Helicobacter pylori Helicobacter pylori Expresses an Expresses an

Autolytic Enzyme: Gene Identification,Autolytic Enzyme: Gene Identification,

Cloning, and Theoretical Protein StructureCloning, and Theoretical Protein Structure

Eleonora Marsich,1 Pierfrancesco Zuccato,1 Sonia Rizzi,1 AmedeEleonora Marsich,1 Pierfrancesco Zuccato,1 Sonia Rizzi,1 Amedeo Vetere,1*Enrico Tonin,2 and Sergio Paoletti1o Vetere,1*Enrico Tonin,2 and Sergio Paoletti1

Journal of Bacteriology, Nov.2002,vol Journal of Bacteriology, Nov.2002,vol 184 184 : 6270~6279 : 6270~6279

Speaker: Hsin-Yi Chen

2002/12/10

In the past few years, we have successfully In the past few years, we have successfully

complete numerous genome sequencing projects.complete numerous genome sequencing projects.

Combining database searches with molecular Combining database searches with molecular

biology techniques, it is possible to provide a biology techniques, it is possible to provide a

complete description of the role of a gene.complete description of the role of a gene.

IntroductionIntroduction

AutolysinsAutolysins Many bacteria contain one or more cell wall lytic Many bacteria contain one or more cell wall lytic

enzymes. These enzymes are called autolysins. enzymes. These enzymes are called autolysins. DiverseDiverse：： NN-acetylglucosaminidases -acetylglucosaminidases

NN-acetylmuramidases -acetylmuramidases transglycosylases etc. transglycosylases etc.

Conjectural functionsConjectural functions ：：involved in cell wall turnover; cell separatioinvolved in cell wall turnover; cell separatio

n, n, competence for genetic transformation, competence for genetic transformation, formation of flagella, and sporulation.formation of flagella, and sporulation.

T4 LysozymeT4 Lysozyme An enzyme found in T4 phage, with splits An enzyme found in T4 phage, with splits

the glycosidic bond between certain residthe glycosidic bond between certain resid

ues in mucopeptides of bacterial cell wallues in mucopeptides of bacterial cell wall

s, resulting in bacteriolysis. s, resulting in bacteriolysis.

A commonA commonββ(1-4)-N-acetylmuramidase.(1-4)-N-acetylmuramidase.

T4 LysozymeT4 Lysozyme

PDB IDPDB ID ：： 2LZM2LZM

164 aa164 aa

catalytic triadcatalytic triad

PurposePurpose

To investigate a gene whose predicted To investigate a gene whose predicted

product exhibits a high level of homology product exhibits a high level of homology

with T4 lysozyme.with T4 lysozyme.

To characterize the gene been found.To characterize the gene been found.

Data bank searchesData bank searches Similarity search genomic databases for Similarity search genomic databases for

phage T4 lysozymephage T4 lysozyme

22 phage lysozymesphage lysozymes

22 hypothetical proteinshypothetical proteins

4 homologues4 homologues

One is putative One is putative HP0339HP0339 gene of gene of H. pylori H. pylori strain ATCC 26695strain ATCC 26695

(highest homology) (highest homology)

putative putative HP0339 HP0339 gene of gene of H. pylori H. pylori strain strain ATCC 26695ATCC 26695

27% identical; 52% similarity

CLUSTALW alignmentCLUSTALW alignment

Asterisks -- Matching residues Colons -- Conserved Dots -- semiconserved Highlighted --catalytic triad

H. pylori H. pylori strain screeningstrain screening

H. pylori H. pylori is characterizedis characterized by the high level oby the high level of genetic variation.f genetic variation.

Examined bacteria by endoscopy from 38 pExamined bacteria by endoscopy from 38 patients with atients with H. pylori H. pylori infections and use infections and use RARAPDPD analysis for genetic typing of the clinical analysis for genetic typing of the clinical isolates.isolates.

Randomly amplified polymorphobic DNA Randomly amplified polymorphobic DNA (RAPD)(RAPD)

2121 HP0339HP0339 positive strainspositive strains

Clinical Clinical H. pyloriH. pylori strains strains from 38 patientsfrom 38 patients

30 RAPD types30 RAPD types

99 HP0339HP0339 negative trainsnegative trains

RAPD & PCR ResultsRAPD & PCR Results

DNA sequence comparisonDNA sequence comparison

Comparison clinically isolated strains and Comparison clinically isolated strains and

HP0339 HP0339 gene showed gene showed 96% identity96% identity in the complete in the complete

sequence, except clinical strains contained an insersequence, except clinical strains contained an inser

tion of tion of 2424 consecutive nucleotides. consecutive nucleotides.

We designated this variant form of the We designated this variant form of the HP0339 HP0339 gegene ne lyslys..

LysLys gene and gene and HP0339HP0339

T4 lysozyme gene sequenceT4 lysozyme gene sequence

HP0339HP0339

Database searches

PCR cloning of PCR cloning of HP0339HP0339 from clinical strainsfrom clinical strains

Lys Lys gene, a gene, a HP0339 HP0339 gene gene carrying 24 extra nucleotidescarrying 24 extra nucleotides

Lysozyme-like gene expression Lysozyme-like gene expression in vivo in vivo ((RT-PCR) