a

topology model proposed by our group, the amino and carboxy terminal ends are located on

the cytoplasmic side of the IM. The wild type (WT) wzySf gene lacks a detectable ribosome

binding site, and has a low G+C %, and a high percentage of minor codons in the first 25

amino acids, contributing to low expression and poor detection of the protein (Daniels et al.,

1998; Morona et al., 1994).

Islam et al. (2011) performed extensive work on Pseudomonas aeruginosa Wzy

(WzyPa) and showed that both PL3 and PL5 of WzyPa contain RX10G motifs, which are

important for the functioning of WzyPa. They also found several Arg residues within these

two motifs are also important for WzyPa function (Islam et al., 2011). However there is little

sequence identity between WzyPa and WzySf. So, it is not possible to predict the functional

amino acid residues of WzySf from another model.

Wzz is a member of the polysaccharide co-polymerase (PCP) family. S. flexneri has two

types of Wzz - chromosomally encoded WzzSf and pHS-2 plasmid encoded WzzpHS2. S.

flexneri 2a has S-LPS with two types of modal chain length: short (S) type (11-17 Oag RUs)

and very long (VL) type (>90 Oag RUs), and the S-type and VL-type Oag chain lengths are

determined by WzzSf and WzzpHS2, respectively. Controlling Oag chain length is crucial for

bacterial virulence, and loss of WzzSf mediated Oag modal chain length regulation affects

virulence due to masking of the OM protein IcsA (Morona et al., 2003; Morona & Van Den

Bosch, 2003b). Daniels and Morona (1999) showed that WzzSf forms a dimer in vivo and may

oligomerise up to a hexamer. Formation of these large complexes is consistent with the

hypothetical complex formation between Wzz and other enzymes of the Oag biosynthesis

pathway, including Wzy (Bastin et al., 1993; Morona et al., 1995). WzzSf and WzzpHS2

compete for the available WzySf (Carter et al., 2009).



77

Woodward et al. (2010) showed that Wzz and Wzy are sufficient to determine the Oag

modal chain length. There are several proposed mechanisms for modal length control by Wzz

and its association with Wzy: a molecular clock model was proposed by Bastin et al. (1993)

and a molecular chaperone model was proposed by Morona et al. (1995). Tocilj et al. (2008)

suggested that Wzz may form a scaffold and recruits Wzy. However, there is no direct

evidence to date on how these proteins are associated with each other in Oag polymerisation

and chain length control.

In this study we were able to overexpress and detect WzySf-GFP expression in S.

flexneri. We performed random mutagenesis on wzySf, and following screening with colicin

E2, for the first time identified amino acid residues important for WzySf function. We

classified the wzySf mutants based on their LPS profiles. We were able to determine mutant

protein expression levels, and also characterised the mutants depending on the phage and

colicin sensitivities they conferred. These findings provided insight to Wzy structure and

function. We further identified WzzSf-dependent WzySf mutants, and identified a novel role

for WzzSf in WzySf Oag polymerisation activity and stability, in addition to its role in

regulating Oag modal chain length.

3.3 Materials and Methods

3.3.1 Bacterial strains and plasmids

The strains and plasmids used in this study are shown in Table 3.1.

3.3.2 Growth media and growth conditions

The growth media used were Lysogeny broth (LB) (10 g/liter tryptone, 5 g/liter yeast extract,

5 g/liter NaCl) and LB agar (LB broth, 15 g/liter bacto agar).



78

Table 3.1 Bacterial strains and plasmids used in this study

Strains or Plasmids Characteristics* Reference Strains S. flexneri PE638 S. flexneri Y rpoB (Rifr) (Morona et al., 1995)

recruiWzy RMM109 PE638!wzy, Rifr (Morona et al., 1994) RMA4337 RMM109 !wzz (Rifr, Tetr) This study PNRM6 RMM109 (pAC/pBADT7-1) This study PNRM11 PNRM6 (pWaldo-TEV-GFP) This study PNRM13 PNRM6 (pRMPN1) This study PNRM75 PNRM6 (pRMPN7) This study PNRM76 PNRM6 (pRMPN8) This study PNRM77 PNRM6 (pRMPN9) This study PNRM78 PNRM6 (pRMPN10) This study PNRM79 PNRM6 (pRMPN11) This study PNRM80 PNRM6 (pRMPN12) This study PNRM81 PNRM6 (pRMPN13) This study PNRM82 PNRM6 (pRMPN14) This study PNRM83 PNRM6 (pRMPN15) This study PNRM84 PNRM6 (pRMPN16) This study PNRM85 PNRM6 (pRMPN17) This study PNRM119 PNRM6 (pRMPN19) This study PNRM120 PNRM6 (pRMPN21) This study PNRM121 PNRM6 (pRMPN22) This study PNRM122 PNRM6 (pRMPN23) This study PNRM123 PNRM6 (pRMPN24) This study PNRM124 PNRM6 (pRMPN25) This study PNRM126 RMA4337 (pAC/pBADT7-1) This study PNRM134 PNRM126 (pRMPN1) This study PNRM131 PNRM126 (pRMPN7) This study PNRM132 PNRM126 (pRMPN15) This study PNRM133 PNRM126 (pRMPN16) This study



79

Table 3.1 continued Strains or Plasmids Characteristics* Reference S. flexneri PNRM136 PNRM126 (pRMPN8) This study PNRM137 PNRM126 (pRMPN10) This study PNRM140 PNRM126 (pRMPN13) This study PNRM141 PNRM126 (pRMPN14) This study PNRM142 PNRM126 (pRMPN9) This study PNRM143 PNRM126 (pRMPN11) This study PNRM144 PNRM126 (pRMPN19) This study PNRM145 PNRM126 (pRMPN24) This study PNRM146 PNRM126 (pRMPN25) This study PNRM147 PNRM126 (pRMPN23) This study PNRM148 PNRM126 (pRMPN21) This study PNRM149 PNRM126 (pRMPN22) This study PNRM150 PNRM126 (pRMPN12) This study PNRM151 PNRM126 (pRMPN17) This study E.coli XL10-Gold

Tetr !(mcrA)183 !(mcrCB-hsdSMR-mrr)173

endA1 supE44 thi-1 recA1 gyrA96 relA1 lac

(F´ proAB lacIqZDM15 Tn10Tetr Cmr)

Stratagene

Lemo21(DE3)

fhuA2 (lon) ompT gal ($ DE3) (dcm) "hsdS/

pLemo(Cmr)

New England Biolabs

PNRM15 Lemo21(DE3) (pRMPN1) This study Plasmids pRMCD6 Source of wzySf (Daniels et al., 1998) pAC/pBADT7-1 Source of T7 RNA polymerase; Cmr (McKinney et al.,

2002) pWaldo-TEV-GFP Cloning vector with GFP tag; Kmr (Waldo et al., 1999) pRMPN1 pWaldo-wzySf-GFP; Kmr This study pRMPN7 pRMPN1 with G130V point mutation in the

wzySf gene

This study

pRMPN8 pRMPN1 with L111I point mutation in the

wzySf gene

This study



80

Table 3.1 continued Strains or Plasmids Characteristics* Reference Plasmids pRMPN9 pRMPN1 with N86K point mutation in the

wzySf gene

This study

pRMPN10 pRMPN1 with L28V point mutation in the

wzySf gene

This study

pRMPN11 pRMPN1 with P165S point mutation in the

wzySf gene

This study

pRMPN12 pRMPN1 with G82C point mutation in the

wzySf gene

This study

pRMPN13 pRMPN1 with N147K point mutation in the

wzySf gene

This study

pRMPN14 pRMPN1 with L191F point mutation in the

wzySf gene

This study

pRMPN15 pRMPN1 with L214I point mutation in the

wzySf gene

This study

pRMPN16 pRMPN1 with P352H point mutation in the

wzySf gene

This study

pRMPN17 pRMPN1 with V92M point mutation in the

wzySf gene

This study

pRMPN19 pRMPN1 with F52Y point mutation in the

wzySf gene

This study

pRMPN21 pRMPN1 with F52C/I242T point mutations in

the wzySf gene

This study

pRMPN22

pRMPN1 with C60F point mutation in the

wzySf gene

This study

pRMPN23 pRMPN1 with Y137H point mutation in the

wzySf gene

This study

pRMPN24 pRMPN1 with L49F/T328A point mutation in

the wzySf gene

This study



81

Table 3.1 continued Strains or Plasmids Strains or Plasmids Strains or Plasmids Plasmids Plasmids Plasmids pRMPN25 pRMPN1 with F54C point mutation in the

wzySf gene

This study

pCACTUS Suicide vector containing sacB, Cmr, and

Orits


pRMA577 Suicide vector contatining SphI-SphI

fragment with the rol gene


pCACTUS-

wzzSf::Tcr

Suicide mutagenesis construct to construct the

strain RMA4337

This study

* Rifr, rifampicin resistant; Kmr, kanamycin resistant; Cmr, chloramphenicol resistant; Tetr,

tetracycline resistant. $ DE3 is $ sBamHIo "EcoRI-B int::(lacI::PlacUV5::T7gene1)i21

"nin5. pLemo is pACYC184-PrhaBAD-lysY.



82

Unless otherwise stated strains were grown in LB broth with aeration for 18 h at 37°C,

then diluted 1/20 into fresh LB broth and grown to mid-exponential phase (OD600 of 0.4 -

0.6). Where required growth medium was supplemented with 0.2% (w/v) glucose to suppress

protein expression. Before induction, cells were centrifuged (2200 x g, SIGMA 3K15 table

top centrifuge, 10 min, 4°C) and washed twice with LB broth to remove glucose. Under

induction conditions, 0.4 mM isopropyl-!-D-thiogalactopyranoside (IPTG) or 0.2% (w/v) or

L-arabinose was added to cultures and grown for 20 h at 20°C. Antibiotics were added as

required to the media at the following final concentrations: kanamycin (Km), 50 µg/ml and

chloramphenicol (Cm), 25 µg/ml.

3.3.3 Construction of expression vector and cloning of wzySf

Primers PN1_wzySfKpnF and PN2_wzySfBamHR (Supplementary Table 3.S1) which

incorporated KpnI and BamHI restriction sites, respectively, were used to amplify the

previously mutated wzySf coding region (GenBank accession number X71970) in the

pRMCD6 plasmid (Table 3.1), possessing three changes at codons 4, 9 and 23 (Daniels et al.,

1998). The rare codons 4, 9 and 23 are present in the translation initiation site of WzySf and

causes lower expression of the wild-type WzySf. The codons ATA, ATA and AGA at 4, 9 and

23 were changed to ATT, ATT and CGT, respectively, and resulted in increased expression of

WzySf (Daniels et al., 1998). Following restriction digestion with enzymes BamHI and KpnI,

the amplified wzySf was ligated into similarly digested pWaldo-TEV-GFP (Waldo et al.,

1999), resulting in the plasmid pWaldo-wzySf-TEV-GFP (denoted pRMPN1). PCR, restriction

enzyme digestion, agarose gel electrophoresis, ligation, and transformation were performed as

described previously (Morona et al., 1994; Morona et al., 1995).



83

3.3.4 WzySf expression in Lemo21(DE3) and In-gel fluorescence

For WzySf expression in Lemo21(DE3) cells (Table 3.1), strains were induced with IPTG and

the over-expressed WzySf-GFP fusion protein was detected by In-gel fluorescence following

the method of Drew et al. (2006) with some modifications. Cells (5X108) were harvested

from the induced culture and washed twice in 200 &l phosphate buffered-saline (PBS). The

pellet was re-suspended in 20 &l PBS and 20 &l Buffer A [200 mM Tris-HCl (pH 8.8), 20%

(v/v) glycerol, 5 mM EDTA (pH 8.0), 0.02% (w/v) bromophenol blue, 4% (w/v) SDS, and

0.05 M DDT] was then added to the cell suspension. The solubilised cell suspension was

incubated at 37°C for 5 min. Samples were then electrophoresed on SDS 15% (w/v)

polyacrylamide gels (SDS-15% PAGE) and BenchMark Pre-Stained Protein Ladder

(Invitrogen) was used as a molecular mass standard. The gel was rinsed with distilled water

and fluorescent imaging of the gel was performed to detect WzySf-GFP protein expression

with a Bio-Rad Gel Doc XR + System using Image Lab software (excitation at 485 nm and

emission at 512 nm).

3.3.5 LPS method

LPS was prepared as described previously (Murray et al., 2003; Tsai & Frasch, 1982). To

prepare LPS samples, 1X109 cells were harvested and resuspended in lysing buffer [Buffer B

- 10% (w/v) glycerol, 2% (w/v) SDS, 4% (w/v) !-mercaptoethanol, 0.1% (w/v) bromophenol

blue, 1 M Tris-HCl, pH 7.6] and incubated with 2 µg/ml of proteinase K for approximately 16

h. The LPS samples were then electrophoresed on an SDS-15% PAGE for 16 to 18 h at 12

mA. The gel was stained with silver nitrate and developed with formaldehyde (Murray et al.,

2003).



84

3.3.6 Random mutagenesis

Random mutagenesis of wzySf was undertaken to obtain a wide range of wzySf mutations in a

non-selected manner. For PCR random mutagenesis the wzySf coding region in plasmid

pRMPN1 was mutagenised by an error prone DNA polymerase using the GeneMorp II EZ-

Clone Domain Mutagenesis Kit (Catalogue number 200552, Stratagene) according to the

manufacturer’s instructions with the primers PN1_wzySfKpnF and PN2_wzySfBamHR

(Supplementary Table 3.S1). The mutagenised plasmids were transformed into competent

Escherichia coli cells, XL10-Gold (Agilent Technologies). Plasmid DNA was then isolated

from randomly chosen transformed mutated colonies and transformed into strain PNRM6

(Table 3.1). Colicin swab assays were performed to screen the mutants (See below). Plasmid

DNA was isolated from putative mutants, transformed into XL10-Gold cells, and subjected to

DNA sequencing (AGRF, Adelaide, Australia).

3.3.7 Construction of the strain RMA4437 (!wzy !wzz)

The S. flexneri Y PE638 "wzy "wzz mutant strain was constructed using allelic exchange

mutagenesis (Morona et al., 1995) to inactivate the wzzSf gene in RMM109 (Morona et al.,

1994). Initially, a tetracycline resistance (tetr) cartridge was inserted into the BglII site of

pRMA577 (Morona et al., 1995) to inactivate wzzSf, and the resulting pCACTUS-wzzSf::tet

(Table 3.1) plasmid was transformed into RMM109 via electroporation (Purins et al., 2008).

Allelic exchange mutagenesis was performed as previously described (Morona et al., 1995).

The wzzSf::tetr mutation in the chromosome was confirmed by PCR with primers ET35 and

ET36 (Supplementary Table 3.S1) to give the PE638 "wzy "wzz mutant RMA4337.



85

3.3.8 Detection of WzySf expression in S. flexneri

For the detection of WzySf expression in S. flexneri, cells were harvested from the 50 ml 0.2%

(w/v) L-arabinose induced culture by centrifugation (9800 x g, Beckman J2-21M Induction

Drive Centrifuge, 10 min, 4 °C) and the cell pellet was resuspended in 4 ml sonication buffer

(Buffer C, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5). The mixture was then lysed by

sonication, followed by centrifugation (2200 x g, SIGMA 3K15 table top centrifuge, 10 min,

4 °C) to remove debris. Ultracentrifugation was performed in a Beckman Coulter Optima

MAX-XP bench top ultracentrifuge (126000 x g for 1 h at 4 °C) to isolate the whole

membrane (WM) fraction. The WM fraction was resuspended in PBS and then solubilised in

Buffer A. Solubilised WM fraction from 3 X 108 cells was electrophoresed on an SDS-15%

PAGE. The gel was rinsed with distilled water and In-gel imaging was performed as

described above. Loading was checked by staining the gel with Coomassie Blue R-250. The

intensity of WzySf-GFP expression for mutant and control strains was measured by Fiji image

processing package (http://fiji.sc/Fiji) and the percent relative WzySf-GFP intensity for each

mutant strain was measured by comparing the WzySf-GFP intensity of each mutant strain with

WzySf-GFP intensity in the control strain PNRM13.


For the colicin sensitivity assay a solution of purified His6-ColE2 (ColE2) with an initial

concentration of 1 mg/ml was used (Tran et al., 2014).

3.3.9.1 ColE2 swab assay

A two fold serial dilution of 1 µg/ml ColE2 was swabbed onto antibiotic selective LB agar

plates containing 0.2% (w/v) L-arabinose with a cotton swab. Plates were left to dry for 1 h at



86

room temperature (RT). Individual 0.2% (w/v) L-arabinose induced mutant and control LB

cultures were then swabbed perpendicular to the ColE2 streak and plates were left to dry for

another 1 h at RT. Plates were then incubated for 16 h at 37°C. The susceptibility of the

mutant strains compared to the control strain was recorded and the image was taken using a

Canon scanner (CanoScan 9000F) against a dark background.

3.3.9.2 ColE2 spot assay

The spot assay was performed using the serial dilutions of ColE2. 100 µl of the individual L-

arabinose induced mutant and control strain cultures were spread onto LB agar plates with

appropriate antibiotics and 0.2% (w/v) L-arabinose. The plates were left to dry for 2 h at RT.

A 2-fold serial dilution of 1 µg/ml of ColE2 [denoted as Neat or (N)] was spotted on the dried

plates, and plates were left to dry for another 3 h at RT. The plates were then incubated for 18

h at 37°C. The end point of the killing zones of mutant strains was compared with the

controls. Images were recorded as above.


Procedures of phage propagation and phage stock preparation have been described previously

(Mavris et al., 1997; Morona et al., 1994). The concentration of the bacteriophage Sf6c stock

used was 8.6 x 107 p.f.u./ml. Mutant and control strains were grown and induced with 0.2%

(w/v) L-arabinose. 100 µl of the individual mutant and control LB cultures were spread onto

LB agar plates with appropriate antibiotics and 0.2% (w/v) L-arabinose. The plates were left

to dry for 2 h at RT. Serial dilutions of the bacteriophage Sf6c stock (undiluted bacteriophage

Sf6c stock was denoted as N) were spotted on the dried plates and the plates were dried for a

further 3 h at RT. The plates were incubated for 18 h at 37°C. Phage sensitivity of the test

strains were compared with the controls. Images were recorded as above.



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3.4 Results

3.4.1 Construction of a WzySf-GFP expression plasmid

A suitable expression system was constructed to express wzySf and to detect WzySf by fusion

to GFP (Drew et al., 2006). S. flexneri 2457T 2a wzySf with three modified codons at postions

4, 9, 23 in pRMCD6 plasmid (Daniels et al., 1998) was PCR amplified and ligated into

pWaldo-TEV-GFP (Table 3.1) (See Materials and Methods). To confirm the construct was

able to express WzySf-GFP-His8 protein, pWaldo-wzySf-TEV-GFP-His8 (denoted as pRMPN1)

was transformed into Lemo21(DE3) cells. Whole cell In-gel fluorescence samples were then

prepared from PNRM15 [Lemo21(DE3), (pRMPN1)] and fluorescent imaging of the gel

detected a fluorescent band at approximately 64 kDa, which corresponded to the predicted

size of the WzySf-GFP protein (Supplementary Fig. 3.S1, lane 1), indicating that the construct

was able to express WzySf-GFP.

3.4.2 Complementation of wzySf deficiency

A complementation assay was performed to confirm the functionality of WzySf-GFP. For this

assay pRMPN1 was co-transformed along with pAC/pBADT7-1 (McKinney et al., 2002) into

a wzySf deficient strain RMM109 (Morona et al., 1994). RMM109 has a frameshift mutation

at position 9214 in the wzySf gene that results in premature termination of WzySf synthesis

(Morona et al., 1994). pAC/pBADT7-1 encodes T7 RNA polymerase, which drives the

expression of wzySf-GFP in pRMPN1. LPS samples were prepared from these control strains.

The silver-stained gel showed that PNRM6 [RMM109 (pAC/pBADT7-1)] had an SR-LPS

profile (Fig. 3.1, lane 3). But the PNRM13 [PNRM6 (pRMPN1)] had an S-LPS profile (Fig.

3.1, lane 5). Hence, pRMPN1 was able to complement the wzySf mutation in RMM109, and

the LPS profile resembled that of the WT strain PE638 (Fig. 3.1, lane 2).



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Figure 3.1 Complementation of wzySf deficiency by WzySf-GFP LPS samples (equivalent to 1X109 bacterial cells) were prepared from the indicated strains by

proteinase K treatment, electrophoresed on an SDS-15% (w/v) PAGE gel, and silver stained

(see Materials and Methods). The positions of S-LPS, SR-LPS, and R-LPS are indicated. The

numbers on the right indicate the Oag RUs.



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3.4.3 Random mutagenesis of wzySf

Nothing is known about the residues important for WzySf function, and there is little sequence

identity between Wzy proteins of different bacterial species. Hence, it is difficult to predict

the functional amino acid residues of the S. flexneri WzySf. To obtain insight into the WzySf

residues needed for function, wzySf coding region in plasmid pRMPN1 was subjected to

random mutagenesis using an error-prone DNA polymerase (See Materials and Methods).

The resulting mutagenised plasmid library was transformed into PNRM6. We screened the

transformants to find mutants using ColE2. The basis for this is that the R-LPS strains are

more susceptible to the killing by colicins than the S-LPS strains (van der Ley et al., 1986),

and we recently found that there is a strong correlation between LPS Oag modal chain length

and susceptibility to ColE2 (Tran et al., 2014). A ColE2 swab assay (See Materials and

Methods) was used to screen and detect mutants that had a different sensitivity to ColE2

compared to the positive control strain PNRM13 (Supplementary Table 3.S2). Interestingly,

the WT strain PE638 was slightly more resistant to ColE2 compared to the complemented

positive control strain PNRM13 (Supplementary Table 3.S2) (Table 3.2). The wzySf mutant

RMM109 (SR-LPS) was highly sensitive to ColE2 (Supplementary Table 3.S2) (Table 3.2).

Transformants that were either more resistant or more sensitive to ColE2 than PNRM13 were

selected (Supplementary Table 3.S2), and the plasmids isolated and transformed into the

XL10-Gold strain. Plasmid DNA from these isolates was subjected to DNA sequencing to

identify mutational alterations in wzySf. The wzySf mutants had the following substitutions:

P352H, V92M, Y137H, L214I, G130V, N147K, P165S, L191F, C60F, L49F/T328A, L28V,

N86K, F54C, F52Y, L111I, G82C, and F52C/I242T (Supplementary Table 3.S3). The

mutations were present in PL1, 2, 3, and 6; TM 2, 4, 5, 7, 8, and 9; and Cytoplasmic Loops

(CL) 1 and 5 of the WzySf topology map (summarised in Fig. 3.2 and Table 3.2). After

sequence confirmation, the mutated plasmids were transformed into PNRM6 (Table 3.1) for

detailed characterisation.



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Figure 3.2 Locations of mutations on the topology map of WzySf Mutational alterations are indicated by arrows on the WzySf topology map [adapted from

Daniels et al. (1998)]. The positions of the periplasmic loops (PL 1-5), transmembrane

regions (TM 1-12), and cytoplasmic loops (CL 1-5) are indicated. Mutations (shaded circles)

are located in PL 1, 2, 3, and 6; TM 2, 4, 5, 7, 8, and 9; and CL 1 and 5.

Mutational analysis of the Shigella flexneri O antigen polymerase Wzy; identification of Wzz-dependent Wzy mutants

91

Table 3.2 ColE2 and bacteriophage Sf6c sensitivities and WzySf-GFP expression of controls and different classes of mutants Mutant class Sensitivity*

Strain Relevant details

!wzy

background

!wzy/!wzz

background

ColE2 Sf6c Relative WzySf-GFP (%) Topology map

location#

!wzy

background

!wzy/!wzz

background

!wzy

background

!wzy/!wzz

background

!wzy

background

!wzy/!wzz

background

RMM109 wzySf mutants 1/256 - R - - - PE638 WT R - 10-6 - - - PNRM13 Positive control 1/2 - 10-5 - 100 - PNRM6 Negative control 1/256 - R - - - PNRM11 Negative control 1/256 - R - - - RMA4337 wzySf and wzzSf mutant - 1/256 - R - - PNRM126 Negative control - 1/256 - R - - PNRM134 Positive control - R - 10-6 - 17 P352H A B PL6 1/64 1/128 R R 36 38 V92M A E PL2 1/32 1/64 R R 84 76 Y137H A E TM5 1/32 1/64 R R 87 97 L214I B C TM8 1/128 1/128 R R 1.4 0.03 G130V C F TM5 1/512 1/128 R R 1.60 28.50 N147K D E PL3 R 1/16 10-6 R 21 52 P165S D E PL3 1/4 1/64 10-5 N 7 125 L191F D E TM7 R 1/16 10-6 N 162 64 C60F D E TM1 R 1/64 10-6 N 30 66 L49F/T328

A

D E TM2/CL5 R 1/64 10-6 R 65 68 L28V D E PL1 R 1/8 10-6 N 55 80 N86K D E PL2 R 1/64 10-6 R 84 82 F54C D E CL1 R 1/64 10-6 R 42 71 F52Y D E TM2 R 1/64 10-6 R 47 63 L111I D E TM4 R 1/8 10-6 N 41 51 G82C D E PL2 R 1/64 10-6 R 40 47 F52C/I242T D E TM2/TM9 R 1/64 10-6 N 82 81



92

# PL, periplasmic loop; TM, transmembrane region; CL, cytoplasmic loop (Fig. 3.2). *R,

resistant; N, plaques detected with undiluted Sf6c stock. The numbers represent the highest

dilution showing the zone of inhibition or plaques formation.



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3.4.4 LPS phenotype conferred by WzySf mutants

The effect of mutations on WzySf LPS Oag polymerisation activity was determined by SDS-

PAGE and silver staining. Following comparison of the resulting degree of LPS Oag

polymerisation of the mutant strains with the relevant positive control PNRM13, the mutants

were grouped into four different phenotypic classes: A-D (Fig. 3.3 and Supplementary Fig.

3.S2). Three of the 17 mutants had reduced degrees of polymerisation compared to PNRM13

and were classified as class A. Class A mutants had following alterations in WzySf: P352H,

V92M, Y137H (Fig. 3.3, lanes 2-4). One mutant (with L214I alteration in WzySf) exhibited

LPS banding pattern with only a few Oag RUs and was classified as class B (Fig. 3.3, lane

5). Another mutant had SR-LPS (with a G130V alteration in WzySf) and was categorised into

class C (Fig. 3.3, lane 6). The other 12 mutants conferred a LPS profile nearly similar to the

positive control PNRM13 and were classified as class D. Members of this class had the

following mutations: N147K, P165S, L191F, C60F, L49F/T328A, L28V, N86K, F54C,

F52Y, L111I, G82C, F52C/I242T (Fig. 3.3, lanes 7-10 and Supplementary Fig. 3.S2, Lanes 1-

8). For rest of the paper the mutant WzySf proteins will be referred according to their

conferred LPS classes.

3.4.5 WzzSf dependence

Since the Wzy-dependent model of LPS assembly suggests a potential interaction between

Wzy and Wzz, we investigated if the LPS profile conferred by mutated WzySf proteins was

dependent on the presence of WzzSf. All the plasmids encoding mutated WzySf proteins were

transformed into RMA4337 carrying pAC/pBADT7-1 (strain PNRM126) (Table 3.1).

RMA4337 is a wzySf and wzzSf double mutant. The LPS profiles conferred in the PNRM126

(!wzy !wzz) were directly compared with the LPS profile conferred in the !wzy background

(PNRM6). The control strain PNRM134 [PNRM126 (pRMPN1)] had S-LPS without Oag

modal chain length control (Fig. 3.4, lane 3), as expected, and was classified as class E for the

purpose of comparison. PNRM126 with mutated wzySf plasmids that conferred a class A LPS



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Figure 3.3 LPS phenotypes conferred by different WzySf mutants expressed in PNRM6

[!wzySf (pAC/pBADT7-1)]

Plasmid-encoded mutated WzySf proteins were expressed in PNRM6. The strains were grown

and induced with arabinose as described in Materials and Methods. LPS samples were

prepared, electrophoresed on SDS-15% (w/v) PAGE gels, and silver stained (see Materials

and Methods). The strains were divided into various mutant classes (A-D) based on their LPS

phenotypes. Lane 1, positive-control strain PNRM13 [PNRM6 (pRMPN1)]; lanes 2 to 10,

"wzy strain (PNRM6) with plasmids encoding mutated WzySf proteins, as indicated. The

position of R-LPS is indicated. The numbers on the left indicate the Oag RUs.



95

Figure 3.4 Comparison of the LPS phenotypes conferred by the WzySf mutants

expressed in the !wzy and !wzy !wzz backgrounds

Plasmids encoding mutated WzySf proteins were expressed in PNRM126 [RMA4337

(pAC/pBADT7-1)] and PNRM6 [RMM109 (pAC/pBADT7-1)]. The strains were grown and

induced as described in Materials and Methods. LPS samples were electrophoresed on SDS-

15% (w/v) PAGE gels and silver stained (see Materials and Methods). Lanes1 to 3 contain the

indicated strains; lanes 4 to 21 contain the "wzy (PNRM6) or "wzy "wzz (PNRM126) strain

with plasmids encoding the indicated mutated WzySf proteins.



96

profile in the !wzy background had a class E LPS profile similar to PNRM134, except

PNRM126 expressing WzyP352H had LPS with few Oag RUs (class B) (Fig. 3.4, lane 5).

PNRM126 expressing WzyL214I had SR-LPS (Fig. 3.4, lane 11). However, in comparison

PNRM6 (!wzy) expressing WzyL214I conferred a class B LPS profile (Fig. 3.4, lane 10).

PNRM126 with a mutated wzySf plasmid that conferred a class C LPS profile in the !wzy

background (PNRM6 expressing G130V), had S-LPS without modal length control and a

reduced degree of polymerisation (designated class F) compared to PNRM134 (Fig. 3.4, lane

13). PNRM126 with mutated wzySf plasmids from the strains with class D LPS profile in the

!wzy background, had class E LPS profile, similar to PNRM134 (Fig. 3.4, lanes 14-21 and

Supplementary Fig. 3.S 3, lanes 1-16). Hence certain WzySf mutants conferred dramatically

different LPS profiles depending on the presence and absence of WzzSf.

3.4.6 ColE2 and bacteriophage Sf6c sensitivities

To confirm the LPS profiles determined above using assays of LPS Oag function, the ColE2

and bacteriophage Sf6c sensitivities of the mutant strains were investigated. ColE2 sensitivity

(summarised in Table 3.2) was determined by spot testing as described in the Materials and

Methods. Strains RMM109, PNRM6, PNRM11, RMA4337, and PNRM126 showed killing

zones at a dilution of 1/256. The WT strain PE638 was resistant to the highest concentration

of ColE2 used. The relevant positive control strain in the !wzy background (PNRM13)

showed a killing zone at a dilution of 1/2 but the relevant positive control strain in the !wzy

!wzz background (PNRM134) was resistant to the tested highest concentration of ColE2.

Strains conferring class A LPS profile in the !wzy background were sensitive to ColE2

(killing zone at 1/32 or 1/64). However, !wzy !wzz strains with mutated wzySf plasmids

conferring the class A LPS profile in the !wzy background were two-fold more sensitive to

ColE2 (killing zone at 1/64 or 1/128) compared to the !wzy background. The !wzy strain

expressing WzyL214I (class B), and the !wzy !wzz strain expressing WzyL214I (class C)

had similar ColE2 sensitivity (1/128). The strain with the class C LPS profile (!wzy

expressing WzyG130V) had the greatest sensitivity to ColE2 (1/512), greater than RMM109

and the negative control strains (PNRM6, PNRM11, PNRM126). However, the !wzy !wzz

strain expressing WzyG130V (class F) was more resistant (1/128) to ColE2 compared to the



97

!wzy background. Strains with a class D LPS profile in the !wzy background were resistant

to the tested highest concentration of ColE2, except !wzy expressing WzyP165S showed

slight sensitivity (1/4). Hence, almost all the strains with class D LPS profile in the !wzy

background were more resistant to ColE2 compared to PNRM13 and were similar to PE638.

The !wzy !wzz strains with mutated wzySf plasmids from the strains with class D LPS profile

in the !wzy background were more sensitive to ColE2 (killing zone at 1/8 to 1/64) than the

control PNRM134 [!wzy !wzz (pRMPN1)], suggesting they have slight defect in Oag

polymerisation in the absence of WzzSf. Hence, ColE2 resistance correlated with degree of

Oag polymerisation and degree of LPS capping with Oag.

The bacteriophage Sf6c sensitivity of the strains (summarised in Table 3.2), carrying

mutated wzySf plasmids, was determined by spot testing as described in the Materials and

Methods. The SR-LPS strains RMM109, PNRM6, PNRM11, RMA4337, and PNRM126

were resistant to bacteriophage Sf6c. The S-LPS strains WT PE638, and the controls,

PNRM13 and PNRM134, were bacteriophage Sf6c sensitive, and plaques were detected at

10-6, 10-5, and 10-6 dilutions, respectively. Strains with class A, B, and C LPS profiles in the

!wzy background were resistant to the highest concentration of bacteriophage Sf6c tested and

were similar to the strains with SR-LPS (RMM109, PNRM6, PNRM11, RMA4337, and

PNRM126). Similarly, the !wzy !wzz strains with mutated wzySf plasmids from the strains

with class A, B, and C LPS profiles in the !wzy background were also resistant to the highest

concentration of bacteriophage Sf6c tested. Strains with a class D LPS profile in the !wzy

!wzz background were bacteriophage Sf6c sensitive (plaques at 10-6), except that the strain

expressing WzyP165S was slightly less sensitive (plaque at 10-5). Hence, the bacteriophage

Sf6c sensitivities of the strains with a class D LPS profile in the !wzy background were

greater than that of PNRM13 and similar to that of PE638. !wzy !wzz strains with mutated

wzySf plasmids from the strains with a class D LPS profile in the !wzy background were more

resistant to bacteriophage Sf6c than the !wzy background. Although they have a class E LPS

profile, similar to PNRM134, they were more resistant to bacteriophage Sf6c than PNRM134.

Hence, bacteriophage Sf6c correlated with degree of Oag polymerisation, and degree of LPS

capping with Oag.



98

3.4.7 WzySf expression level

We determined the level of parental and mutant WzySf-GFP expressed in !wzy and !wzy

!wzz S. flexneri strains. To measure the protein expression level of the mutants, In-gel

fluorescence was performed and the % relative WzySf-GFP expression was calculated (See

Materials and Methods). The expression levels of different WzySf-GFP mutants were

compared with that of WzySf-GFP in PNRM13 (100%). The WzySf-GFP expression level in

PNRM134 was less than PNRM13 (Fig. 3.5A and B, lane 2), with a relative WzySf-GFP level

of 17% (Table 3.2). In the !wzy background, most of the WzySf-GFP mutants were expressed

at a level less than 100% except the mutants L191F (class D) had expression of 162% (Fig.

3.5B, lane 7 and Table 3.2). However, in the !wzy !wzz background, the relative WzySf-GFP

level of L191F was 64% (Fig. 3.5B, lane 8 and Table 3.2), which was less than the control

PNRM13. In the !wzy background, the relative WzySf-GFP levels of P165S, L214I, and

G130V were very low (7%, 1.4%, and 1.6% respectively) (Fig. 3.5B, lane 5; Fig. 3.5A, lanes

9 and 11; and Table 3.2). However, in the !wzy !wzz background, P165S and G130V had

relative WzySf-GFP levels of 125% and 28.50%, respectively (Fig. 3.5B, lane 6; Fig 5A, lane

12; and Table 3.2) but the relative WzySf-GFP level of L214I was almost not detectable at

0.03% (Fig. 3.5A, lane 10 and Table 3.2). In the !wzy !wzz background, the relative WzySf-

GFP level of P165S was higher than the control PNRM13 (100%) (Fig. 3.5B, lane 6 and

Table 3.2). These data indicates that expression of certain WzySf mutants was affected by

WzzSf, but the effect was mutant specific.

3.5 Discussion

In this study, we constructed and characterised a collection of wzySf mutants. We found that

ColE2 screening was an effective method to detect wzySf mutants conferring subtle effects on

LPS structure (Table 3.2). The use of WzySf-GFP allowed comparison of the protein

expression level of different mutants. We also found that bacteriophage Sf6c sensitivity was

dependent on LPS phenotype structure. Strains with mutant WzySf conferring longer Oag

chain and/or a greater degree of Oag polymerisation were more sensitive to bacteriophage



99

Figure 3.5 Protein expression levels of the mutated WzySf-GFP compared to the positive

control

The strains were grown in LB and induced as described in Materials and Methods. In-gel

fluorescence samples were prepared from the mutants in the "wzy and "wzy "wzz

backgrounds and electrophoresed on SDS 15% (w/v) PAGE gels (see Materials and

Methods). (A) Lanes 1 and 2 contain the indicated strains; lanes 3 to 12 contain the "wzy or

"wzy "wzz strain expressing mutated WzySf-GFP, as indicated. (B) Lanes 1 and 2 contain the

indicated strains; lanes 2 to 26 contain the "wzy or "wzy "wzz strain expressing mutated

WzySf-GFP, as indicated.



100

Sf6c; while bacteriophage Sf6c only infected strains with WT (or nearly WT) LPS, both in

degree of Oag polymerisation and apparent level of LPS capping with Oag chains.

Only a few studies have been conducted on Wzy proteins. During characterisation of

Wzy of Francisella tularensis, Kim et al. (2010) reported several amino acid residues

important for Oag polymerisation. Modification of these residues (G176, D177, G323, and

Y324) led to a loss of Oag polymerisation (Kim et al., 2010). Islam et al. (2011) showed that

the PL3 and PL5 of WzyPa have net positive and net negative charges respectively, and they

established their “catch-and-release” model. However, for WzySf, we found that at a

physiological pH both the PL3 and PL5 possess net negative charge (pI of PL3 is 4.65 and

PL5 is 5.09). In Pseudomonas aeruginosa PAO1 there is a uronic acid sugar in the Oag

(Knirel et al., 2006), which is negatively charged. However, the Oag of WzySf is neutral. So,

the charged property of the substrate for WzySf is different from the WzyPa.The RX10G motifs

of the PL3 and PL5 of WzyPa (Islam et al., 2011) are also absent in WzySf. However, both PL3

and PL5 of WzySf contained RX15G motifs (starting from R164 in PL3 and R289 in PL5)

(Fig. 3.2). So, perhaps a modified version of the “catch-and-release” mechanism (Islam et al.,

2011) exists for S. flexneri. Moreover, addition of either glucosyl or O-acetyl groups or PEtN

residue by various linkages to the sugars within the tetrasaccharide repeats of serotype Y

creates 17 different serotypes of the S. flexneri (Allison & Verma, 2000; Sun et al., 2012;

Wang et al., 2010b). As the polymerisation of all these Oags is done by WzySf, and we

conclude that WzySf must be quite flexible for its substrate recruitment. Due to scant

homology between wzy proteins of different bacterial species, we were unable to create any

directly comparable mutation in WzySf with respect to other systems. This led us to perform

random mutagenesis on wzySf followed by screening with ColE2.

The mutations we found are present in a wide region (PL1, 2, 3, and 6; TM 2, 4, 5, 7,

8, and 9; and CL 1 and 5) of the WzySf topological model experimentally determined by

Daniels et al. (1998). We were able to locate a number of mutations in the TM regions and

interestingly the G130V mutation, which resulted in the complete loss of polymerisation

activity of WzySf in the !wzy background, was also located in TM5. Topological models are

not very accurate, but due to a lack of crystal structure we used the WzySf topology model

previously determined by our group to locate the mutational alterations. Furthermore, we



101

reassessed our topological model using 5 different topology prediction programs

(SPOCTOPUS, MEMSAT, HMMTOP, TOPCONS, and TMHMM) (Supplementary Table

S4). The results showed that until TM 6 all the programs are consistent with our topological

model. Three of them also validate TM10-TM11 of our topological model. So, the topological

model of WzySf determined by our group is nearly consistent with the topological models

predicted by the programs. The mutations, which resulted in the partial loss of polymerisation

in the !wzy background, were V92M, Y137H, L214I, and P352H. Among them V92M and

Y137H are present in the PL2 and TM5. These regions were validated by 5 of the programs.

P352H is present in the PL6 and this region was validated by 3 of the programs (MEMSAT,

HMMTOP, and TOPCONS). L214I is present in the TM8 and the region (amino acid 209-

226) was different compared to the computer prediction (amino acid 227-247). However,

G130V is present in the TM5, which was validated by 5 of the programs. Recently, Reddy et

al. (2014) reported that different topology prediction programs are suitable for different

families of proteins. So, non-prediction of TM7--TM9 by some programs does not invalidate

the presence of these regions in our model.

In the !wzy background, strains with the class A LPS profile had S-LPS with reduced

degree of Oag polymerisation (Fig. 3.3, lanes 2-4), mutants with the class B LPS profile had

LPS with few Oag RUs (Fig. 3.3, lane 5), and the mutant with class C LPS profile had SR-

LPS (Fig. 3.3, lane 6). As a result, the class A mutants were more sensitive to ColE2 than

PNRM13 and the class B mutants were even more sensitive to ColE2 than the mutants with

the class A LPS profile. The mutant with class C LPS had the highest ColE2 sensitivity. From

these three classes it is clear that the LPS with shorter Oag chains were more sensitive to

ColE2. Strains with class A, B, and C LPS profiles in the !wzy background were resistant to

bacteriophage Sf6c. The lack of bacteriophage Sf6c sensitivity indicates that bacteriophage

Sf6c only infects if the LPS has WT or close to a WT level of Oag polymerisation. In this

study, we found that the WzySf mutants conferring the class D LPS profile in the !wzy

background had a similar level of Oag polymerisation compared to the positive control strain

PNRM13, as determined by SDS-PAGE and silver staining (Fig. 3.3, lanes 7-10 and

Supplementary Fig. 3.S2, lanes 1-8) but they were more resistant to ColE2 and more sensitive

to bacteriophage Sf6c (Table 3.2) (except !wzy strain with WzyP165S). The ColE2 and



102

bacteriophage Sf6c sensitivities of these strains were similar to WT strain PE638. We

conclude that the Oag polymerisation activity of the mutants with class D LPS profile in the

!wzy background, was similar to the WT strain (PE638). Hence, the WT WzySf-GFP protein

is not 100% active, and the mutant proteins conferring the class D LPS profile are more active

and/or are better exported or folded or assembled to the IM. The level of Oag polymerisation

of the strains with class D LPS profile was more than PNRM13, although this was not

distinguishable by silver staining. Hence, the ColE2 assay was more sensitive than silver

staining and SDS-PAGE in determining the degree of Oag polymerisation.

Mutation in wzz resulted in Oag without modal chain length control in different

organisms (Bastin et al., 1993; Morona et al., 1995). Recently, Kenyon and Reeves (2013)

showed that Wzy of Yersinia pseudotuberculosis also needs Wzz for complete Oag

polymerisation. Here we examined the polymerisation activity of different WzySf mutants in

the absence of WzzSf. In the !wzy !wzz background, the strains with mutated wzySf plasmids

from the strains having class B LPS profiles in the !wzy background had an SR-LPS profile,

and ColE2 and bacteriophage Sf6c sensitivities were similar to the !wzy background.

Interestingly, the !wzy !wzz strain with WzyG130V had S-LPS without modal length control

(class F) where as the !wzy strain with WzyG130V had SR-LPS (class C) and this correlated

with an increased resistance to ColE2. In the !wzy !wzz background, strains with a class E

LPS profile (S-LPS without modal length control) had increased sensitivity to ColE2 and

increased or similar resistance to bacteriophage Sf6c compared to PNRM134 [!wzy !wzz

(pRMPN1)] and when in the !wzy background (strains with class A and class D LPS profiles)

(Table 3.2). We speculate that these strains (strains with a class E LPS profile) had subtle

alterations in the level of Oag polymerisation and/or capping of LPS with Oag, and were

unable to act efficiently as a bacteriophage Sf6c receptor and, correspondingly, were also

more sensitive to ColE2. It is known that phage adsorption to the cells of E. coli K12

increases with increase in density of receptor protein at the cell surface (Schwartz, 1976).

Based on our study, the relationship between ColE2 and Oag density/concentration seems

linear. Deceasing Oag progressively increases sensitivity to ColE2. However, the relationship

between bacteriophage Sf6c and Oag density/concentration seems non-linear. Decreasing Oag

rapidly decreases sensitivity to bacteriophage Sf6c. This may be because bacteriophage Sf6c



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interaction with its receptor is complex and most likely requires multi-receptor binding to

bacteriophage Sf6c tail spike proteins (TSPs) to achieve irreversible binding and hence

activation of bacteriophage Sf6c.

According to the proposed molecular clock model, Wzz acts as a molecular clock and

regulates Wzy activity between two states: “E” or extension state favors polymerisation and

“T” or transfer state favors the ligation reaction (Bastin et al., 1993). The molecular

chaperone model describes Wzz as a typical molecular chaperone, which regulates the overall

ratio of Wzy and WaaL in a complex, and controls the enzyme kinetics of the ligation reaction

to define the modality (Morona et al., 1995). However, Woodward et al. (2010) suggested

that there is an interaction between Wzy and Wzz and they showed that these proteins are

enough to shape the Oag modal chain length. Islam et al. (2013) suggested that the chain

length of the Oag is determined by the interaction of Wzz and Wzy. Recent work of Taylor et

al. (2013) also suggested the direct interaction of Wzz and Wzy in the Oag biosynthesis

pathway. In this study, for the first time we were able to give an insight in the association of

WzySf and WzzSf in the Oag biosynthesis. We found WzySf mutants where polymerisation

activity was dependent on WzzSf (WzyP352H and WzyL214I), and some other mutants where

polymerisation activity was repressed in the presence of WzzSf (WzyV92M, WzyY137H, and

WzyG130V). The "wzy "wzz strain, with WzyV92M or WzyY137H had S-LPS without

modal length control (class E) but in the "wzy background, they showed class A LPS profile

(a greatly reduced degree of Oag polymerisation) (Fig 4, lanes 6-7 and lanes 8-9). The "wzy

strain with WzyG130V had class C LPS profile (SR-LPS) but remarkably the "wzy "wzz

strain with WzyG130V had S-LPS without modal length control, albeit with a reduced degree

of polymerisation (class F) (Fig 4 lanes 12-13). Oag polymerisation by these WzySf mutants

was repressed by WzzSf, in the "wzySf background; their LPS had shorter Oag chains. The

"wzy strain with WzyP352H had class A LPS profile, and the "wzy strain with WzyL214I had

class B LPS profile but in the "wzy/"wzy background they had shorter Oag chains (class B

and C respectively) compared to the "wzy background (Fig. 3.4, lanes 4-5 and 4 lanes 10-11).

So, these WzySf mutants need WzzSf for their Oag polymerisation activity. These findings

suggest that WzzSf is associated with WzySf not only for Oag modal chain length control but



104

also for polymerisation activity, and amino acids V92, G130, Y137, L214, and P352 have role

in the association of WzySf and WzzSf during the Oag polymerisation mediated by WzySf.

The relative WzySf-GFP level in PNRM134 ("wzy "wzz) was lower than in PNRM13

("wzy) (Fig. 3.5A and B, lanes 1-2), suggesting that the WT WzySf-GFP had better expression

in the presence of WzzSf. WzySf mutants conferring class A LPS profile ("wzy background)

had WzySf-GFP levels less than the WzySf-GFP in PNRM13 (Fig. 3.5A, lanes 3, 5, 7 and

Table 3.2). WzyL214I was not detectable either in the "wzy (class B) or "wzy "wzz (class C)

backgrounds (Fig. 3.5A, lanes 9-10 and Table 3.2). Hence, amino acid L214 is important for

WzySf-GFP production. WzyG130V was not detectable in the "wzy background (class C) but

was detected in the "wzy "wzz background (class F) (Fig. 3.5A, lanes 11-12 and Table 3.2).

Except for the strain "wzy with WzyL191F, all the mutants with class D LPS profile ("wzy

background) had a lower protein expression level than WzySf-GFP in PNRM13 (Fig. 3.5B and

Table 3.2) by some unknown mechanism. However, the mutants with a class D LPS profile

("wzy background) had S-LPS and were more resistant to ColE2 and more sensitive to

bacteriophage Sf6c than PNRM13. In the "wzy "wzz background, WzyL191F had an

expression level less than WzySf-GFP in PNRM13 (Fig. 3.5B, lane 8 and Table 3.2).

WzyP165S was not detectable in PNRM6 ("wzy) but strain PNRM126 ("wzy "wzz) with

WzyP165S present at a higher level than WzySf-GFP in PNRM13 (Fig. 3.5B, lanes 5-6 and

Table 3.2). Apparently, there are more than one underlying mechanism for the class D (!wzy

background) to class E ("wzy "wzz background) LPS profile conversion. Two of the

mutations (P165S and G130V) resulted in decreased WzySf-GFP levels in the presence of

WzzSf. This suggested that the presence of WzzSf destabilises WzyP165S and WzyG130V,

which resulted in lower WzySf-GFP levels. The mutation L191F resulted in decreased WzySf-

GFP level in the absence of WzzSf. Therefore, in this case WzzSf stabilises the protein

WzyL191F resulting in better WzySf-GFP expression. This finding suggests that G130, P165,

and L191 are important for the stabilisation of WzySf through interaction with WzzSf.

In conclusion, our findings identified amino acid residues on the WzySf important for

its polymerisation function and interaction with WzzSf. Residues, which are important for the

polymerisation and interaction of WzzSf and WzySf are present in the PL 2, 6 and TM 5, 8.

These regions may contribute in the interaction of WzySf with substrates and also to the



105

interaction with WzzSf. We identified a number of amino acids (G130, P165, L191), which

are important for the stabilisation of WzySf in association with WzzSf. Hence, our data

suggested that WzzSf also has a role in WzySf stability.

3.6 Acknowledgements

We thank Dr. Daniel O. Daley for the pWaldo-TEV-GFP plasmid. Funding for this work is

provided by a program grant to R.M. from the National Health and Medical Research Council

(NHMRC) of Australia. P.N. is the recipient of an international postgraduate research

scholarship from the University of Adelaide.



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3.7 Supplementary information

Table 3.S1 Different primers made in this study

Primer* Sequence (5’-3’) Purpose PN1_wzySfKpnF 5’–GGCGGTACC ATGAATAATATTA

ATAAAATTTTTATTACA - 3’ amplification Of wzySf

PN2_wzySfBamHR 5’–GCGGGATCC TTTTGCTCCAGAA GT GAGGTTA - 3’

amplification of wzySf

ET35_F 5’-AGAGTAGAAAATAATAATGTTTC T- 3’


ET35_R 5’–GGCAAGCTTTTACTTCGCGTTGT AATTACG - 3’


*F, Forward; R, Reverse.



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Table 3.S2 Colicin E2 (ColE2) sensitivity (performed by swab assay) of different WzySf mutants during screening

Strain Relevant details ColE2 sensitivity*^ RMM109 Mutant wzySf ++++ PE638 WT R PNRM13 Positive control + PNRM6 Negative control ++++ PNRM11 Negative control ++++ Mutant # Mutant class

ColE2 sensitivity*^ P352H class A ++

V92M class A ++ Y137H class A ++ L214I class B +++ G130V class C ++++ N147K class D R P165S class D R L191F class D R C60F class D R L49F/T328A class D R L28V class D R N86K class D R F54C class D R F52Y class D R L111I class D R G82C class D R F52C/I242T class D R

*R, Resistant; ^Relative sensitivity to ColE2: ++++ > +++ > ++ > +; # Mutants are in

PNRM6.



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Table 3.S3 Nucleotide base change in the WzySf mutants

Mutations Site of point mutation in the WzySf sequence (5’-3’)

P352H 5’-CTTTTTACACCCATGGGAATTTTT-3’ V92M 5’-CTGTTTGATGTGCAACCATACCTT-3’

Y137H 5’-TATATTTCATACATAATTTTGTTG-3’ L214I 5’-TTTACAACATTATCACTGTTATTA-3’

G130V 5’-GCATTATATGGATCACTGTTATAT-3’

N147K 5’-GGTTTGTTAAATTTTAATTTAATT-3’

P165S 5’-TTTTTTCGTCCCGATGGGGCTTTT-3’ L191F 5’-AAAAAATATTTAAAATGTCTCATA-3’

C60F 5’-ACCCAAAAATGTGTCAGTCTTTTT-3’

L49F/T328A 5’-ATTTTTAATTTAGTTACATTCAAT-3’ 5’-AGTACAAAAACATCATTAATGATG-3’

L28V 5’-CTGGAGCCATTGGGAATATTCCCT-3’ N86K 5’-AATAAAATAAATGATATACTGTTT-3’

F54C 5’-ACATTCAATTTTTCAATCACCCAA-3’

F52Y 5’-TTAGTTACATTCAATTTTTCAATC-3’

L111I 5’-AGATATACTTTAAAAGTATTTTCA-3’ G82C 5’-TTATTAGCTGGTAATAAAATAAAT-3’

F52C/I24T 5’-TTAGTTACATTCAATTTTTCAATC-3’ 5’-CTATTTATAATTATGCTTTACATG-3’



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Table 3.S4 Verification of the WzySf topological model using topological prediction programs

Models Daniels et al.

1998 SPOCTOPUS MEMSAT HMMTOP TOPCONS TMHMM

Total TM 12 10 12 12 12 11

TM1 4 - 22 5 - 25 7 - 25 7 - 25 5 - 25 7 - 24 TM2 35 - 54 32 - 52 35 - 52 36 - 55 32 - 52 34 - 56

TM3 63 - 81

58 - 78 60 - 82 62 - 81 61 - 81 63 - 82 TM4 95 - 112

94 - 114 95 - 111 96 - 115 87 - 107 92 - 111

TM5 126 - 146

123 -1 43 122 - 146 122 - 146 132 - 152 118 - 140 TM6 168 - 186

170 - 190 169 - 186 167 - 186 167 - 187 167 - 186

TM7 190 - 206

196 - 216 194 - 218 193 - 217 196 - 216 193 - 215 TM8 209 - 226

227 - 247 226 - 247 230 - 248 226 - 246 230 - 249

TM9 230 - 247

298 - 318 269 - 285 269 - 285 270 - 290 262 - 281

TM10 301 - 318

349 - 369 302 - 218 302 - 321 298 - 318 301 - 323

TM11 330 - 349

---- 328 - 344 328 - 345 330 - 350 335 - 368

TM12 353 - 370 ---- 352 - 368 350 - 368 353 - 373

Consistency with current model

----- Nearly consistent

Nearly consistent

Nearly consistent

Nearly consistent

Nearly consistent



110

Figure 3.S1 WzySf expression

Plasmid pRMPN1 (pWaldo-wzySf-TEV-GFP-His8) was transformed into Lemo21(DE3) cells.

Strain was grown and induced as described in the Materials and Methods. To perform In-gel

fluorescence, whole cell In-gel fluorescence samples were prepared and electrophoresed on

SDS 15% (w/v) PAGE gel and the BenchMark protein ladder (Invitrogen) was used as a

molecular mass standard (See Materials and Methods). Samples in each lane are as follows: 1.

PNRM15 [Lemo21(DE3) (pRMPN1)] and 2: Lemo21(DE3).



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Figure 3.S2 LPS phenotype conferred by the class D WzySf mutants expressed in

PNRM6 ["wzy (pAC/pBADT7-1)]

Strains were grown and induced with arabinose as described in the Materials and Methods.

LPS samples were prepared, electrophoresed on an SDS-15% (w/v) PAGE gel, and silver

stained (See Materials and Methods). The class D WzySf mutants in each lane are: 1.

L49F/T328A, 2. L28V, 3. N86K, 4. F54C, 5. F52Y, 6. L111I, 7. G82C, 8. F52C/I242T.



112

Figure 3.S3 Comparison of the LPS phenotype conferred by the class D WzySf mutants

expressed in the "wzy and "wzy "wzz backgrounds

Plasmids encoded mutated WzySf proteins were expressed in PNRM6 (!wzy) and PNRM126

(!wzy !wzz). Strains were grown and induced as described in the Materials and Methods. LPS

samples were electrophoresed on an SDS-15% (w/v) PAGE gel and silver stained (See



113

Materials and Methods). Lanes 4-36 are the "wzy or "wzy "wzz strains with plasmids

encoding mutated WzySf protein. The class D WzySF mutants in each lane are as follows: 1.

L49F/T328A (!wzy), 2. L49F/T328A ("wzy "wzz), 3. L28V ("wzy), 4. L28V ("wzy "wzz), 5.

N86K ("wzy), 6. N86K ("wzy "wzz), 7. F54C ("wzy), 8. F54C ("wzy "wzz), 9. F52Y ("wzy),

10. F52Y ("wzy "wzz), 11. L111I ("wzy), 12. L111I ("wzy "wzz), 13. G82C ("wzy), 14.

G82C ("wzy "wzz), 15. F52C/I242T ("wzy), 16. F52C/I242T ("wzy "wzz).



114

3.7.1 Construction of pWaldo-wzySf-GFP-His81

Primers PN1_wzySfKpnF and PN2_wzySfBamHR (Table 3.S1) which incorporated the KpnI

and BamHI restriction sites, respectively, were used to amplify the previously mutated S.

flexneri 2457T 2a wzySf coding region in pRMCD6 plasmid (Table 2.2) (Daniels & Morona,

1999). Codons 4, 9, and 23 are rare codons present in the translation initiation regions of WT

WzySf and cause lower expression of WT WzySf (Daniels et al., 1998). Hence, the codons

ATA, ATA, and AGA at 4, 9, and 23 were altered to ATT, ATT, and CGT, respectively, and

result in the increased expression of WzySf (Daniels et al., 1998). The amplified wzySf was

cloned into pGEM-T-Easy vector and generated the plasmid p-GEM-T-Easy-wzySf. Following

digestion of the p-GEM-T-Easy-wzySf with BamHI and KpnI, the generated fragment

containing wzySf was ligated into similarly digested pWaldo-TEV-GFP (Waldo et al., 1999).

pWaldo-TEV-GFP contains T7 promoter, ribosomal binding site, and T7 terminator for the

regulation of gene expression, Km selection marker, GFP moiety for detection of the protein,

C-terminal His8 tag for protein purification, and tobacco etch virus (TEV) protease

recognition site for removal of the GFP-His8 moiety (Drew et al., 2006; Waldo et al., 1999).

The insertion of wzySf in the resulting plasmid pWaldo-wzySf-GFP-His8, (Fig. 3.S4) also

denoted as pRMPN1 (Table 2.2), was confirmed by DNA sequencing. The sequence of the

insert in pWaldo-wzySf-GFP-His8 is shown in Fig. 3.S5.

1 Sections 3.7.1 onwards were not part of the original manuscript and were included as additional information for

the thesis chapter.



115

Figure 3.S4 Construction of pWaldo-wzySf-GFP-His8 (pRMPN1) plasmid The plasmid pWaldo-wzySf-GFP-His8 was constructed as described in section 6.2. Briefly,

forward and reverse primers incorporating KpnI and BamHI restriction sites, respectively,

were used to amplify wzySf (Genbank accession number X71970.1) from plasmid pRMCD6

(Daniels & Morona, 1999) (A). The amplified fragment was then ligated into pGEM-T-Easy

(B), excised from the plasmid with KpnI/BamHI double digest (C), and ligated into the

similarly digested vector pWaldo-TEV-GFP (Waldo et al., 1999) (D).



116

1 atgaataatattaataaaatttttattacatttttatgtattgaactgattattggtggt 1 M N N I N K I F I T F L C I E L I I G G 61 ggtggacgtttactggagccattgggaatattccctttgcgatatttattatttgtattt 21 G G R L L E P L G I F P L R Y L L F V F 121 agttttatacttttaatttttaatttagttacattcaatttttcaatcacccaaaaatgt 41 S F I L L I F N L V T F N F S I T Q K C

181 gtcagtctttttatatggttgcttttatttcctttttatggcttctttgtcggcttatta 61 V S L F I W L L L F P F Y G F F V G L L

241 gctggtaataaaataaatgatatactgtttgatgtgcaaccatacctttttatgctgtca 81 A G N K I N D I L F D V Q P Y L F M L S

301 cttatatatctatttacactaagatatactttaaaagtattttcatgtgagatttttatt 101 L I Y L F T L R Y T L K V F S C E I F I

361 aaaatagttaatgcatttgcattatatggatcactgttatatatttcatacataattttg 121 K I V N A F A L Y G S L L Y I S Y I I L

421 ttgaatttcggtttgttaaattttaatttaatttatgaacacttatcattgactagcgag 141 L N F G L L N F N L I Y E H L S L T S E

481 ttcttttttcgtcccgatggggcttttttttccaaatccttctacttttttggtgtcggt 161 F F F R P D G A F F S K S F Y F F G V G

541 gcgattatcagttttgtcgacaaaaaatatttaaaatgtctcataatagtgcttgcgata 181 A I I S F V D K K Y L K C L I I V L A I

601 ttattgacagaatcaagaggtgtattactttttacaacattatcactgttattagccagt 201 L L T E S R G V L L F T T L S L L L A S

661 tttaaattacataagctatatttaaatactattataataatattgggcagcgttctattt 221 F K L H K L Y L N T I I I I L G S V L F

721 ataattatgctttacatggtcggatcacgcagtgaagattctgactctgttagatttaat 241 I I M L Y M V G S R S E D S D S V R F N



117

781 gatttatatttttattataaaaatgttgatttagcgacgttcttgtttggaagaggattt 261 D L Y F Y Y K N V D L A T F L F G R G F

841 ggttcatttatattagatcgattaaggattgaaatagtacctcttgagatacttcagaaa 281 G S F I L D R L R I E I V P L E I L Q K

901 acaggcgttattggtgtatttatatcattagttcctatgttgcttatctttttgaaaggc 301 T G V I G V F I S L V P M L L I F L K G

961 tattttttaaatagtacaaaaacatcattaatgatgtcgttaatactttttttcagtatt 321 Y F L N S T K T S L M M S L I L F F S I

1021 accgtttctataactaatccatttctttttacacccatgggaatttttattataggcgtt 341 T V S I T N P F L F T P M G I F I I G V

1081 gtagttttatgggtattttctatagaaaatatccaaattagtaataacctcacttctgga 361 V V L W V F S I E N I Q I S N N L T S G

1141 gcaaaataaGGATCC 381 A K - BamHI

Figure 3.S5 DNA and predicted amino acid sequence of the inserted WzySf sequence in pWaldo-TEV-GFP plasmid The DNA sequence and the amino acid sequence of the wzySf fragment inserted in pWaldo-

TEV-GFP plasmid are shown. The wzySf (GenBank accession number X71970.1) sequence is

highlighted in yellow, the sart codon is highlighted in green, and the stop codon is highlighted

in pink. The BamHI sequence is shown in grey. The alterations in codons 4, 9 and 23 of wzySf

are indicated in red text.

118

119

Chapter 4

Mutational analysis of the major periplasmic loops of Shigella

flexneri Wzy: identification of the residues affecting O antigen

modal chain length control, and Wzz-dependent polymerisation

activity

Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of the residues affecting O antigen modal chain length control, and Wzz-dependent polymerisation activity

120

Mutational analysis of the major periplasmic loops of

Shigella flexneri Wzy: identification of the residues

affecting O antigen modal chain length control, and Wzz-

dependent polymerisation activity

Pratiti Nath and Renato Morona

Discipline of Microbiology and Immunology, School of Molecular and

Biomedical Science, University of Adelaide, Adelaide 5005, Australia

Microbiology. 2015 Apr;161(Pt 4):774-85. doi: 10.1099/mic.0.000042. Epub 2015 Jan 27.


121


Title of Paper Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of the residues affecting O antigen modal chain length control, and Wzz dependent polymerisation activity.

Publication Status Published

Publication Details Microbiology. 2015 Apr;161(Pt 4):774-85. doi: 10.1099/mic.0.000042. Epub 2015 Jan 27.


Pratiti Nath

Contribution to the Paper Performed all experiments, performed analysis on all

samples, interpreted data, and wrote manuscript.

Signature

Date 20.5.15

Name of Co-Author Renato Morona

Contribution to the Paper Supervised development of work, helped in data

interpretation, manuscript evaluation and editing.

Signature

Date


122

Chapter 4: Second paper

4.1 Abstract

The O antigen (Oag) component of LPS is a major Shigella flexneri virulence determinant.

Oag is polymerised by WzySf, and its modal chain length is determined by WzzSf and

WzzpHS2. Site-directed mutagenesis was performed on wzySf in pWaldo-wzySf-TEV-GFP to

alter Arg residues in WzySf’s two large periplasmic loops (PLs) (PL3 and PL5). Analysis of

the LPS profiles conferred by mutated WzySf proteins in the wzySf deficient ("wzy) strain

identified residues that affect WzySf activity. The importance of the guanidium group of the

Arg residues was investigated by altering the Arg residues to Lys and Glu, which generated

WzySf mutants conferring altered LPS Oag modal chain lengths. The dependence of these

WzySf mutants on WzzSf was investigated by expressing them in a wzySf and wzzSf deficient

("wzy "wzz) strain. Comparison of the LPS profiles identified a role for the Arg residues in

the association of WzySf and WzzSf during Oag polymerisation. Colicin E2 and bacteriophage

Sf6c susceptibility supported this conclusion. Comparison of the expression levels of different

mutant WzySf -GFPs with the wild-type WzySf-GFP showed that certain Arg residues affected

production levels of WzySf in a WzzSf -dependent manner. To our knowledge, this is the first

report of S. flexneri WzySf mutants having an effect on LPS Oag modal chain length, and

identified functionally significant Arg residues in WzySf.

4.2 Introduction

Shigella flexneri is the main causative agent for the disease shigellosis or bacterial dysentery.

Approximately, 125 million shigellosis cases occur annually in Asia, with nearly 14000

fatalities (Bardhan et al., 2010). The O antigen (Oag) component of the lipopolysaccharide

(LPS) of Shigella flexneri plays an important role in the pathogenesis of the bacteria. Oag is

composed of oligosaccharide repeat units (RUs) or O units. Oag is linked to the hydrophobic

anchor of the LPS (Lipid A) by the non-repeating oligosaccharide domain known as the core

sugar region (Raetz & Whitfield, 2002; Sperandeo et al., 2009). The complete LPS structure

with Oag chains is termed smooth LPS (S-LPS). However, the LPS structure devoid of Oag is


123

termed rough LPS (R-LPS), and LPS with only one O unit is termed semi-rough LPS (SR-

LPS) (Morona et al., 1994).

Oag is the serotype determinant and also the protective antigen of the bacteria (Jennison

& Verma, 2004; Stagg et al., 2009; Sun et al., 2013b). On the basis of the composition of

Oag, S. flexneri is divided into 17 serotypes (Sun et al., 2013b). Except serotype 6, all the

serotypes share a basic polysaccharide backbone containing three L-rhamnoses (Rha), and

one N-acetylglucosamine (GlcNAc). This basic Oag structure is known as serotype Y. The

differences between the serotypes are conferred by addition of glucosyl, O-acetyl, or

phosphoethanolamine (PEtN) functional groups by various linkages to the sugars of the basic

tetrasaccharide RU (Allison & Verma, 2000; Sun et al., 2012; Wang et al., 2010b). Oag

restricts the accessibility of the colicin to their outer membrane (OM) receptor protein (Tran

et al., 2014; van der Ley et al., 1986). In addition, the bacteriophage Sf6 uses Oag as a

receptor and forms plaques on serotype Y and X strains (Lindberg et al., 1978).

S. flexneri LPS biosynthesis occurs mainly by two separate pathways: 1) lipid A and

core biosynthesis and 2) Oag biosynthesis. Oag biosynthesis occurs on either side of the inner

membrane (IM) and it is mediated by the Wzy-dependent pathway (Allison & Verma, 2000;

Morona et al., 1995). S. flexneri Oag biosynthesis starts by the transfer of GlcNAc phosphate

from an uridine diphosphate-GlcNAc (UDP-GlcNAc) to undecaprenol phosphate (Und-P) at

the cytoplasmic side of the IM by WecA (Guo et al., 2008; Liu et al., 1996; Wang et al.,

2010b). The rhamnosyl transferases (RfbG and RfbF) then add sequential Rha residues to the

GlcNAc to form the O unit (Morona et al., 1994). Translocation of the O unit to the

periplasmic side is mediated by the protein Wzx. At the periplasmic side O units are

polymerised by Wzy to form the Oag. The chain length of the Oag is regulated by Wzz

(Daniels et al., 1998; Morona et al., 1994). Finally, the Oag chains are transferred to the core-

lipid A by the ligase WaaL. The Lpt proteins (Lpt A-G) facilitate the transport of the LPS

from the IM to the OM (Ruiz et al., 2008; Sperandeo et al., 2009).

S. flexneri Wzy (WzySf) is a 43.7 kDa hydrophobic integral membrane protein. It has 12

transmembrane (TM) segments and two large periplasmic (PL) domains (PL3 and PL5)

(Daniels et al., 1998; Morona et al., 1994). Previously we were able to identify some of the


124

key functional amino acid residues of WzySf, providing insight into WzySf structure and

function (Nath et al., 2015). WzySf has an RX15G motif in both of the PL3 and PL5 starting

from R164 in PL3 and from R289 in PL5. There are several Arg residues between these two

motifs. In the Pseudomonas aeruginosa Wzy (WzyPa), it was found that the PL3 and PL5

have RX10G motifs, which are important for Oag polymerisation activity. There are several

Arg residues within these two motifs, which play an important role in the Oag polymerisation

(Islam et al., 2011). However, there is little sequence identity between WzySf and WzyPa.

(Islam et al., 2013) performed extensive work on WzyPa and conducted a “jackhammer”

search to find the homologues of WzyPa. However, their results showed that WzyPa is not

related to Wzy from Enterobacteriaceae.

The modal length of the Oag chain is regulated by Wzz proteins, members of the

polysaccharide co-polymerase (PCP) family (Morona et al., 2000b). S. flexneri 2a has S-LPS

with two types of modal chain length: short (S) type (11-17 Oag RUs) and very long (VL)

type (>90 Oag RUs), and the S-type and VL-type Oag chain lengths are determined by WzzSf

and WzzpHS2, respectively (Morona et al., 2003; Morona & Van Den Bosch, 2003b).

(Woodward et al., 2010) proposed that Wzy and Wzz have an interaction during Oag

biosynthesis and that these two proteins are enough to shape the Oag modal chain length.

Several other research groups have also suggested that Wzz and Wzy interact during Oag

biosynthesis (Islam et al., 2013; Marolda et al., 2006; Taylor et al., 2013; Tocilj et al., 2008).

However, there is a lack of direct evidence on the association of Wzz and Wzy in Oag

polymerisation and chain length control. Recently, we identified the WzzSf dependent WzySf

mutants, and showed that WzzSf has a novel role in the stability of WzySf and also in the Oag

polymerisation activity of WzySf (Nath et al., 2015).

In this study we performed site-directed mutagenesis on Arg residues in the PL3 and

PL5 of WzySf and identified key Arg residues (R164, R250, R258, and R289) important for

WzySf polymerisation activity and Oag modal chain length control. Several Arg residues have

a role in the association of WzzSf and WzySf during Oag biosynthesis and the WzzSf dependent

stability of WzySf.


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4.3.1 Bacterial strains and plasmids

The strains and plasmids used in this study are detailed in Table 4.1.

4.3.2 Growth media and growth conditions

The growth media used were lysogeny broth (LB) broth (10 g/liter tryptone, 5 g/liter yeast

extract, 5 g/liter NaCl) and LB agar (LB broth, 15 g/liter bacto agar).

Strains were grown in LB broth with aeration for 18 h at 37°C. 18 h cultures were

diluted 1/20 into fresh LB broth and grown to mid-exponential phase (OD600 of 0.4-0.6). To

suppress protein expression, growth medium was supplemented with 0.2% (w/v) glucose

where required. Cells were centrifuged (2200 x g, SIGMA 3K15 table top centrifuge, 10 min,

4°C) and washed twice with LB broth to remove glucose. To induce protein expression, 0.2%

(w/v) L-Arabinose was added to cultures and grown for 20 h at 20°C. Antibiotics were added

as required to the media at the following final concentrations: 50 µg/ml kanamycin (Km) and

25 µg/ml chloramphenicol (Cm).

4.3.3 LPS method

LPS was prepared as described previously (Nath et al., 2015). Cells (1X109) were harvested

and resuspended in lysing buffer and incubated with proteinase K for approximately 16 h. The

LPS samples were then separated by SDS-PAGE on 15% (w/v) gels for 16.5 h at 12 mA.

Silver nitrate was used to stain the gels and finally the gels were developed with

formaldehyde (Murray et al., 2003).


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Strains or Plasmids Characteristics* Reference Strains S. flexneri PE638 S. flexneri Y rpoB (Rifr) (Morona et al., 1994) RMM109 PE638"wzy, Rifr (Morona et al., 1994) RMA4337 RMM109 "wzz (Rifr, Tetr) (Nath et al., 2015) PNRM6 RMM109 (pAC/pBADT7-1) (Nath et al., 2015) PNRM13 PNRM6 (pRMPN1) (Nath et al., 2015) PNRM16 PNRM6 (pRMPN2) This study PNRM17 PNRM6 (pRMPN3) This study PNRM18 PNRM6 (pRMPN4) This study PNRM19 PNRM6 (pRMPN5) This study PNRM20 PNRM6 (pRMPN6) This study PNRM126 RMA4337 (pAC/pBADT7-1) (Nath et al., 2015) PNRM134 PNRM126 (pRMPN1) (Nath et al., 2015) PMRM127 PNRM126 (pRMPN2) This study PMRM128 PNRM126 (pRMPN3) This study PMRM129 PNRM126 (pRMPN5) This study PMRM130 PNRM126 (pRMPN6) This study PMRM153 PNRM126 (pRMPN4) This study PNRM190 PNRM6 (pRMPN27) This study PNRM192 PNRM6 (pRMPN28) This study PNRM194 PNRM6 (pRMPN29) This study PNRM196 PNRM6 (pRMPN30) This study PNRM198 PNRM6 (pRMPN31) This study PNRM216 PNRM6 (pRMPN32) This study PNRM218 PNRM6 (pRMPN33) This study PNRM220 PNRM6 (pRMPN34) This study PNRM222 PNRM6 (pRMPN36) This study PNRM232 PNRM6 (pRMPN35) This study


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Table 4.1 continued Strains or Plasmids Characteristics* Reference E. coli XL10-G Tet r"(mcrA)183 "(mcrCB-hsdSMR-

mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F´ proAB lacIqZ"M15Tn10 (Tetr) Camr]

Stratagene

Plasmids pAC/pBADT7-1 Source of T7 RNA polymerase; Cmr (McKinney et al., 2002)

pWaldo-TEV-GFP Cloning vector with GFP tag; Kmr (Waldo et al., 1999) pRMPN1 pWaldo-wzySF-GFP; Kmr (Nath et al., 2015) pRMPN2 pRMPN1 with WzyR164A This study pRMPN3 pRMPN1 with WzyR250A This study pRMPN4 pRMPN1 with WzyR258A This study pRMPN5 pRMPN1 with WzyR278A This study pRMPN6 pRMPN1 with WzyR289A This study pRMPN27 pRMPN1 with WzyR164K This study pRMPN28 pRMPN1 with WzyR250K This study pRMPN29 pRMPN1 with WzyR258K This study pRMPN30 pRMPN1 with WzyR278K This study pRMPN31 pRMPN1 with WzyR289K This study pRMPN32 pRMPN1 with WzyR164E This study pRMPN33 pRMPN1 with WzyR250E This study pRMPN34 pRMPN1 with WzyR258E This study pRMPN35 pRMPN1 with WzyR278E This study pRMPN36 pRMPN1 with WzyR289E This study

* Rifr, rifampicin resistant; Kmr, kanamycin resistant; Cmr, chloramphenicol resistant; Tetr,

tetracycline resistant.


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4.3.4 Site-directed mutagenesis

Site-directed mutagenesis on wzySf in plasmid pRMPN1 (Nath et al., 2015; Waldo et al.,

1999) was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit

(Catalogue number 210518, Stratagene) following the manufacturer’s instructions.

Mutagenised plasmids were transformed into XL10-Gold. Plasmids were isolated and the

mutations within the coding region were identified by DNA sequencing (AGRF, Adelaide,

Australia). The oligonucleotide primers used for site-directed mutagenesis are listed in

Supplementary Table 4.S1.

4.3.5 Detection of WzySf expression in S. flexneri

Procedure of WzySf-GFP expression in S. flexneri has been described previously (Nath et al.,

2015). Cells were harvested from the 50 ml L-arabinose induced culture. Then the cell pellet

was resuspended in 4 ml sonication buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) and

lysed by sonication. Cell debris was removed by centrifugation (2200 x g, SIGMA 3K15 table

top centrifuge, 10 min, 4 °C). The whole membrane (WM) fraction was isolated by

ultracentrifugation (Beckman Coulter Optima L-100 XP bench top ultracentrifuge, 126 000 X

g, 1 h, 4 °C). The WM fraction was resuspended in PBS and then solubilised in Buffer A [200

mM Tris-HCl (pH 8.8), 20% (v/v) glycerol, 5 mM EDTA (pH 8.0), 0.02% (w/v)

bromophenol blue, 4% (w/v) SDS, and 0.05 M DTT]. Solubilised WM fractions (from 3 X

108 cells) were electrophoresed on SDS-15% (w/v) PAGE gels. Gels were rinsed with

distilled water, and fluorescent imaging of the gels was performed to detect wild type (WT)

and mutant WzySf-GFP protein expression with a Bio-Rad Gel Doc XR + System using Image

Lab software (excitation at 485 nm and emission at 512 nm). Loading was checked by

staining the gels with Coomassie Blue R-250. The intensity of WT and mutant WzySf-GFP

expression in control and mutant strains was measured by Fiji image processing package

(http://fiji.sc/Fiji) and the percent relative WzySf-GFP intensity for each mutant was measured

by comparing with WT WzySf-GFP intensity in the control strain PNRM13.


129


For the colicin sensitivity assay, His6-colicin E2 (ColE2) with an initial concentration of 1

mg/ml was used (Tran et al., 2014). The procedure of ColE2 spot assay has been described

previously (Nath et al., 2015). The ColE2 spot assay was performed for all strains expressing

WT and mutant WzySf-GFP and the other control strains. The end point of the killing zones of

mutant strains was compared with the controls.


The procedures used for phage propagation and phage stock preparation have been described

previously (Mavris et al., 1997; Morona et al., 1994), and the bacteriophage Sf6c sensitivity

assay was as described previously (Nath et al., 2015). Phage sensitivity of all strains

expressing mutant WzySf-GFP was compared with the strains expressing WT WzySf-GFP and

the other controls.

4.4 Results

4.4.1 Site-directed mutagenesis of Arg residues in PL3 and PL5 of WzySf

In a previous study on WzySf, random mutagenesis failed to detect any functional residue in

PL5 (Nath et al., 2015). However, Islam et al. (2011) found that the Arg residues in the two

principal PLs (PL3 and PL5) of WzyPa are important for Oag polymerisation activity. The

RX10G motifs of the PL3 and PL5 of WzyPa (Islam et al., 2011) are absent in WzySf.

However, both PL3 and PL5 of WzySf contained RX15G motifs (starting from R164 in PL3

and R289 in PL5) (Fig. 4.1) (Table 4.S2), and there are also several Arg residues between

these two motifs. So, site-directed mutagenesis on wzySf in the pRMPN1 was performed to

change the basic polar and positively charged Arg residues (R164, R250, R258, R278, and

R289) to Ala (no-npolar and neutral substitution), Lys (basic polar and positively charged

substitution), and Glu (acidic polar and negatively charged substitution) (Fig. 4.1) (See


130

Figure 4.1 Location of the mutations constructed in this study on the topology map of WzySf Mutational alterations were indicated by arrows on the WzySf topology map [adapted from

Daniels et al. (1998)]. The positions of PL1-5, TM1-12, and cytoplasmic loops (CL) 1-5 are

indicated. The residues mutated in this study (dark grey circles) are located in PL3 and 5. The

position of RX15G motifs (light grey circles) in PL3 and PL5 of WzySf, starting from R164 and

R289 respectively, are indicated.


131

Materials and Methods). Mutated plasmids were transformed into PNRM6 [RMM109

(pAC/pBADT7-1)] (!wzy) (Table 4.1) for phenotypic analysis (Nath et al., 2015).

4.4.2 LPS phenotype conferred by the WzySf mutants

LPS profiling by SDS-PAGE and silver staining were used to detect the effect of the WzySf

mutations on the LPS Oag polymerisation. Mutants were initially grouped into five different

phenotypic classes (A, B, C, D, and F) (Fig. 4.2 and Table 4.2) by comparing the LPS profiles

of the mutant strains with the WT positive control PNRM13. Based on the number of Oag

RUs in the Oag modal chain length (WzzSf regulated S-type) the class A mutants were further

subdivided into subclasses A1, A2, and A3 (Table 4.2). The mutational alteration R164A

resulted in complete loss of Oag polymerisation activity (SR-LPS or class C) (Fig. 4.2, lane

3), R164K resulted in an S-LPS with reduced Oag polymerisation (<30 Oag RUs) and lacking

modal chain length control (class F LPS) (Fig. 4.2, lane 5; and Table 4.2), and R164E resulted

in complete loss of polymerisation activity (SR-LPS or class C) (Fig. 4.2, lane 7), similar to

R164A. Mutational alterations R250A and R250E resulted in decreased Oag polymerisation

activity (LPS with <11 Oag RUs or class B) (Fig. 4.2, lanes 9 and 13). However, the

mutational alteration R250K resulted in an S-LPS with reduced Oag polymerisation, and the

modal chain length was reduced to 8-11 RUs (subclass A1) (Fig. 4.2, lane 11; and Table 4.2)

and was just detectable compared to the positive control (PNRM13). Similar to mutational

alterations of R164, both R258A and R258E resulted in an SR-LPS (class C) (Fig. 4.2, lanes

15 and 19). The mutational alteration R258K resulted in an S-LPS with reduced

polymerisation and the modal chain length was reduced to 9-14 RUs (subclass A2) (Fig. 4.2,

lane 17; and Table 4.2). For residue R278, the mutational alterations investigated had no

detectable effect on the LPS profiles, and all strains had LPS profiles (class D) similar to the

relevant WT control (PNRM13) (Fig. 4.2, lanes 21, 23, and 25). For residue R289, mutational

alteration R289A resulted in a class A LPS profile and the LPS Oag modal chain length of


132

Figure 4.2 Comparison of the LPS phenotype conferred by the WzySf mutants expressed in the "wzy and "wzy "wzz backgrounds The plasmids encoded mutant and WT WzySf proteins were expressed in PNRM6 [RMM109

(pAC/pBADT7-1)] and PNRM126 [RMA4337 (pAC/pBADT7-1)]. Strains were grown and

induced as described in the Materials and Methods. LPS samples were electrophoresed on a

SDS-15% (w/v) PAGE gel and silver stained (See Materials and Methods). Strains were

grouped into various mutant classes (A-F) and subclasses (A1-3) based on their LPS profiles

as described in the text (Table 4.2). Lanes 1-2 are: 1. PNRM13 [PNRM6 (pRMPN1)]; 2.

PNRM134 [PNRM126 (pRMPN1)]. Lanes 3-32 are the "wzy or "wzy "wzz strains with

plasmids encoding mutated WzySf proteins. The WzySf mutants in each lane are as follows: 3.

R164A ("wzy), 4. R164A ("wzy "wzz), 5. R164K ("wzy), 6. R164K ("wzy "wzz), 7. R164E

("wzy), 8. R164E ("wzy "wzz), 9. R250A ("wzy), 10. R250A ("wzy "wzz), 11. R250K

("wzy), 12. R250K ("wzy "wzz), 13. R250E ("wzy), 14. R250E ("wzy "wzz), 15. R258A

("wzy), 16. R258A ("wzy "wzz), 17. R258K ("wzy), 18. R258K ("wzy "wzz), 19. R258E


133

("wzy), 20. R258E ("wzy "wzz), 21. R278A ("wzy), 22. R278A ("wzy "wzz), 23. R278K

("wzy), 24. R278K ("wzy "wzz), 25. R278E ("wzy), 26. R278E ("wzy "wzz), 27. R289A

("wzy), 28. R289A ("wzy "wzz), 29. R289K ("wzy), 30. R289K ("wzy "wzz), 31. R289E

("wzy), 32. R289E ("wzy "wzz). The position of R-LPS is indicated. The numbers on the left

and right indicate the Oag RUs. Letters (A1-3, B-F) at the bottom indicate the mutant class

(Table 4.2).


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Table 4.2 LPS profiles of different WzySf mutant phenotypic classes

WzySf mutant Class LPS profile A1 S-LPS with reduced Oag polymerisation, and the modal

chain length was reduced to 8-11 RUs A2 S-LPS with reduced polymerisation and the modal chain

length was reduced to 9-14 or 8-14 Oag RUs A3 S-LPS with reduced polymerisation (<22 Oag RUs) and

the modal chain length was similar to the WT control (PNRM13)

B LPS with few Oag RUs (<11 Oag RUs) C SR-LPS D LPS profile similar to the WT control PNRM13 E S-LPS lacking Oag modal chain length control F S-LPS with reduced Oag polymerisation and lacking Oag

modal chain length control (<30 Oag RUs)


135

this strain was similar to PNRM13 (Fig. 4.2, lane 27; and Table 4.2) but lacked S-LPS with

Oag >22 RUs; the LPS profile of this strain was further classified as subclass A3. The

mutational alteration R289E resulted in a class A LPS profile with an Oag modal length of 8-

14 RUs (Subclass A2) (Fig. 4.2, lane 31; and Table 4.2) that was shorter than that seen in the

positive control (PNRM13). The mutational alteration R289K resulted in a class D LPS

profile (Fig. 4.2, lane 29). So, except R278 the other Arg residues (R164, R250, R258, and

R289) were found to be important for WzySf Oag polymerisation activity. For the positions

R164, R1250, and R258 the guanidium functional group of Arg is important as Lys

substitution resulted in partial WzySf activity. In particular, certain substitutions [R164K (no

modal chain length), R250K (8-11 RUs), R258K (9-14 RUs), and R289E (8-14 RUs)] (Table

4.2) resulted in an S-LPS with a decreased Oag modal chain length.

4.4.3 WzzSf dependence and polymerisation activity

Previously we found an effect of WzzSf on WzySf Oag polymerisation activity (Nath et al.,

2015). We investigated the dependence of the mutant WzySf proteins generated above on

WzzSf for their Oag polymerisation activity. All the plasmids encoding the mutated WzySf

proteins were transformed into the strain PNRNM126 [RMA4337 (pAC/pBADT7)], which

has both wzySf and wzzSf genes inactivated. LPS profiles conferred in the PNRM126

background (!wzy !wzz) were directly compared with the LPS profiles conferred in the

PNRM6 background (!wzy). The positive control strain in the !wzy !wzz background,

PNRM134 [PNRM126 (pRMPN1)] (Table 4.1) had a class E LPS profile (S-LPS without

Oag modal length control) (Fig. 4.2, lane 2; and Table 4.2). WzySf with Ala, Lys, and Glu

substitutions of R164 resulted in similar LPS profiles both in the !wzy and !wzy !wzz

backgrounds (Fig. 4.2, lanes 3-4, 5-6, and 7-8). The WzySf mutations R250A, R250E, and

R258A resulted in similar LPS profiles both in the !wzy and !wzy !wzz backgrounds (Fig.

4.2, lanes 9-10, 13-14, and 15-16). Interestingly, WzyR250K resulted in LPS with greatly

reduced Oag polymerisation (class B) in the !wzy !wzz background (Fig. 4.2, lane 12)

compared to the !wzy background (class A1) (Fig. 4.2, lane 11). In contrast, WzyR258E


136

resulted in dramatic increase in Oag polymerisation in the !wzy !wzz background (class E)

compared to the !wzy background (Class C) (Fig. 4.2, lanes 19-20). However, WzyR258K

resulted in a class F LPS profile (Fig. 4.2, lane 18) in the !wzy !wzz background. For residue

R278, all changes resulted in class E LPS profiles (Fig. 4.2, lanes 22, 24, and 26) in the !wzy

!wzz backgrounds, as expected. WzyR289A and WzyR289E resulted in class F LPS profiles

(Fig. 4.2, lanes 28 and 32) in the !wzy !wzz background. In contrast, the !wzy !wzz strain

with WzyR289K resulted in an S-LPS lacking Oag modal chain length control (class E LPS

profile) (Fig. 4.2, lane 30) and was similar to the control PNRM134 (Fig. 4.2, lane 2). Hence,

some of the WzySf mutants showed remarkably different LPS profiles in the absence of

WzzSf, indicating WzzSf dependence of their Oag polymerisation activity as previously

reported for other WzySf mutants (Nath et al., 2015).

4.4.4 ColE2 sensitivity of strains with WzySf mutants

The ColE2 sensitivity of the strains expressing WzySf mutants was investigated to verify the

LPS profiles determined by SDS-PAGE and silver staining. The ColE2 sensitivity

(summarised in Table 4.3) was determined by spot testing as described in the Materials and

Methods.

As expected, the negative control strains RMM109, PNRM6, PNRM11, RMA4337,

and PNRM126 had the highest sensitivity to ColE2 (killing zone at a dilution of 1/256) (Table

4.3). The WT strain PE638 and the positive control with WzySf-GFP in the !wzy !wzz

background (PNRM134) were resistant to the highest concentration of ColE2 used. However,

the positive control with WzySf-GFP in the !wzy background (PNRM13) showed a killing

zone at a dilution of 1/2 (Table 4.3), as previously reported (Nath et al., 2015). Strains with a

class A LPS profile in the !wzy background was sensitive to ColE2. Among them, strains

with decreased Oag modal chain length (subclass A1) were relatively more sensitive to ColE2

(killing zone at 1/64). However, the strains with more Oag (subclass A2 and A3) showed a

Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of residues affecting O antigen modal chain length control and Wzz-dependent polymerisation

137

Table 4.3 ColE2 and bacteriophage Sf6c sensitivities, and WzySf-GFP expression levels Sensitivity* Mutant class ColE2

Sf6c Relative WzySf-GFP (%)

Strain or mutants

Relevant details !wzy background

!wzy !wzz background

Topology map location#

!wzy background


!wzy background


!wzy background


Strains RMM109 wzySf mutant 1/256 - R - - - PE638 Wild type R - 10-6 - - - PNRM13 Positive control 1/2 - 10-5 - 100 - PNRM6 Negative control 1/256 - R - - - PNRM11 Negative control 1/256 - R - - - RMA4337 wzySf and wzzSf

mutant - 1/256 - R - -

PNRM126 Negative control - 1/256 - R - - PNRM134 Positive control - R - 10-6 - 17 Mutants R164A C C PL3 1/256 1/256 R R 132 87 R250A B B PL5 1/128 1/128 R R 81 55 R258A C C PL5 1/256 1/256 R R 62 200 R278A D E PL5 R 1/16 10-6 N 18 14 R289A A3 F PL5 1/32 1/128 R R 14 82 R164K F F PL3 1/64 1/128 R R 120 125 R250K A1 B PL5 1/64 1/128 R R 142 11 R258K A2 F PL5 1/32 1/64 N R 104 0.02 R278K D E PL5 1/2 1/16 10-5 10-1 46 36 R289K D E PL5 1/4 1/64 10-5 N 60 38 R164E C C PL3 1/256 1/256 R R 80 99 R250E B B PL5 1/128 1/128 R R 133 143 R258E C E PL5 1/256 1/64 R R 16 38 R278E D E PL5 R 1/64 10-6 R 135 28 R289E A2 F PL5 1/32 1/64 N R 21 140

Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of residues affecting O antigen modal chain length control and Wzz-dependent polymerisation activity

138

# PL - Periplasmic loop (See Fig. 4.1).

* R, Resistant; N, plaques detected with undiluted Sf6c stock; the numbers represent the highest

dilution showing the zone of inhibition or plaques formation.


139

killing zone at 1/32. Strains with a class B LPS profiles (both !wzy and !wzy !wzz backgrounds)

showed a killing zone at 1/128. As expected, the strains with class C LPS profiles (SR-LPS) (both

!wzy and !wzy !wzz backgrounds) showed the highest sensitivity to ColE2 (killing zone at 1/256),

and their sensitivity to ColE2 was similar to the negative control strains. Strains with WT like class

D LPS profiles (!wzy background) were more resistant to ColE2 (killing zone R-1/4). Among them

the !wzy strains with WzyR278A and WzyR289E showed ColE2 sensitivity similar to the WT strain

PE638, greater than the relevant positive control PNRM13. Strains with a class E LPS profile (!wzy

!wzz background) showed greater sensitivity to ColE2 (killing zone 1/16-1/64) compared to the

relevant positive control PNRM134, suggesting that they had a decreased level of Oag

polymerisation. Strains with a class F LPS profile in the !wzy and !wzy !wzz backgrounds were also

very sensitive to ColE2, and showed a killing zone at 1/64 or 1/128. Hence, as reported previously

(Nath et al., 2015), the ColE2 assay detects subtle differences in LPS Oag chain length and density,

which are consequences of difference in Oag polymerisation.

4.4.5 Bacteriophage Sf6c sensitivity of strains with WzySf mutants

The bacteriophage Sf6c sensitivity of the strains expressing WzySf mutants was investigated to

further verify the LPS profiles determined by SDS-PAGE and silver staining. The bacteriophage

Sf6c sensitivity of the strains (summarised in Table 4.3), carrying mutated wzySf plasmids were

determined by spot testing (see Materials and Methods).

The negative control strains (with an SR-LPS profile) RMM109, PNRM6, PNRM11,

RMA4337, and PNRM126 were all resistant to the highest concentration of bacteriophage Sf6c

tested, as expected. The WT strain PE638 and the positive control with WzySf-GFP in the !wzy !wzz

background (PNRM134) showed the highest sensitivity to bacteriophage Sf6c and showed plaques at

10-6 dilution. However, for the positive control with WzySf-GFP in the !wzy background (PNRM13)

the phage showed plaques at 10-5 (Table 4.3) (Nath et al., 2015). Strains with class A, B, C, and F

LPS profiles in the !wzy and !wzy !wzz backgrounds were resistant to the highest concentration of


140

bacteriophage Sf6c tested, similar to the negative control strains, except !wzy strain with

WzyR258K and WzyR289E showed plaques for undiluted Sf6c stock. However, the strains with

class D LPS profiles were very sensitive to bacteriophage Sf6c (plaques at 10-5 or 10-6). Among them

the !wzy !wzz strain with WzyR278A and WzyR278E showed the highest sensitivity to Sf6c and

their sensitivity to bacteriophage Sf6c was greater than the relevant positive control PNRM13, and

similar to the positive control with WzySf-GFP in the !wzy !wzz background (PNRM134). The

strains with class E LPS profile were relatively more resistant to bacteriophage Sf6c (resistant or

plaques at 10-1 or N) compared to the relevant positive control PNRM134, indicating a difference in

Oag density. Similar to our previous data (Nath et al., 2015), the bacteriophage Sf6c assays above

indicated that the degree of Oag polymerisation and density is correlated with bacteriophage Sf6c

sensitivity.

4.4.6 Protein expression levels of the WzySf mutants

We measured parental and mutant WzySf-GFP expression in the !wzy and !wzy !wzz backgrounds

by in-gel fluorescence and then calculated the % relative WzySf-GFP expression of all the mutant

strains by comparing expression levels of different WzySf-GFP mutants with the WzySf-GFP in

PNRM13 (100%) (See Materials and Methods). The positive control in the !wzy !wzz background

(PNRM134) had WzySf-GFP expression level (relative WzySf-GFP level 17%) less than the positive

control in the !wzy background (PNRM13) (Fig. 4.3A, B, and C, lanes 1 and 2, Table 4.3).

In both the !wzy and !wzy !wzz backgrounds, with some exceptions, most of the WzySf

mutants were expressed at a level less than 100%. The !wzy strain with WzyR164A had expression

of 132% (Fig. 4.3A, lane 3; and Table 4.3) but the !wzy !wzz strain with WzyR164A had a relative

WzySf-GFP level of 87% (Fig. 4.3A, lane 4; and Table 4.3). Both of these strains had an SR-LPS

profile. The !wzy !wzz strain with WzyR258A had an SR-LPS but the relative WzySf-GFP level was


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Figure 4.3 Protein expression level of the WzySf-GFP mutants The strains were grown in LB and induced as described in the Materials and Methods. In-gel

fluorescence samples were prepared from the mutants in the "wzy and "wzy "wzz backgrounds,

and electrophoresed on SDS 15% (w/v) PAGE gels (See Materials and Methods). (A) Strains in

lane 1 and 2 are as follows: 1. PNRM13 [PNRM6 (pRMPN1)]; 2. PNRM134 [PNRM126

(pRMPN1)]. Lanes 3-12 are the "wzy or "wzy "wzz strains expressing mutated WzySf-GFP


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proteins. The WzySf mutants in each lane are as follows: 3. R164A ("wzy), 4. R164A ("wzy

"wzz), 5. R250 ("wzy), 6. R250 ("wzy "wzz), 7. R258A ("wzy), 8. R258A ("wzy "wzz), 9.

R278A ("wzy), 10. R278A ("wzy "wzz), 11. R289A ("wzy), 12. R289A ("wzy "wzz). (B) Strains

in lane 1 and 2 are as follows: 1. PNRM13; 2. PNRM134. Lanes 3-10 are the "wzy or "wzy "wzz

strains expressing mutated WzySf-GFP proteins. The WzySf mutants in each lane are as follows:

3. R164K ("wzy), 4. R164K ("wzy "wzz), 5. R164E ("wzy), 6. R164E ("wzy "wzz), 7. R250K

("wzy), 8. R250K ("wzy "wzz), 9. R250E ("wzy), 10. R250E ("wzy "wzz). (C) Strains in lane 1

and 2 are as follows: 1. PNRM13; 2. PNRM134. Lanes 3-14 are the "wzy or "wzy "wzz strains

expressing mutated WzySf-GFP proteins. The WzySf mutants in each lane are as follows: 3.

R258K ("wzy), 4. R258K ("wzy "wzz), 5. R258E ("wzy), 6. R258E ("wzy "wzz), 7. R278K

("wzy), 8. R278K ("wzy "wzz), 9. R278E ("wzy), 10. R278E ("wzy "wzz), 11. R289K ("wzy),

12. R289K ("wzy "wzz), 13. R298E ("wzy), 14. R289E ("wzy "wzz). In each panel, the relative

WzySf-GFP level of all the mutants were measured by considering the WzySf-GFP in PNRM13 in

lane 1 as 100%. Letters (A1-3, B-F) at the bottom indicate the mutant class (Table 4.2).


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200% (Fig. 4.3A, lane 8; and Table 4.3), which is double than the WzySf-GFP level in PNRM13.

The !wzy strain with WzyR258A had a relative WzySf-GFP level of 62% (Fig. 4.3A, lane 7; and

Table 4.3). Both the !wzy and !wzy !wzz strains with WzyR164K expressed at a level more than

100% (120% and 125%, respectively) (Fig. 4.3B, lanes 3 and 4; and Table 4.3). In the !wzy !wzz

background both WzyR250K and WzyR258K were expressed at a very low level (11% and

0.02%, respectively) (Fig. 4.3B, lane 8; Fig. 4.3C lane 4; and Table 4.3). However, in the !wzy

strain, WzyR250K and WzyR258K were expressed at high levels (142% and 104%, respectively)

(Fig. 4.3B, lane 7; Fig. 4.3C, lane 3; and Table 4.3). Interestingly, the !wzy and !wzy !wzz

strains with WzyR250E had class B LPS profile but their relative WzySf-GFP levels were 133%

and 143%, respectively (Fig. 4.3B, lanes 9 and 10; and Table 4.3). The !wzy strain with

WzyR278E and the !wzy !wzz strain with WzyR289E had very high relative WzySf-GFP levels

(135% and 140%, respectively) (Fig. 4.3C, lanes 9 and 14; and Table 4.3). However, the !wzy

!wzz strain with WzyR278E and the !wzy strain with WzyR289E had low relative WzySf-GFP

levels (28% and 21% respectively) (Fig. 4.3C, lanes 10 and 13; and Table 4.3). Comparison of %

WzySf-GFP expression levels of different mutants in the !wzy and !wzy !wzz backgrounds

indicates that the expression of certain WzySf mutant proteins was affected by WzzSf.

4.5 Discussion

Wzy proteins are essential for the synthesis of many Oags that are virulence determinants of the

Gram-negative bacteria. WzySf has two large PLs (PL3 and PL5) (Daniels et al., 1998). During

mutational characterisation of WzySf we found that the amino acid P165 in PL3 is important for

the stabilisation of WzySf through interaction with WzzSf (Nath et al., 2015). However, through

random mutagenesis we were unable to identify any other amino acid residues in PL3 and PL5

important for the Oag polymerisation activity and association with WzzSf. In this study site-

directed mutagenesis of these two loops generated mutants that were then characterised based on


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their LPS profiles, ColE2 and bacteriophage Sf6c sensitivities, and WzySf-GFP expression to

reveal novel mutant phenotypes.

Islam et al. (2011) showed that the mutational alteration of Arg to Ala in the WzyPa PL3

and PL5 within the two RX10G motifs resulted in either complete or partial loss of Oag

polymerisation activity and alterations of some of the Arg residues to Lys resulted in LPS

profiles similar to their Ala substitution. In S. flexneri, site-directed mutation of R164, R250,

R258, and R289 to Ala also resulted in the complete or partial loss of Oag polymerisation activity

in the "wzy background. The Arg residues in PL3 and PL5 were changed to Ala, Lys, and Glu to

determine the importance of the guanidium functional group of Arg at these positions. In the

!wzy background, Ala, Lys, and Glu substitutions of R164, R250, and R258 resulted in complete

or partial loss of polymerisation (Fig. 4.2). Lys substitutions at these three positions resulted in S-

LPS with reduced degree of Oag polymerisation (class F or class A) but with different Oag modal

chain lengths [Class F (without modal chain), class A1 (8-11 RUs), class A2 (9-14 RUs)] (Fig.

4.2) compared to the relevant positive control PNRM13. These WzySf mutants (WzyR164K,

WzyR250K, and WzyR258K) resulted in LPS with different Oag modal chain lengths. So,

guanidium functional group of Arg residues at these positions had a position-specific effect on

Oag polymerisation and modal chain length control. This effect has not been reported previously

for S. flexneri wzySf mutations.

Previously, we found Wzz-dependent WzySf mutants (Nath et al., 2015). In this study, we

found several new examples of WzzSf-dependent WzySf mutants. The !wzy !wzz strain with

WzyR250K had decreased Oag polymerisation in the absence of WzzSf and the !wzy !wzz strain

with WzyR258E had increased Oag polymerisation in the absence of WzzSf, even though

WzyR258E was inactive in the !wzy background (Fig. 4.2). Hence, residues R250 and R258

have roles in the association of WzySf and WzzSf during the WzySf mediated Oag polymerisation.

These and our previous results (Nath et al., 2015) suggest that the interactions between WzySf

and WzzSf are complex.


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The ColE2 and bacteriophage Sf6c sensitivity assays supported the LPS profiles of the

WzySf mutants. We found that the WzySf mutants with shorter Oag chains in the LPS were more

sensitive to ColE2 (Table 4.3), consistent with our previous results (Nath et al., 2015). Here we

found that in the !wzy background the strains with different Oag modal chain lengths showed

different sensitivities to ColE2, with an increase in resistance correlated with an increase in Oag

RUs in the LPS Oag modal chain. Strains with class A-C LPS profiles in the !wzy background

were resistant to bacteriophage Sf6c (Table 4.3). This result was consistent with our previous

findings (Nath et al., 2015) that bacteriophage Sf6c only infects if the S-LPS has WT or nearly

WT level of Oag polymerisation. In our previous study we found that while the strains with class

D LPS had S-LPS profiles very similar to the relevant positive control strain (PNRM13), they

were more resistant to ColE2 and more sensitive to bacteriophage Sf6c compared to PNRM13

(Nath et al., 2015). Here the !wzy strain with WzyR278A and WzyR278E showed a similar

phenotype (Fig. 4.3, and Table 4.3).

Previously, we found that WzzSf is not only associated with Oag modal chain length

control but also affects the level of WzySf (Nath et al., 2015). In the !wzy and !wzy !wzz

backgrounds most of the mutant WzySf-GFP proteins had expression levels less than the WzySf-

GFP in PNRM13 (Table 4.3). However, in the !wzy background the expression level of

WzyR164A and in the !wzy !wzz background the expression level of WzyR258A were greater

than the WzySf-GFP in PNRM13 (Table 4.3). We speculate that residues R164 and R258 are

important for the stabilisation of WzySf through a potential interaction with WzzSf. The !wzy

strain with WzyR164A and the !wzy !wzz strain with WzyR258A had SR-LPS profiles (Fig.

4.2). So, the absence of Oag polymerisation activity is not due to a lack of protein expression.

The !wzy and !wzy !wzz strains with WzyR164K and WzyR250E had a higher level of

expression compared to WzySf-GFP in PNRM13 but the LPS profiles of these strains indicated

that the mutant proteins had decreased Oag polymerisation activity compared to the relevant

positive controls (Table 4.3 and Fig. 4.3). So, these mutations in some way stabilised the protein,

both in the presence and absence of WzzSf.


146

The !wzy strain with WzyR250K and WzyR258K had high levels of expression but !wzy

!wzz strain with WzyR250K and WzyR258K had very low levels of expression (Fig. 4.3 and

Table 4.3), suggesting that the presence of WzzSf stabilises these WzySf mutants. The !wzy strain

with WzyR250K had LPS with an increased degree of Oag polymerisation compared to !wzy

!wzz strain with WzyR250K, and the !wzy and the !wzy !wzz strains with WzyR258K had

nearly similar LPS profiles (class A2 and class F), but the !wzy strain with WzyR258K had LPS

with an Oag modal chain length of 9-14 RUs (Fig. 4.2). However, all these strains had LPS with

a decreased level of Oag polymerisation compared to the relevant positive controls. These results

again suggest that Oag polymerisation activity of WzySf is not correlated with the expression

level of the protein. The !wzy strain with WzyR278E and the !wzy !wzz strain with WzyR289E

(Fig. 4.3 and Table 4.3) had higher levels of expression compared to the WzySf-GFP in PNRM13.

Hence, residues R278 and R289 are also important for the stabilisation of WzySf through a

potential interaction with WzzSf.

According to the model proposed by Islam et al. (2011), at physiological pH, WzyPa PL3

and PL5 possess a net positive charge and a net negative charge, respectively. PL3, the “capture

arm”, catches incoming negatively charged Oag subunit for subsequent transfer to PL5, which

acts as a “retention arm”. It involves a relatively transient interaction with the Oag and is

responsible for the constant binding and release of growing Oag chain. They proposed that these

characteristics of PLs support their roles in the “catch- and-release” mechanism during Oag

polymerisation by Wzy (Islam et al., 2011). Zhao et al. (2014) found that Escherichia coli O86

Wzy (WzyEc) has a different number of TM and different amino acid sequence compared to

WzyPa but the pI values of PL3 and PL4 (the two largest PLs) of WzyEc are equivalent to PL3 and

PL5 of WzyPa. At physiological pH, PL3 and PL4 of WzyEc possess a net positive charge and a

net negative charge, respectively, which led them to conclude that WzyEc follows a similar

catalytic mechanism to WzyPa (Zhao et al., 2014). For WzySf, we found that the pI values of PL3

and Pl5 were 4.65 and 5.09, respectively, using the ExPASy pI calculator

(http://web.expasy.org/compute_pi/). Hence, at physiological pH both the PL3 and PL5 of WzySf


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possess net negative charge. While the P. aeruginosa PAO1 Oag contains negatively charged

uronic acid (Knirel et al., 2006), S. flexneri Oag is neutral. So, the charge property of the substrate

for WzySf is different from the WzyPa. Polymerisation of the Oag of all the serotypes of S. flexneri

is conducted by a single type of WzySf, which defines the flexibility of substrate recruitment of

WzySf. The RX10G motifs of WzyPa contain several other Arg residues within the motifs (R176,

R180 in PL3 and R291 in PL5) (Islam et al., 2011; Islam et al., 2013) but the WzySf had no Arg

residues within the RX15G motifs of PL3 and PL5 (Fig. 4.1). The RX10G motifs of WzyPa starts in

the PL and ends in the PL (Islam et al., 2011; Islam et al., 2013) but the RX15G motifs of WzySf

start in the PL and end in the TM (Fig. 4.1). Nevertheless, we found that the Arg residues in the

PL3 and PL5 have roles in Oag polymerisation, association with WzzSf, and WzySf expression

level. Hence, a modified version of “catch-and-release” mechanism (Islam et al., 2011) may

exist for S. flexneri Oag synthesis.

In conclusion, we identified key Arg residues in PL3 and PL5 of WzySf that are important

for the polymerisation activity, association with WzzSf during polymerisation, and WzzSf

dependent stabilisation of the protein. The WzySf mutants that confer altered Oag modal chain

length suggest that WzzSf functions to alter the activity of WzySf and this is mimicked by certain

mutational alterations, leading to change in the Oag modal chain length. The current findings

extended the previous finding (Nath et al., 2015), and we conclude that a wider region (PL 2, 3,

5, 6 and TM 5, 8) is involved in the Oag polymerisation activity and potential interaction with

WzzSf. We hypothesize that these regions may contribute to the catalytic site of WzySf.


148


Funding for this work is provided by a Program Grant to R.M. from the National Health and

Medical Research Council (NHMRC) (Grant number: 565526) of Australia. P.N. is the recipient

of an international postgraduate research scholarship (Adelaide Scholarship International) from

the University of Adelaide.


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Table 4.S1 Primers used in this study

Primer *

Sequence (5’-3’) # Mutation Arg to Ala mutation

PN5_R164A_F AGCGAGTTCTTTTTTGCCCCCGATGGGGC R164A PN6_R164A_R GCCCCATCGGGGGCAAAAAAGAACTCGCT R164A PN9_R250A_F ATGCTTTACATGGTCGGATCAGCCAGTGAAGAT

TCTGAC R250A

PN10_R250A_R GTCAGAATCTTCACTGGCTGATCCGACCATGTAAAGCAT

R250A

PN11_R258A_F GTGAAGATTCTGACTCTGTTGCCTTTAATGATTTATATTTTTATTATAAAAATGTTG

R258A

PN12_R258A_R CAACATTTTTATAATAAAAATATAAATCATTAAAGGCAACAGAGTCAGAATCTTCAC

R258A

PN13_R278A_F GCGACGTTCTTGTTTGGAGCCGGATTTGGTTCATTTATATTAG

R278A

PN14_R278A_R CTAATATAAATGAACCAAATCCGGCTCCAAACAAGAACGTCGC

R278A

PN15_R289A_F TCATTTATATTAGATCGATTAGCCATTGAAATAGTACCTCTTGAG

R289A

PN16_R289A_R CTCAAGAGGTACTATTTCAATGGCTAATCGATCTAATATAAATGA

R289A

Arg to Lys mutation PN27_R164K_F GACTAGCGAGTTCTTTTTTAAACCCGATGGGGC R164K PN28_R164K_R GCCCCATCGGGTTTAAAAAAGAACTCGCTAGTC R164K PN29_R250K_F ATGCTTTACATGGTCGGATCAAAAAGTGAAGAT

TCTGAC R250K

PN30_R250K_R GTCAGAATCTTCACTTTTTGATCCGACCATGTAAAGCAT

R250K

PN31_R258K_F CGGATCACGCAGTGAAGATTCTGACTCTGTTAAATTTAATGATT

R258K

PN32_R258K_R AATCATTAAATTTAACAGAGTCAGAATCTTCACTGCGTGATCCG

R258K

PN33_R278K_F GCGACGTTCTTGTTTGGAAAAGGATTTGGTTCATTTATATTAG

R278K


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Table 4.S1 continued Primer *

Sequence (5’-3’) # Mutation Arg to Lys mutation PN34_R278K_R CTAATATAAATGAACCAAATCCTTTTCCAAACA

AGAACGTCGC R278K

PN35_R289K_F GGTTCATTTATATTAGATCGATTAAAAATTGAAATAGTACCTCTTGAG

R289K

PN36_R289K_R CTCAAGAGGTACTATTTCAATTTTTAATCGATCTAATATAAATGAACC

R289K

Arg to Glu mutation PN41_R164E_F GACTAGCGAGTTCTTTTTTGAGCCCGATGGGGC R164E PN42_R164E_R GCCCCATCGGGCTCAAAAAAGAACTCGCTAGTC R164E PN43_R250E_F ATGCTTTACATGGTCGGATCAGAGAGTGAAGAT

TCTGAC R250E

PN44_R250E_R GTCAGAATCTTCACTCTCTGATCCGACCATGTAAAGCAT

R250E

PN45_R258E_F CGGATCACGCAGTGAAGATTCTGACTCTGTTGAGTTTAATGATT

R258E

PN46_R258E_R AATCATTAAACTCAACAGAGTCAGAATCTTCACTGCGTGATCCG

R258E

PN47_R278E_F GCGACGTTCTTGTTTGGAGAGGGATTTGGTTCATTTATATTAG

R278E

PN48_R278E_R CTAATATAAATGAACCAAATCCCTCTCCAAACAAGAACGTCGC

R278E

PN49_R289E_F GGTTCATTTATATTAGATCGATTAGAGATTGAAATAGTACCTCTTGAG

R289E

PN50_R289E_R CTCAAGAGGTACTATTTCAATCTCTAATCGATCTAATATAAATGAACC

R289E

*F, Forward; R, Reverse; # Bold - changed codon.


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Table 4.S2 Periplasmic loop (PL)3 and PL5 of WzySf

PL Sequence of the PL (5’-3’) pI of the PL Sequence of the RX15G motif (5’-3’)

PL3 LNFNLIYEHLSLTSEFFFRPDGA

4.65 RPDGAFFSKSFYFFGVG

PL5 VGSRSEDSDSVRFNDLYFYYKNVDLATFLFGRGFGSFILDRLRIEIVPLEILQKT

5.09 RIEIVPLEILQKTGVIG

152

!!!!!!

153

Chapter 5

Detection of Wzy/Wzz interaction in Shigella flexneri


154


Pratiti Nath and Renato Morona

Department of Molecular and Cellular Biology, School of Biological

Sciences, University of Adelaide, Adelaide 5005, Australia

Microbiology (In Press) Published online 09 July, 2015, doi:10.1099/mic.0.000132


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Title of Paper Detection of Wzy/Wzz interaction in Shigella flexneri

Publication Status Submitted for publication

Publication Details Microbiology (In Press) Published online 09 July, 2015,

doi:10.1099/mic.0.000132


Pratiti Nath

Contribution to the Paper Performed all experiments, performed analysis on all

samples, interpreted data, and wrote manuscript.

Signature

Date 20.5.15

Name of Co-Author Renato Morona

Contribution to the Paper Supervised development of work, helped in data

interpretation, manuscript evaluation and editing.

Signature

Date


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Chapter 5: Third paper

5.1 Abstract

The O antigen (Oag) component of Shigella flexneri lipopolysaccharide (LPS) is important for

virulence and a protective antigen. It is synthesised by the Wzy-dependent mechanism. S. flexneri

Wzy has 12 transmembrane (TM) segments and two large periplasmic loops (PL). The modal

chain length of the Oag is determined by Wzz. Experimental evidence supports multi-protein

interactions in the Wzy-dependent pathway. However, evidence for direct interaction of Wzy

with the other proteins of the Wzy-dependent pathway is limited. Initially, we purified Wzy-

GFP-His8 and detected the presence of a dimeric form. Then in vivo crosslinking was performed

in a S. flexneri wzy mutant strain carrying plasmids encoding Wzy-GFP-His8 and untagged Wzz.

Following solubilisation with n-dodecyl-"-D-maltopyranoside (DDM) and affinity purification of

Wzy-GFP-His8, immunoblotting with Wzz antibody detected co-purification of Wzz; this was

supported by mass spectrometry (MS) analysis. This is the first reported isolation of a complex

between Wzy and Wzz. Wzy mutants (WzyR164A, WzyV92M, WzyY137H, and WzyR250K)

whose properties are affected by Wzz were able to form complexes with Wzz. Their mutational

alterations do not affect the interaction of Wzy with Wzz. Thus the interaction may involve many

regions of Wzy.

5.2 Introduction

The lipopolysaccharide (LPS) of S. flexneri plays an important role in the pathogenesis of the

bacteria (Jennison & Verma, 2004). LPS is composed of three domains - 1) Lipid A - the

hydrophobic anchor of the LPS, 2) Core oligosaccharides - a non-repeating oligosaccharide

domain, and 3) O antigen (Oag) chains - an oligosaccharide repeat domain. Oag tetrasaccharide

repeat units (RUs) are linked to the Lipid A via the Core (Raetz & Whitfield, 2002).

Oag is the most variable domain, serotype determinant, and also the protective antigen of

the bacteria (Jennison & Verma, 2004; Stagg et al., 2009; Sun et al., 2013b). S. flexneri Oag is

synthesised by the Wzy-dependent pathway (Allison & Verma, 2000; Morona et al., 1995). Oag


157

biosynthesis starts by the transfer of N-acetylglucosamine phosphate (GlcNAc-1-P) from an

UDP-GlcNAc to undecaprenol phosphate at the cytoplasmic side of the inner membrane (IM) by

WecA (Guo et al., 2008; Liu et al., 1996; Wang et al., 2010b). Then the rhamnosyl transferases

RfbG and RfbF add sequential rhamnose residues to the GlcNAc to form the RU (Morona et al.,

1994). The flippase protein Wzx translocates the RU to the periplasmic side where the RUs are

polymerised by Wzy to form the Oag. The Oag chain length is determined by Wzz, a

polysaccharide co-polymerase (PCP) type1 protein (Daniels & Morona, 1999; Morona et al.,

1995; Morona et al., 2009). Finally, the ligase WaaL ligates Oag chains to the core-lipid A to

form LPS. The Lpt proteins (Lpt A-G) transport LPS from the IM to the outer membrane (Ruiz et

al., 2008; Sperandeo et al., 2009).

The S. flexneri Oag polymerase protein Wzy (WzySf)2 is a 43.7 kDa hydrophobic integral

membrane protein. It has 12 transmembrane (TM) segments and two large periplasmic (PL)

domains (Daniels et al., 1998; Morona et al., 1994). In S. flexneri 2a Wzy activity is affected by

two types of Wzz - chromosomally encoded WzzSf 3

and pHS-2 plasmid encoded WzzpHS2

(Papadopoulos & Morona, 2010; Purins et al., 2008). This results in LPS with two types of Oag

modal chain lengths: the predominant short (S) type (11 - 17 Oag RUs) determined by Wzz, and

the minor very long (VL) type (>90 Oag RUs) determined by WzzpHS2 (Morona et al., 2003;

Morona & Van Den Bosch, 2003b). The S-type Oag chains are responsible for IcsA mediated

actin-based motility (Van den Bosch & Morona, 2003) and affects virulence (Van den Bosch et

al., 1997), and the VL-type Oag chains are responsible for resistance to complement (Hong &

Payne, 1997). Wzz and WzzpHS2 compete for the available Wzy (Carter et al., 2009).

Recent research supports the presence of multi-protein interactions in the Wzy-dependent

pathway (Marczak et al., 2013; Marczak et al., 2014; Marolda et al., 2006). Woodward et al.

purified Escherichia coli O86 Wzy (WzyEc) and showed using an in vitro assay that Wzz and

Wzy are sufficient to determine the Oag modal chain length (Woodward et al., 2010). Daniels

and Morona (1999) showed that Wzz forms a dimer in vivo and may oligomerise up to a

2 In rest of the paper we refer to WzySf as Wzy. 3 In rest of the paper we refer to WzzSf as Wzz.


158

hexamer. Tocilj et al. (2008) found PCP oligomers in the crystal structures, and suggested that

Wzz may form a scaffold and recruit Wzy. Marolda et al. (2006) provided genetic data

supporting the interaction of Wzx, Wzz, and Wzy in the Oag biosynthesis pathway but Carter et

al. (2009) failed to detect a physical interaction between Wzy and Wzz. Marczak et al. (2013)

showed by using a bacterial two-hybrid system that the Rhizobium leguminosarum PssP, which is

a PCP, interacts with PssL and PssT, which are Wzx and Wzy proteins, respectively. Recently,

they again showed that the PssP2 protein, which is also a PCP, interacts with PssT using a

bacterial two-hybrid system (Marczak et al., 2014). However, to date there is a lack of evidence

on the interaction of Wzy with other proteins of the Wzy-dependent Oag biosynthesis pathway

using more direct approaches.

In this study our aim was to detect the interaction of Wzy and Wzz by use of chemical

cross-linking of intact cells, and purification of tagged Wzy. Our western immunoblotting and

MS data support the first isolation of a Wzy and Wzz complex. Hetero-oligomeric complex

formation between Wzy mutants whose properties are Wzz-dependent and Wzz were then

investigated by cross-linking. The data indicated that these Wzy mutants could be cross-linked to

Wzz, however, there was no consistent correlation with an effect on Wzy mutant properties.


5.3.1 Ethics Statement

The Wzz antibody was produced under the National Health and Medical Research Council

(NHMRC) Australian Code of Practice for the Care and Use of Animals for Scientific Purposes

and was approved by the University of Adelaide Animal Ethics Committee.

5.3.2 Bacterial strains, growth media and growth conditions

The strains used in this study are shown in Table 5.1.


159

The growth media used were LB broth (10 g/liter tryptone, 5 g/liter yeast extract, 5 g/liter

NaCl) and LB agar (LB broth, 15 g/liter bacto agar).

Strains to be induced were initially grown in LB broth with aeration for 18 h at 37°C.

Cultures were then diluted 1/20 into fresh LB broth and grown to mid-exponential phase (OD600

of 0.4 - 0.6). To suppress Wzy-GFP-His8 expression, growth medium was supplemented with

0.2% (w/v) glucose where required. Cells were collected by centrifuged (9600 x g, Beckman

Coulter Avanti J-26XP centrifuge, 8 min, 4°C) and washed twice with LB broth to remove

glucose. To induce protein expression either 0.4 mM IPTG or 0.2% (w/v) L-Arabinose was

added and cultures grown for 20 h at 20°C. Strain PNRM271 did not require induction and was

grown in LB broth with aeration for 18 h at 37°C. Antibiotics were added as required to the

media at the following final concentrations: kanamycin (Km), 50 µg/ml; chloramphenicol (Cm)

25 µg/ml; and ampicillin (Amp), 100 µg/ml.

5.3.3 Plasmids and DNA methods

The plasmids used in this study are shown in Table 5.1. Plasmid constructs were prepared from

E. coli DH5# strains using the QIAprep Spin Miniprep kit (QIAGEN). Preparation of

electrocompetent cells and the electroporation method was described previously (Morona et al.,

2003).


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Strains or Plasmids Characteristics* Reference Strains S. flexneri RMM109 PE638"wzy, Rifr (Morona et al., 1994) RMA4337 RMM109 "wzz (Rifr, Tetr) (Nath et al., 2015) PNRM6 RMM109 (pAC/pBADT7-1) (Nath et al., 2015) PNRM13 PNRM6 (pRMPN1) (Nath et al., 2015) PNRM16 PNRM6 (pRMPN2) (Nath & Morona, 2015) PNRM85 PNRM6 (pRMPN17) (Nath et al., 2015) PNRM122 PNRM6 (pRMPN23) (Nath et al., 2015) PNRM126 RMA4337 (pAC/pBADT7-1) (Nath et al., 2015) PNRM134 PNRM126 (pRMPN1) (Nath et al., 2015) PNRM159 PNRM13 (pWSK29-wzz) This study PNRM192 PNRM6 (pRMPN28) (Nath & Morona, 2015) PNRM271 RMM109 (pWSK29-wzz) This study PNRM289 PNRM16 (pWSK29-wzz) This study PNRM293 PNRM122 (pWSK29-wzz) This study PNRM299 PNRM85 (pWSK29-wzz) This study PNRM301 PNRM192 (pWSK29-wzz) This study E. coli Lemo21(DE3) fhuA2 (lon) ompT gal ($ DE3)

(dcm) !hsdS/ pLemo (Cmr) New England Biolabs

PNRM15 Lemo21 (DE3) (pRMPN1) This study Plasmids pAC/pBADT7-1 Source of T7 RNA polymerase; Cmr (McKinney et al., 2002) pRMPN1 pWaldo-wzy-GFPe; Kmr (Nath et al., 2015) pWSK29-wzz pWSK29 with S. flexneri 2a wzz (Murray et al., 2006) pRMPN2 pRMPN1 with WzyR164A (Nath & Morona, 2015) pRMPN17 pRMPN1 with WzyV92M (Nath et al., 2015) pRMPN23 pRMPN1 with WzyY137H (Nath et al., 2015) pRMPN28 pRMPN1 with WzyR250K (Nath & Morona, 2015)

*Rifr, rifampicin; Kmr, kanamycin resistant; Cmr, chloramphenicol resistant; Tetr, tetracycline-

resistant. $ DE3 is # sBamHIo !EcoRI-B int:(lacI::PlacUV5::T7 gene1)i21!nin5. pLemo is

pACYC184-PrhaBAD-lysY.


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5.3.4 Purification of Wzy from Lemo21(DE3) strain

Wzy-GFP-His8 was purified from PNRM15 [Lemo21(DE3) (pRMPN1)] strain following the

method of Woodward et al. (2010) with some modifications. Cells were harvested from 200 ml

induced culture by centrifugation (9600 x g, Beckman Coulter Avanti J-26XP centrifuge, 8 min,

4°C) and the cell pellet was washed with sonication buffer (20 mM Tris-HCl, 150 mM NaCl, pH

7.5) followed by disruption of the cell by sonication (Branson B15). Cell debris was removed by

centrifugation (2200 X g, SIGMA 3K15 table top centrifuge, 10 min, 4°C), then the whole

membrane (WM) fraction was collected by ultracentrifugation (Beckman Coulter Optima L-100

XP ultracentrifuge, 250,000 X g, 1 h, 4 °C), resuspended in 500 µl Milli Q water, and 500 µl 2X

solubilisation buffer [40 mM Tris-HCl, 300 mM NaCl, 10% (w/v) sodium dodecanoyl sarcosine

(SDDS) (Anatrace), pH 7.5] added, and left 16 h at 4°C. Unsolubilised material was removed by

ultracentrifugation (Beckman Coulter Optima Max-XP tabletop ultracentrifuge, 160,000 X g, 1 h,

4°C) and the solubilised supernatant was incubated with 100 µl IMAC Ni-Charged Resin (Bio-

Rad) pre-equilibrated with equilibration buffer [20 mM Tris-HCl, 150 mM NaCl, 5 mM

imidazole, 0.1% (w/v) n-dodecyl-"-D-maltopyranoside (DDM) (Anatrace), 10% (v/v) glycerol,

pH 7.5] for 1 h at room temperature (RT). The Ni-NTA resin was washed with wash buffers [20

mM Tris-HCl, 150 mM NaCl, 0.1% (w/v) DDM, 10% (v/v) glycerol, pH 7.5] with different

imidazole concentrations (10 mM, 20 mM, and 50 mM). Finally, Wzy-GFP-His8 was eluted in

200 µl elution buffer [20 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, 0.1% (w/v) DDM,

10% (v/v) glycerol, pH 7.5].

5.3.5 In vivo protein cross-linking

In vivo cross-linking with dithiobis(succinimidyl propionate) (DSP) was performed as described

by Daniels and Morona (1999) with some modifications. Cells were harvested from 200 ml

induced cultures by centrifugation as above and the pellets were washed with DSP cross-linking

buffer [150 mM NaCl, 20 mM sodium phosphate buffer (Na2PO4/NaH2PO4), pH 7.2]. The pellets

were then resuspended in 200 ml of DSP cross-linking buffer followed by incubation with 0.2


162

mM DSP (Thermo Fischer Scientific) for 45 min at 37°C [Treated (T) samples]. A duplicate of

each sample was incubated without DSP [Untreated (UT) samples]. Excess DSP was quenched

with 20 mM Tris-HCl, pH 7.5, and the cells were washed again with DSP cross-linking buffer.

Harvested cells were then either stored at -20°C or immediately disrupted by sonication as

described below.

5.3.6 Isolation of Wzy from S. flexneri strains

Following DSP cross-linking, sonication, and isolation of WM as above; the WM fraction was

solubilised in 1 ml 1X solubilisation buffer but with 0.5 M NaCl, 20 mM imidazole, 4% (w/v)

DDM, and 10% (v/v) glycerol. After separating the unsolubilised material as above, the soluble

material was then incubated with 200 µl of Ni-NTA resin (QIAGEN) pre-equilibrated with

equilibration buffer but with 500 mM NaCl and 20 mM imidazole. The Ni-NTA resin was

washed with wash buffers with 500 mM NaCl and different imidazole concentrations (20 mM, 30

mM, 50 mM, and 80 mM). Finally, the proteins were eluted from the resin with 200 µl of elution

buffer but with 500 mM NaCl.

5.3.7 SDS-PAGE and Western Immunoblotting

Purified proteins samples were mixed 1:1 with 2X sample buffer (Lugtenberg et al., 1975)

without "-mercaptoethanol ("-ME) and heated for 5 mins at 37°C prior to electrophoresis on SDS

15% (w/v) polyacrylamide gels (SDS-15% PAGE). Protein gels were either stained with

Coomassie R-250, or subjected to western immunoblotting on nitrocellulose membrane with

either rabbit polyclonal Wzz (Daniels & Morona, 1999) at 1:750 dilution or mouse monoclonal

His6 antibodies (Genscript) at 1:50000 dilution in 2.5% (w/v) skim milk. Goat anti-rabbit

horseradish-peroxidase (HRP)-conjugate or a goat anti-mouse HRP-conjugate (KPL) was used as

secondary antibody. Blots were developed using chemiluminescence reagents (Sigma) as

described by the manufacturer (Tran et al., 2014). BenchMark protein ladder (Invitrogen) was

used as molecular mass standard.


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5.3.8 In-gel fluorescence

In-gel fluorescence was performed as previously described with some modifications (Nath et al.,

2015). Purified proteins samples were mixed 1:1 with Buffer A [200 mM Tris-HCl (pH 8.8), 20%

(v/v) glycerol, 5 mM EDTA (pH 8.0), 0.02% (w/v) bromophenol Blue, 4% (w/v) SDS, and 0.05

M dithiothreitol (DTT)]. Then the samples were heated for 5 mins at 37°C prior to

electrophoresis on SDS-15% PAGE gels and BenchMark Fluorescent Protein Standard

(Invitrogen) was used as a molecular mass standard. Gels were rinsed with distilled water and

fluorescent imaging of the gel was performed to detect Wzy-GFP-His8 protein expression with a

Bio-Rad Gel Doc XR + System using Image Lab software (excitation at 485 nm and emission at

512 nm).

5.3.9 Liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-

MS/MS)

LC-ESI-MS/MS was performed at the Adelaide Proteomics Centre. Protein samples were

electrophoresed on 4-12% (w/v) SDS PAGE gels (Invitrogen no. NPO322BOX) followed by

staining with Brilliant Blue G (Sigma). Novex sharp unstained protein standard (Thermo Fisher

Scientific) was used as molecular mass standard. Regions and bands of interest were excised and

destained with 100 mM ammonium bicarbonate (NH4HCO3) in 30% (v/v) acetonitrile (ACN).

Samples were then washed with 50 mM NH4HCO3, reduced with 0.5 µmol DTT in 50 mM

NH4HCO3, and alkylated with 2.75 µmol iodoacetamide in 100 mM NH4HCO3 followed by

digestion with 100 ng trypsin (Promega) in 5 mM NH4HCO3 in 10% (v/v) ACN. Resulting

peptides were extracted using three washes of 1% (v/v) formic acid (FA) in water, 1% (v/v) FA

in 50% (v/v) ACN, and 100% (v/v) ACN respectively. The volumes of the resulting peptide

extracts were reduced by vacuum centrifugation to approximately 1 µl and resuspended with

0.1% (v/v) formic acid in 2% (v/v) ACN to a total volume of 10 µl prior to LC-ESI-MS/MS

analysis. LC-ESI-MS/MS was performed using an Ultimate 3000 nano-flow system (Dionex)

coupled to an Impact II QTOFmass spectrometer (Bruker Daltonics) via an Advance


164

CaptiveSpray source (Bruker Daltonics). Post acquisition, acquired spectra were subjected to

peak detection and de-convolution using Compass Data Analysis for OTOF (Version 1.7, Bruker

Daltonics). Processed MS/MS spectra were then exported to Mascot generic format and

submitted to Mascot (Version 2.3.02) for identification.

5.4 Results

5.4.1 Purification of Wzy-GFP-His8 from Lemo21(DE3)

Several studies provided evidence for complex formation between proteins of the Wzy-dependent

Oag biosynthesis pathway and previously we identified an association of Wzz and Wzy by

finding Wzy mutants whose properties were Wzz dependent (Nath et al., 2015). Prior to

performing experiments to investigate the interaction of Wzy and Wzz in S. flexneri, we initially

optimised the purification of Wzy-GFP-His8 from Lemo21(DE3) strain carrying pRMPN1

(denoted as PNRM15) as described in the Materials and Methods. The purified protein from

PNRM15 was separated on an SDS-15% PAGE gels followed by Coomassie Blue staining and

in-gel fluorescence. Both the Coomassie Blue stained gel and in-gel fluorescence image showed a

band at ~64 kDa corresponding to the monomer of Wzy-GFP-His8 (Fig. 5.1A and B, lane 1). The

Coomassie Blue stained gel and in-gel fluorescence image also showed a band with an apparent

molecular weight of ~115 kDa (Fig. 5.1A and B, lane 1), which is potentially a Wzy-GFP-His8

dimer. The concentration of purified Wzy-GFP-His8 was 1 mg/ml and the purified protein was

approximately 90% pure.


165

Figure 5.1 Purification of Wzy-GFP-His8 Wzy-GFP-His8 was purified from PNRM15 [Lemo21(DE3) (pRMPN1)] as described in the

Materials and Methods. Purified protein and solubilised whole membrane fraction (WM) from

PNRM15 were electrophoresed on SDS 15% polyacrylamide gels followed by in-gel

fluorescence (A) and Coomassie Blue staining (B) (See Materials and Methods). The migration

positions of the molecular mass standards (in kDa) are indicated on the LHS.


166

5.4.2 Detection of complex formation between Wzy and Wzz by immunoblotting

After establishing a method to purify a high level of Wzy-GFP-His8, we modified this method for

S. flexneri by using different detergent, salt, and imidazole concentrations, and a different Ni-

NTA resin, to investigate the potential interaction in S. flexneri of Wzy and Wzz by in vivo

chemical cross-linking with DSP, which reacts with the lysine residues and is a fixed arm length

(12 Å), mercaptoethanol cleavable cross-linking reagent (See Materials and Methods).

The S. flexneri strains used for this cross-linking study were: 1) PNRM159 which is a wzy

mutant ("wzy) strain with plasmids pRMPN1 (encodes Wzy-GFP-His8) (Nath et al., 2015) and

pWSK29-wzz (encodes untagged Wzz) (Murray et al., 2006), 2) PNRM134 which is a wzy and

wzz double mutant ("wzy "wzz) strain with plasmid pRMPN1, and 3) PNRM271 which is a wzy

mutant strain with plasmid pWSK29-wzz. After in vivo cross-linking and affinity purification,

samples were analysed by SDS-PAGE, western immunoblotting, and in-gel fluorescence (See

Materials and Methods). The protein sample from cross-linked PNRM159 ["wzy + Wzz + Wzy-

GFP-His8] cells when subjected to western immunoblotting with anti-Wzz showed bands ranging

from ~82 kDa to above ~180 kDa (Fig. 5.2A, lane 2). However, there was no detectable band in

the equivalent regions for the protein sample from the non-cross-linked PNRM159 cells (Fig.

5.2A, lane 1). There were no detectable bands for the protein samples from non-cross-linked and

cross-linked PNRM134 ["wzy "wzz + Wzy-GFP-His8] cells (Fig. 5.2A, lanes 3 and 4).

Furthermore, the protein samples from non-cross-linked and cross-linked PNRM271 ["wzy +

Wzz] cells also had no detectable bands in the region ~82 kDa to ~180 kDa (Fig. 5.2A, lanes 5

and 6). The presence of bands ranging from ~82 kDa to above ~180 kDa in protein sample from

the cross-linked PNRM159 cells (Fig. 5.2A, lane 2) suggested that a Wzy and Wzz complex

could only be detected when DSP cross-linking was performed, and when both Wzy and Wzz

were present.

Western immunoblotting of protein samples from the cross-linked PNRM159 and

PNRM134 cells with anti-His to detect Wzy-GFP-His8 detected bands ranging from ~64 kDa to

above ~180 KDa (Fig. 5.2B, lanes 2 and 4). The bands for the protein sample from the cross-


167

Figure 5.2 In vivo cross-linking with DSP In vivo cross-linking was performed using DSP as described in the Materials and Methods.

Proteins were purified from DSP treated (T) and untreated (UT) PNRM159 ["wzy + Wzz + Wzy-

GFP-His8], PNRM134 ["wzy "wzz + Wzy-GFP-His8], and PNRM271 ["wzy + Wzz] cells.

Samples were electrophoresed on SDS 15% (w/v) polyacrylamide gels followed by western

immunoblotting with Wzz antibody (A) and His tag antibody (B), and in-gel fluorescence (C)

(See Materials and Methods). Purified Wzy-GFP-His8 from PNRM15 [Lemo21(DE3)

(pRMPN1)] was used as a positive control (lane 7) for the anti-His Western blot. The migration


168

positions of the molecular mass standards (in kDa) are indicated on the LHS. The symbols in the

figure are as follows:

Cross-linked Wzy-GFP-His8 + Wzz

Cross-linked Wzy-GFP-His8 + Wzz

Wzy-GFP-His8 monomer

Wzy-GFP-His8 dimer


169

linked PNRM134 cells (Fig. 5.2B, lane 4) were absent for the same sample probed with anti-Wzz

(Fig. 5.2A, lane 4). The bands ranging from ~82 kDa to above ~180 kDa for the protein sample

from the cross-linked PNRM159 cells detected with anti-Wzz (Fig 2A, lane 2) were also detected

with anti-His (Fig. 5.2B, lane 2). The band at ~ 64 kDa in Fig. 5.2B, lanes 1 and 3 is the Wzy-

GFP-His8 monomer, as seen for the positive control (purified Wzy-GFP-His8 from PNRM15)

(Fig. 5.2b, lane 7 and Fig. 5.1, lane 1). A band at ~115 kDa in Fig. 5.2B, lanes 1 and 3 is likely to

be a Wzy-GFP-His8 dimer, as seenas seen for the positive control (purified Wzy-GFP-His8 from

PNRM15) (Fig. 5.2B, lane 7 and Fig. 5.1, lane 1). As expected the protein samples from cross-

linked and non-cross-linked PNRM271 cells had no detectable bands when probed with anti-His

(Fig. 5.2B, lanes 5 and 6). In-gel fluorescence band profiles were similar to that detected in the

anti-His western blot (Fig. 5.2C).

5.4.3 MS analysis of protein samples following DSP cross-linking

Our western immunoblotting detected the oligomer formation between Wzz and Wzy, and to

confirm this observation MS analysis was performed on the protein bands. For MS analysis in

vivo cross-linking with DSP of the S. flexneri strains PNRM159 ["wzy + Wzz + Wzy-GFP-His8],

PNRM134 ["wzy "wzz + Wzy-GFP-His8], and PNRM271 ["wzy + Wzz] was performed

followed by purification of Wzy-GFP-His8 by affinity chromatography (a mock purification was

performed for the control strain PNRM271). The protein samples were then separated on SDS 4-

12% gels followed by Coomassie Blue staining. The bands and regions of interest were excised

from the Coomassie Blue stained gels for MS analysis (See Materials and Methods).

A section of the gel equivalent to the region (~82 kDa to above ~180 kDa) we detected

above in protein sample from the cross-linked PNRM159 cells (Fig. 5.2A, lane 2) was excised

from the Coomassie Blue stained gel (region 2 of Fig. 5.3B, lane 2), and after MS analysis the

presence of peptides from Wzy and Wzz were detected (Table 5.2). However, in the equivalent

region of the protein sample from the non-cross-linked PNRM159 cells (region 1 of Fig. 5.3B,

lane 1) had peptides from Wzy but no peptides from Wzz were detected (Table 5.2). The


170


171

Figure 5.3 Analysis of protein bands by MS Strains were grown as described in the Materials and Methods. In vivo cross-linking was

performed using DSP. Samples were prepared and were subjected to SDS-PAGE as described in

the Materials and Methods. Proteins were purified from DSP treated (T) and untreated (UT)

PNRM159 ["wzy + Wzz + Wzy-GFP-His8], PNRM134 ["wzy "wzz + Wzy-GFP-His8], and

PNRM271 ["wzy + Wzz] cells. Protein samples were solubilised and electrophoresed on SDS

15% (w/v) polyacrylamide gels (A) and on SDS 4-12% (w/v) gels (B) followed by Coomassie

Blue staining. Bands and regions of interest were excised from the Coomassie Blue stained SDS

4-12% (w/v) gel (B) for MS analysis. The excised regions were marked by numbers (1-8) in the

boxes on the figure. A section of the bands between ~82 kDa to above ~180 kDa corresponding

to the region showing the oligomer formation in Fig. 5.2A was excised from the Coomassie Blue

stained gel (region 2). Equivalent regions were excised for the other samples from the Coomassie

Blue stained gel (regions 1, 3, 4, 5, and 6). The ~50 kDa band (monomeric Wzy) and ~90 kDa

band (dimeric Wzy) were excised (regions 8 and 7, respectively). The migration positions of the

molecular mass standards (in kDa) are indicated on the LHS.


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Table 5.2 Detected peptides by MS analysis

Excised region ¶

Detected Wzz peptides Detected Wzy peptides Location of Wzy peptides on the topology map* #

1 No Wzz peptide GFGSFILDR PL5 NVDLATFLFGR PL5 LLEPLGIFPLR PL1 FNDLYFYYK PL5 IEIVPLEILQK PL5 LRIEIVPLEILQK PL5

2 QNLLDIEK GFGSFILDR PL5 VSDLQETLIGR NVDLATFLFGR PL5 NQQLPLTVSYVGQTAEGAQMK LLEPLGIFPLR PL1 FSSAFSALAETLDNQEEPEKLTIEPSVK FNDLYFYYK PL5 IEIVPLEILQK PL5 LRIEIVPLEILQK PL5

3 No Wzz peptide GFGSFILDR PL5 NVDLATFLFGR PL5

4 No Wzz peptide NVDLATFLFGR PL5 IEIVPLEILQK PL5 LRIEIVPLEILQK PL5

5 No Wzz peptide No Wzy peptide 6 No Wzz peptide No Wzy peptide 7 No Wzz peptide GFGSFILDR PL5

VFSCEIFIK CL2 NVDLATFLFGR PL5 LLEPLGIFPLR PL1 FNDLYFYYK PL5 IEIVPLEILQK PL5 LRIEIVPLEILQK PL5 SEDSDSVRFNDLYFYYK PL5

8 No Wzz peptide GFGSFILDR PL5 NVDLATFLFGR PL5 LLEPLGIFPLR PL1 FNDLYFYYK PL5 IEIVPLEILQK PL5 LRIEIVPLEILQK PL5 SEDSDSVRFNDLYFYYK PL5

¶ Regions refer to Fig. 5.3; * Wzy topology map is provided as Supplementary Fig. 5.S1;

# PL, periplasmic loop; CL, cytoplasmic loop.


173

equivalent regions of the protein samples from the non-cross-linked and cross-linked PNRM134

cells (region 3 of Fig. 5.3B, lane 3 and region 4 of Fig. 5.3B, lane 4) had peptides from Wzy but

no Wzz peptides were detected (Table 5.2). As expected, the equivalent regions of the protein

samples from the non-cross-linked and cross-linked PNRM271 cells (region 5 of Fig. 5.3B, lane

5 and region 6 of Fig. 5.3B, lane 6) had only a few non-relevant peptides (data not shown).

MS identification was carried out on the band corresponding to the monomeric Wzy (Fig.

5.1, lane 1 and Fig. 5.2B, lanes 1, 3, and 7). The Wzy-GFP-His8 migrated as a triplet at ~50 kDa

in the SDS 4-12% gels and was denoted as region 8 (Fig. 5.3B, lane 1). MS analysis of region 8

detected Wzy as the predominant protein (Table 5.2). The ~115 kDa band (Fig. 5.1, lane 1 and

Fig. 5.2B, lanes 1, 3, and 7) proposed to be a Wzy-GFP-His8 dimer migrated as a doublet at ~90

kDa in the SDS 4-12% gels, and was denoted as region 7 (Fig. 5.3B, lane 1). MS analysis of

region 7 also detected Wzy as the predominant protein (Table 5.2). No Wzz peptides were

detected in these bands. Hence, the MS analysis confirmed the result observed by western

immunoblotting and in-gel fluorescence.

It is important to mention that in the bands excised above from protein samples obtained

from cross-linked and non-cross-linked S. flexneri strains expressing Wzy-GFP-His8 (PNRM159

and PNRM 134), no other protein known to be involved in Wzy-dependent Oag biosynthesis was

detected by MS analysis.

5.4.4 Analysis of the Wzy mutants with Wzz dependent properties

Previously we identified a number of Wzz-dependent Wzy mutants (Nath & Morona, 2015; Nath

et al., 2015). These Wzy mutants had either partial or null polymerisation activity and their

activity or expression was altered by the presence or absence of Wzz. The Wzy mutants that

expressed mutant Wzy-GFP-His8 at a level comparable to the WT Wzy-GFP-His8 in the positive

control strain (PNRM13) were selected for in vivo cross-linking assay. Plasmid pWSK29-wzz

was transformed into PNRM6 ("wzy) with pWaldo-TEV-GFP plasmids encoding WzyR164A-

GFP-His8, WzyV92M-GFP-His8, WzyY137H-GFP-His8, and WzyR250K-GFP-His8 (Table 5.1).


174

Figure 5.4 Chemical cross-linking of WzySf mutants In vivo cross-linking was performed using DSP as described in the Materials and Methods.

Proteins were purified from DSP treated (T) and untreated (UT) PNRM159 ["wzy + Wzz + Wzy-

GFP-His8], and PNRM6 ("wzy) with pWSK29-wzz and expressing mutated Wzy proteins as

indicated. WT and Wzy-GFP-His8 mutant proteins were purified and electrophoresed on SDS

15% (w/v) polyacrylamide gels followed by western immunoblotting with Wzz antibody. The

migration positions of the molecular mass standards (in kDa) are indicated on the LHS.


175

Then in vivo crosslinking was performed followed by purification of mutant Wzy-GFP-His8 (See

Materials and Methods). The proteins were analysed by SDS-PAGE followed by western blotting

with Wzz antibody. The protein samples of the cross-linked Wzy mutants showed bands from

~82 kDa to above ~180 kDa (Fig. 5.4, lanes 3, 5, 7, and 9) similar to the protein sample from

cross-linked PNRM159 ["wzy + Wzz + Wzy-GFP-His8] cells (Fig. 5.4, lane 1). There were no

detectable bands for the protein samples from the non-cross-linked mutant Wzy strains (Fig. 5.4,

lanes 4, 6, 8, and 10). Hence, all Wzy mutants could be cross-linked to Wzz.

5.5 Discussion

The Wzy-dependent pathway is one of the most widely distributed polysaccharide biosynthesis

pathway in the nature. Oag is one of the important virulence determinants for a number of

bacteria including S. flexneri. S. flexneri Oag polymerisation protein Wzy was identified more

than 20 years ago (Morona et al., 1994) but its low expression and detectability (Daniels et al.,

1998) impeded its purification and characterisation. Recently, we were able to identify functional

amino acid residues of Wzy (Nath & Morona, 2015; Nath et al., 2015) but further understanding

of Wzy and its interaction with other Oag biosynthesis proteins require a method to purify and

isolate Wzy. In this work we were consistently able to purify high levels of S. flexneri Wzy

protein. For a long time, multi-protein complex formation among the proteins of the Wzy

dependent Oag biosynthesis pathway has been suggested (Marolda et al., 2006; Whitfield, 2006;

Whitfield, 2010; Woodward et al., 2010).

However, here for the first time we were able to isolate a complex between Wzz and Wzy,

and found that the Wzy mutants with Wzz dependent properties can also form complexes with

Wzz following chemical cross-linking.Wzy is a membrane spanning protein, and in common

with many membrane proteins this made purification of Wzy difficult. Woodward et al. (2010)

for the first time were able to purify a Wzy protein, WzyEc. They detected a band (36 kDa)

corresponding to the WzyEc monomer and they also detected a band at a higher molecular weight

(58 kDa) which they proposed to be the dimer of WzyEc (Woodward et al., 2010). We were able


176

to obtain high yield of ~90% pure monomeric Wzy-GFP-His8 which migrated as band at ~64 kDa

(Fig. 5.1A and B, lane 1). Previously, Daniels et al. (1998) showed by western immunoblotting

using anti-PhoA serum that Wzy::PhoA fusion protein was able to form a dimer. Our purified

protein also showed a band ~115 kDa (Fig. 5.1A and B, lane 1) which we proposed was the

dimer of Wzy. Both the monomeric and proposed dimeric forms of Wzy showed positive signals

in the anti-His western blot (Fig. 5.2b, lane 7). The MS analysis for this proposed dimeric Wzy

confirmed the prevalence of Wzy peptides. So, Wzy may form dimers but the in vivo correlation

of the dimeric form with the polymerisation activity is yet unknown.

In a previous study to detect Wzy and Wzz complex Carter et al. (2009) inserted a 3xFLAG

tag at the 3’ end of the chromosomal wzy and wzz, resulting in cells expressing C-terminal

3xFLAG fusion Wzy and Wzz proteins. Then they performed in vivo cross-linking with DSP and

disuccinimidyl glutarate (DSG) followed by sonication to obtain WM fractions. The WM

fractions were separated on 4-12% Tris-Glycine gels and then probed by western immunoblotting

followed by infrared imaging to detect the protein complex. However, they were unable to detect

interaction between Wzy and Wzz and they suggested that Wzy and Wzz may not physically

interact (Carter et al., 2009). Wzy is expressed at a very low level in S. flexneri. So, the detection

of the interaction of this protein with the other proteins of the Wzy-dependent pathway may be

difficult. In our study we used a high level of Wzy and Wzz. The MS analysis of the ~82 kDa to

~180 kDa bands detected peptides for Wzy and Wzz (region 2, Fig. 5.3B, lane 2) (Table 5.2), and

hence supported the detection of Wzy and Wzz complex formation by western immunoblotting.

Our MS analysis of the ~82 kDa to ~180 kDa region of the gel (region 2, Fig. 5.3B, lane 2),

detected peptides of Wzz and Wzy but we were unable to detect peptides from the other proteins

of the Wzy-dependent Oag biosynthesis pathway. Furthermore, except for one peptide, the Wzy

peptides we detected were from the periplasmic regions of Wzy (Table 5.2) (Fig. 5.S1, in the

Supplemental Material). So, the standard trypsin digestion and associated methods used for MS

analysis (See Materials and Methods) may be limited in their applicability to detect the

membrane spanning proteins of the Wzy-dependent Oag biosynthesis pathway.

Previously we found that Wzy needs Wzz both for its activity and expression level /stability


177

and we identified a number of Wzy mutants where either the polymerisation activity or the

expression of these mutants was influenced by Wzz. These Wzy mutants had partial or null

polymerisation activity (Nath & Morona, 2015; Nath et al., 2015). Many of these Wzz-dependent

Wzy mutants had a very low-level of expression. Hence, purification of these mutant proteins

was not possible. We selected some of these Wzy mutants with a better expression level and

assessed their ability to form complexes with Wzz. Following DSP crosslinking all the mutants

formed complexes with Wzz similar to the control (Fig. 5.4). Hence, for these Wzy mutants no

correlation between complex formation and the Wzz dependence could be detected. Wzy

interaction may involve many regions of Wzy and single point mutation may not be enough to

interrupt the interactions.

This study for the first time provides direct evidence of complex formation between Wzy

and Wzz, proteins of the Wzy-dependent Oag biosynthesis pathway. The molecular details

regarding the interaction of these proteins still need to be revealed including, the nature of the

interaction sites, and how this contributes to Oag biosynthesis. This study provides the

foundation to investigate this interaction.


Funding for this work is provided by a program grant to R.M. from the National Health and

Medical Research Council (NHMRC) (Grant number: 565526) of Australia. P.N. is the recipient

of an international postgraduate research scholarship (Adelaide scholarship International) from

the University of Adelaide. The University of Adelaide Research Centre for Infectious Disease

provided travel funding support for P.N.


178


Figure 5.S1 Topology map of WzySf S. flexneri Wzy has 12 TMs and two large PLs. The positions of PL1 to 5, TM 1 to 12, and

cytoplasmic loops (CL 1 to 5) are indicated on the Wzy topology map [adapted from Daniels et

al. (1998)].


179

5.7.1 Optimisation of WzySf-GFP-His8 purification 4

Purification of WzySf-GFP-His8 was performed after confirming that the construct pRMPN1 was

able to express WzySf-GFP-His8 and was able to complement the wzySf deficiency in a wzySf

deficient strain (RMM109).

5.7.1.1 Cell fractionation

Strain PNRM15 was grown and induced with IPTG for 20 h at 20°C. Cells were harvested from

200 ml induced culture by centrifugation (9600 x g, Beckman Coulter Avanti J-26XP centrifuge,

8 min, 4°C). The harvested cell pellets was washed with sonication buffer (20 mM Tris-HCl, 150

mM NaCl, pH 7.5) and was disrupted by sonication (Branson B15) followed by centrifugation

(2200 X g, SIGMA 3K15 table top centrifuge, 10 min, 4°C) to remove cell debris. Then the

whole membrane (WM) fraction was isolated by ultracentrifugation (Beckman Coulter Optima L-

100 XP ultracentrifuge, 250,000 X g, 1 h, 4 °C) and was used for the purification of WzySf-GFP-

His8.

5.7.1.2 Optimisation of whole membrane fraction solubilisation

To optimise the solubilisation of WM, the WM fractions were resuspended in 500 µl Milli Q

water and then solubilised in 500 µl 2X solubilisation buffer (40 mM Tris-HCl, 300 mM NaCl,

pH 7.5) containing different detergents [2% (w/v) DDM, 2% (w/v) Lauryldimethylamine-oxide

(LDAO), 2% (w/v) SDS, 5% (w/v) Zwittergent 3-14, 2% (w/v) Sodium lauroyl sarcosine

(Sarkosyl), and 10% (w/v) SDDS] at different temperatures (RT or 4°C). Unsolubilised material

was separated by ultra centrifugation (Beckman Coulter Optima Max-XP tabletop ultracentrifuge,

4 Section 5.7.1 onwards were not part of the original manuscript and were added as additional information for the

thesis chapter.


180

Figure 5.S2 Optimisation of the whole membrane solubilisation Whole membrane fractions of PNRM15 were solubilised in 2x solubilisation buffer containing

2% (w/v) SDS, 2% (w/v) LDAO, 2% (w/v) sarkosyl, 5% (w/v) Zwittergent 3-14, 2% (w/v) DDM

for 1h at room temperature (RT); and 10% (w/v) SDDS for overnight at 4°C as described in

section 5.7.1.2. Unsolubilised material was separated by ultra centrifugation. Supernatants and

unsolubilised pellets were collected. In-gel fluorescence was performed to assess the amount of

WzySf-GFP-His8 in the supernatants (A) and pellets (B).


181

160,000 X g, 1 h, 4°C). Supernatants and unsolubilised pellets were collected. In-gel fluorescence

was performed to check the amount of WzySf-GFP- His8 in the supernatants and pellets (Fig.

5.S2). In the in-gel fluorescence image the WM solubilised with SDDS showed the highest

WzySf-GFP-His8 expression for the supernatant (Fig. 5.S2A, lane 6) and the lowest WzySf-GFP-

His8 expression for the unsolubilised pellet (Fig. 5.S2B, lane 6). Hence, the detergent SDDS was

selected for WM solubilisation and the WM was resuspended in 500 µl of Mili Q water followed

by solubilisation with 500 µl of 2X solubilisation buffer [40 mM Tris-HCl, 300 mM NaCl, 10%

(w/v) SDDS, pH 7.5] at 4°C for overnight. The solubilised supernatant was used for eltuion of

WzySf-GFP-His8.

5.7.1.3 Metal affinity purification of WzySf-GFP-His8

100 µl IMAC Ni-Charged Resin (Bio-Rad) was equilibrated with equilibration buffer [20 mM

Tris-HCl, 150 mM NaCl, 5 mM imidazole, 0.1% (w/v) DDM, 10% (v/v) glycerol, pH 7.5] and

incubated with solubilised supernatant in a 10 ml Falcon tube for 1 h at RT with gentle shaking.

The mixture was centrifuged (Labofuge 400R Heraeus Instrument, 3000 rpm, 10 min), the

supernatant was discarded, and the Ni-NTA beads were washed with 5 ml wash buffer [20 mM

Tris-HCl, 150 mM NaCl, 0.1% (w/v) DDM, 10% (v/v) glycerol, pH 7.5] with different imidazole

concentrations (10 mM, 20 mM, and 50 mM). The beads were incubated at RT for 5 min with

gentle rotation for each wash. Finally, the His8 tagged WzySf-GFP protein was eluted in 200 µl

elution buffer [20 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, 0.1% (w/v) DDM, 10%

(v/v) glycerol, pH 7.5] at RT for 1 h with gentle rotation.

5.7.2 Negative dominance

To check the importance of the dimerisation for the functioning of WzySf the negative dominance

study was performed. For this study the WzySf mutants (WzyR164A, WzyR250A, WzyR278A, WzyR289A, WzyG130V, WzyL11I, WzyL214I, WzyP352H) (See Chapter 3 and 4) were co-


182


183

Figure 5.S3 Negative dominance Plasmids pRMPN1 with mutations in the wzySf gene were transformed into WT S. flexneri strain

PE638 as described in section 1.7.2. Strains were grown and LPS samples were prepared

followed by electrophoresis on an SDS-15% PAGE gel. The gel was stained with silver nitrate

and developed with formaldehyde. Lane 1, PE638; Lane 2, Strain PNRM12 [PE638 (pWaldo-

TEV-GFP) (pAC/pBADT7-1)]; Lane 3, Strain PNRM14 [PE638 (pRMPN1) (pAC/pBADT7-1)];

Lanes 4 to 11, PE638 strain with pAC/pBADT7-1 and plasmids encoding mutated WzySf

proteins, as indicated.


184

-expressed with WT WzySf. If the dimerisation is important for functioning then the formation of

mixed oligomer of the mutant WzySf and WT WzySf might cause the inactivation of WT WzySf

protein due to the presence of compromised WzySf protein in the oligomer.

Plasmid pRMPN1 having mutations in the wzySf gene expressing mutated WzySf proteins

(WzyR164A, WzyR250A, WzyR278A, WzyR289A, WzyP352H, WzyL214I, WzyL111I, and

WzyG130V) (Table 2.2) was co-transformed with pAC/pBADT7-1 into the WT S. flexneri strain

PE638 (Table 2.1). pAC/pBADT7-1 encodes T7 RNA polymerase, which drives the expression

of wzySf-gfp in pRMPN1. Strains were grown in LB broth with aeration for 18 h at 37°C and

diluted 1/20 into fresh LB broth and grown to mid-exponential phase (OD600, 0.4 to 0.6). To

suppress protein expression growth medium was supplemented with 0.2% (w/v) glucose. Cells

were centrifuged (2,200 X g; Sigma 3K15 tabletop centrifuge; 10 min; 4°C) and washed twice

with LB broth to remove glucose. To induce protein expression 0.2% (w/v) L-Arabinose was

added to the cultures and grown for 20 h at 20°C. Antibiotics Km (50 µg/ml) and Cm (25 µg /ml)

was added to the medium. LPS samples were prepared as described previously (Chapter 2). The

LPS samples were then electrophoresed on an SDS-15% PAGE gel for 16 to 18 h at 12 mA. The

gel was stained with silver nitrate and developed with formaldehyde.

Strain PE638 had S-LPS profile. After transformation of the plasmids expressing mutated

WzySf proteins into PE638, the generated strains also had S-LPS profiles similar to the WT

PE638 (Fig. 5.S3). Interestingly, some of theses mutated WzySf proteins had partial (WzyR289A,

WzyP352H, and WzyL214I) and some had null (WzyR164A, WzyR250A, and WzyG130V)

activity. Hence, the dimer formation of WzySf may be not essential for the functioning of WzySf.

185

Chapter 6

Conclusion

Conclusion

186

Chapter 6: onclusion

6.1 Introduction

According to a review of literature from 1966-1997, annually ~164.7 million shigellosis cases

occurred worldwide, of which 163.2 million cases were in developing countries with 1.1 million

deaths (Kotloff et al., 1999). However, a current review of literature from 1990-2009 suggested

that ~125 million shigellosis cases occur annually in Asia with 14,000 fatalities (Bardhan et al.,

2010). The later author suggested that the number of deaths by shigellosis was reduced by 98%

possibly due to improved nutrition, vitamin A supplementation, less virulent strains, and

availability of antimicrobial drugs (Bardhan et al., 2010; Kotloff et al., 1999). Due to

antimicrobial resistance, load of disease, and clinical severity, Shigella is a significant target of

vaccine development (Livio et al., 2014), however there is no available vaccine for shigellosis

(Stagg et al., 2009).

The immunity against S. flexneri is serotype specific and different serotypes of S. flexneri are

generated due to different chemical structure of the LPS Oag RUs (Jennison & Verma, 2004;

Stagg et al., 2009) (detailed in Chapter 1). However, the Oags of all the S. flexneri serotypes are

polymerised by a single type of Oag polymerase WzySf (Daniels et al., 1998; Morona et al.,

1994). Hence, the characterisation and purification of WzySf is potentially useful for in vitro

synthesise of Oag, which will be a great resource for S. flexneri vaccine development in future.

Several proteins are involved in the Wzy-dependent Oag biosynthesis pathway. For a long

time, researchers speculated that these Oag biosynthesis proteins are associated with each other

and may form a multi-protein complex (Marolda et al., 2006; Whitfield, 2006; Whitfield, 2010;

Woodward et al., 2010) (detailed in Chapter 1 and 5). Hence, identification of these associations

will help to understand the Wzy-dependent Oag biosynthesis pathway.

Below is an overview of the outcomes of this thesis. The results characterised WzySf,

identified the association of WzySf and WzzSf during Oag biosynthesis pathway; and established a

foundation to understand the Wzy-dependent Oag biosynthesis pathway.

Conclusion

187

6.2 Residues important for polymerisation function of WzySf

Only a few studies have been conducted to characterise the Wzy proteins of different bacterial

species. Kim et al. (2010) characterised WzyFt and predicted it has 11 TM segments. Islam et al.

(2010) created a topology map of WzyPa, which had 14 TM segments. Previous experimental data

on WzySf topological mapping identified 12 TM segments (Daniels et al., 1998) which is

different to that found in WzyFt and WzyPa. There is also little sequence identity between wzy

genes and Wzy proteins of different bacterial species (Morona et al., 1994). Hence, random

mutagenesis (Chapter 3) was performed on the wzySf and identified a number of residues (V92,

G130, L214, and P352) important for the polymerisation function of WzySf. These residues are

present in the PL2, TM5, TM8, and PL6. Previous characterisation of the Wzy from the other

bacteria identified important functional residues only in the PLs (Islam et al., 2013; Kim et al.,

2010). However, this study for the first time was able to identify functional residues in the TMs

of WzySf. In Chapter 3, the ColE2 sensitivity assay was used for screening of mutants, and also to

verify the LPS profiles of the mutant strains. This method was found to be an effective method to

identify subtle differences in the LPS profiles of the mutant strains. The bacteriophage Sf6c

sensitivity assay was also used for the first time as an effective method to characterise the WzySf

mutants based on their LPS profiles. Significantly, this approach found mutations in WzySf that

resulted in different LPS phenotypes (SR LPS, LPS with few Oag RUs, and reduced

polymerisation) and also generated strains (Class D) which had LPS profiles nearly similar to the

positive control strain but their Oag density was more than that of the positive control strain.

Other than this study on WzySf, the most characterised Wzy is WzyPa. In WzyPa the Arg

residues (R175, R176, R180, R290, and R291) in the PL3 and PL5 are important for the function

(Islam et al., 2011). Site-directed mutagenesis of the Arg residues in the PL3 and PL5 of WzySf

also identified several functionally important Arg residues (R164, R250, R258, and R289). The

PL3 and PL5 of WzyPa has RX10G motifs which are important for the “catch-and-release”

mechanism (Islam et al., 2011). Zhao et al. (2014) found that WzyEc has a different number of

TM and different amino acid sequence compared to WzyPa but the pI values of PL3 and PL4 (the

two largest PLs) of WzyEc are equivalent to PL3 and PL5 of WzyPa, which led them to conclude

Conclusion

188

that WzyEc follows a similar catalytic mechanism to WzyPa. Marczak et al. (2013) found that

PssT has RX10G motifs in the two PLs similar to WzyPa. They proposed that these RX10G motifs

might be associated with the polymerisation activity (Marczak et al., 2013).

Comparison of WzySf with WzyPa shows a number of striking differences: comparatively

different pI values for PL3, different number of TMs, and absence of RX10G motifs and instead

the presence of RX15G motifs in the PL3 and PL5; different arrangement of the Arg residues in

the motifs; and different substrate specificity of WzySf compared to WzyPa (Table 6.1). The

unique characteristics of WzySf also suggest that it follows a different or a modified mechanism

for its polymerisation function compared to WzyPa and other Wzy proteins (including WzyEc and

PssT).

6.3 Purification of WzySf

Due to the transmembrane nature, the purification of Wzy was always challenging (Woodward et

al., 2010). WzySf is a hydrophobic protein with a low percentage of G + C content in the coding

region (Daniels et al., 1998; Morona et al., 1994). The first evidence of the purification of Wzy

protein was the work of Woodward et al. (2010) (detailed in Chapter 1 and 5). WzySf was

purified using the Woodward et al. protocol with some modifications (Chapter 5), that include

using a different cloning vector, overexpression system [Lemo21(DE3)], Ni-beads, centrifugation

speed, and avoiding the concentration and desalting steps, FPLC, and gradient elution. The

method purified 1 mg/ml of ~90% pure monomeric WzySf (Fig. 5.1). This is the first evidence of

purification of S. flexneri WzySf. Woodward et al. (2010) used further purification steps and

concentrated their purified protein but the yield of WzyEc was almost undetectable in a

Coomassie Blue stained gel [see Supplementary Fig. 2.b of Woodward et al. (2010)]. Without

Conclusion

189

Table 6.1 Comparison of WzySf and WzyPa

Characteristics WzySf WzyPa

Number of TM 12 14 pI value of PL3 4.65 8.59

Charge property of PL3 at pH 7.4 Negatively charged Positively charged Motifs in PL3 and PL5 RX15G RX10G

Arrangement of the Arg residues in the motifs

No Arg residue within the RX15G motif

Arg residue within the RX10G motif

Charge property of substrate Neutral Negative

Conclusion

190

further purification [see Supplementary Fig. 2.a of Woodward et al. (2010)] their purified protein

was highly contaminated and the yield was also less compared to purified WzySf although

minimum steps were used during WzySf purification. Woodward et al. (2010) suggested that

WzyEc is able to form a dimer; and previously Daniels et al. (1998) showed by Western

immunoblotting using anti-PhoA serum that Wzy::PhoA fusion protein was able to form a dimer.

The data in Chapter 5 shows that WzySf is able to form a dimer, which also supported by

proteomics analysis. However, the experiments to determine negative dominance (Chapter 5)

suggest that the dimer formation has no correlation with the functioning of the protein. Further

investigation is needed to identify the connection of dimer formation with the functioning of

WzySf.

6.4 Understanding the association of the Oag biosynthesis proteins

The data in Chapters 3 and 4 for the first time provide the insight into the association of WzzSf

and WzySf in the Oag biosynthesis pathway. Seven WzySf amino acid residues (V92, Y137,

G130, L214, R250, R258, and P352) were identified that are important for the polymerisation

function of WzySf through the interaction with WzzSf (Table 6.2). For four of the WzySf mutants

(WzyV92M, WzyG130V, WzyY137H, and WzyR258E) the presence of WzzSf repressed WzySf

polymerisation activity and for three of the WzySf mutants (WzyL214I, WzyR250K, and

WzyP352H) the presence of WzzSf increased WzySf polymerisation activity. WzzSf also has role

in the stability of the WzySf protein. Eight amino acid residues (G130, R164, P165, L191, R250,

R258, R278, and R289) were identified which are important for the WzzSf dependent stabilisation

of WzySf (Table 6.2). For five WzySf mutants (WzyR164A, WzyL191F, WzyR250K,

WzyR258K, and WzyR278E) the presence of WzzSf stabilises WzySf but for four WzySf mutants

(WzyG130V, WzyP165S, WzyR258A, and WzyR289E) the absence of WzzSf stabilises the

protein. Residue R258 is important for stabilisation of WzySf but exhibited opposite effects

depending on the nature of the amino acid substitutions. The amino acid residues important for

Conclusion

191

Table 6.2 LPS profiles of the Wzz-dependent WzySf mutants in the presence and absence of

WzzSf

Mutation LPS profile WzzSf present WzzSf absent

R164A C (SR-LPS) C R164K F [S-LPS with reduced Oag polymerisation

and lacking Oag modal chain length control (<30 Oag RUs)]

F

R164E C C R250A B [LPS with few Oag RUs (<11)] B R250K A1 [S-LPS with reduced Oag polymerisation,

and modal chain length reduced to 8-11 RUs] B

R250E B B R258A C C R258K A2 [S-LPS with reduced polymerisation and

modal chain length reduced to 9-14 or 8-14 Oag RUs]

F

R258E C E [S-LPS lacking Oag modal chain length control] R278A D [LPS profile similar to the WT control

PNRM13] E

R278K D E R278E D E R289A A3 [S-LPS with reduced polymerisation (<22

Oag RUs) and modal chain length similar to the WT control (PNRM13)]

F

R289K D E R289E A2 F P352H A [S-LPS with reduced polymerisation (<20

Oag RUs)] B

V92M A E Y137H A E L214I B C G130V C F N147K D E P165S D E L191F D E

Conclusion

192

the association with WzzSf are not only present in the PL (PL2, 3, 5, and 6) but also present in the

TM (TM 5, 7, and 8). Noticeably, the amino acid G130 present in the TM5 has roles both in the

polymerisation activity of WzySf and association with WzzSf. WzzSf has both negative and

positive effects on the polymerisation ability and stability of WzySf.

Although several authors had suggested the complex formation by the proteins of the

Wzy-dependent Oag biosynthesis pathway, only Marolda et al. (2006) provided genetic evidence

about the association of Wzx, Wzy, and Wzz; and Marczak et al. showed by using a bacterial

two-hybrid system, that the R. leguminosarum PssP interacts with PssL and PssT (Marczak et al.,

2013; Marczak et al., 2014). However, there is no evidence about the direct physical interactions

of these proteins. In vivo chemical cross-linking followed by purification of WzySf identified

WzySf and WzzSf complex formation. Proteomics analysis also supported this data (Chapter 5).

The detected WzySf and WzzSf complex formation provides the first evidence of a close physical

interaction of the proteins of the Wzy-dependent Oag biosynthesis pathway. Although the

mutational data indicate that PL2, 3, 5, 6, and TM 5, 7, and 8 are involved in the association of

WzySf and WzzSf, the complex formation ability of the Wzz-dependent WzySf mutants with WzzSf

suggests that more extensive interactions between WzySf and WzzSf also occur.

6.5 Mechanism of the association of Wzz and Wzy during the O antigen

polymerisation in S. flexneri

There are several proposed models on the interaction of Wzy and Wzz, and on the Wzy-

dependent Oag polymerisation (described in Chapter 1). Some of them considered the direct

interaction of Wzz and Wzy. However, some models described the association of these proteins

through the interaction with the Oag polymer. Considering the merits and limitations of all the

proposed models and the result of this thesis I propose an “activation and inactivation”

mechanism (Fig. 6.1) to explain the polymerisation of Oag by WzySf and the association of WzzSf

and WzySf during the Oag biosynthesis process.

Conclusion

193

It is known that certain Wzy proteins such as Yersinia pseudotuberculosis (WzyYp), have

an absolute requirement for Wzz for polymerisation activity (Kenyon & Reeves, 2013). However,

WzySf has activity without WzzSf but the Oag lacks modal chain length control. In this model,

WzySf has two forms: activated (or stabilised) and inactivated (or destabilised). These two forms

switch spontaneously. WzzSf acts as a molecular chaperone protein and promotes a certain

frequency of switching likely controlling the polymerisation activity of WzySf and the modal

chain length of the growing Oag chain. For WzyYp and the other Wzy proteins that need Wzz

strictly for polymerisation activity, there is no spontaneous switching and Wzz is essential to

control the switching between the activated and inactivated forms of Wzy. Previously, Morona et

al. (1995) also proposed that Wzz acts as a molecular chaperone to facilitate the interaction of

WaaL, Wzy, and lipid-linked Oag chain (Morona et al., 1995).

Data generated from the mutational study suggests that in S. flexneri WzzSf binds at

different regions of WzySf during the Oag polymerisation process. Some of the regions can be

speculated through this mutational study but a number of other regions may be involved. The

presence of WzzSf stabilises WzyR164A, WzyL191F, WzyR250K, WzyR258, and WzyR278E;

and increased polymerisation activity of WzyL214I, WzyR250K, and WzyP352H. However,

presence of WzzSf destabilises WzyG130V, WzyP165S, WzyR258A, and WzyR289E and

repressed polymerisation activity of WzyV92M, WzyG130V, WzyY137H, and WzyR258E.

Hence, initially, WzzSf binds at a region (including R164, L191, R250, R258, and R278) of WzySf

which facilitates the switching towards the “activated” (or stabilised) form of WzySf, and the

activation of WzySf turns on the rapid polymerisation and WzySf starts to synthesise the nascent

Oag chain (the spontaneous polymerisation of WzySf is a slow process). Then WzzSf binds to

other regions (including L214 and P352) of WzySf as well. Mechanical feedback of this binding

facilitates the Oag synthesis and WzzSf controls the Oag modal chain length by interacting with

it. The WzzSf binding regions (denoted as “activation region”) that have roles in activating WzySf

and polymerisation are present in close proximity within the WzySf quaternary structure.

Conclusion

194

Figure 6.1 “Activation and inactivation” mechanism The activated or stabilised, and inactivated or destabilised forms of WzySf interconvert/switch

spontaneously. However, WzzSf acts as a molecular chaperone protein and promotes a certain

frequency of switching which controls the polymerisation activity of WzySf at certain level and

also determines the modal chain length of the growing Oag. Activated WzySf synthesises Oag,

and finally binding of WzzSf at certain regions of WzySf generates mechanical feedback which

releases the synthesised Oag from the substrate binding site of WzySf. Residues in PL2, 3, 5, 6

and TM5, 7, 8 of WzySf are involved in the switching between the two forms by possible

interactions with WzzSf.

Conclusion

195

Next, WzzSf moves to bind to the other regions (including G130, P165, and R289) of WzySf

which facilitates the switching towards the “inactivated” (or destabilised) form of WzySf, and the

inactivated WzySf turns off polymerisation. Then WzzSf binds to the other regions of WzySf

(including V92 and Y137) as well. Mechanical feedback of this binding releases the Oag chain

from the substrate binding domain of WzySf. WzzSf binding regions (denoted as “inactivation

region”) that have roles in inactivating WzySf and releasing the growing Oag chain may be

present in close proximity within the WzySf quaternary structure.

The mutational study showed that amino acid R258 of WzySf has a role in the WzzSf-

dependent stabilisation and destabilisation of WzySf, and R258 also has role in the WzzSf

dependent repression of polymerisation activity of WzySf depending on the mutational

substitutions (R258A, R258K, and R258E). Residue R258 is critical for interaction with WzzSf

and exhibits allele specific effects on Wzz-dependence, polymerisation activity, and protein

stability. The activation and inactivation regions are present on the PL2, 3, 5, 6, and TM5, 7, and

8. The residue R258 may be present in the middle of the activation and inactivation regions

within the WzySf quaternary structure.

6.6 Conclusion and future work

Collectively, this thesis identified and characterised functionally important amino acid

residues of WzySf, identified several novel LPS phenotypes conferred by the WzySf mutants, and

found that WzzSf affects the functioning and stability of WzySf, both positively and negatively.

The work also first time identified direct physical interaction of WzzSf and WzySf, and developed

a purification method for WzySf. We speculate that the PL2, 3, 5, 6 and TM5, 7, 8 may form the

catalytic site of the protein. However, the interaction of WzzSf and WzySf may be conducted on a

wider region of WzySf.

WzySf mutants were generated by mutagenesis on wzySf in pRMPN1. Hence, the effect of

mutations was measured for the overexpressed protein. Repetition of the mutagenesis on

Conclusion

196

chromosomal wzySf can be performed to understand if there is any effect of the overexpression on

the functioning of WzySf mutants.

During characterisation of WzySf and identification of the association of WzzSf and WzySf

(Chapter 3 and 4) all the mutational studies were conducted in S. flexneri Y serotype. However,

for all the S. flexneri serotypes the Oag polymerisation is conducted by a single type of WzySf.

Although the WT WzySf protein is identical in all serotypes, it will be meaningful to investigate

the effect of the mutation on WzySf functioning and association with WzzSf in different serotypes

to know if these mutations have any serotype specific effects.

Woodward et al. (2010) performed the first in vitro polymerisation assay and showed that

WzySf and WzzSf are sufficient to shape Oag. The purified WT WzySf protein can be used in an in

vitro polymerisation assay. Purified WzzSf can also be added to the in vitro system to control the

chain length. After optimisation of the protocol, different purified mutated WzySf proteins can be

used instead of the WT protein to understand the actual mechanism of Oag polymerisation and

association with WzzSf.

Preparing a safe and efficient vaccine against S. flexneri infection is the only way to control

the disease. As the S. flexneri infection is serotype specific, the conjugated vaccines using

detoxified LPS can be used as a vaccine. However, the conjugated vaccines have deficiencies

including accurate control of the detoxification step (Phalipon et al., 2006). Hence, purified

WzySf and WzzSf may be useful to produce Oag in vitro for vaccine development against S.

flexneri infection.

WzzSf determines the S-type (11-17 Oag RUs) Oag and previous study indicated that S-type

Oag is required for maintaining the polar localisation of IcsA for efficient actin-based motility

and cell-to-cell spreading (Morona & Van Den Bosch, 2003b; Van den Bosch & Morona, 2003).

Control over the functioning of WzySf and WzzSf can stop the infection caused by S. flexneri and

these two proteins are potential targets for anti-infection drug development.

197

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198

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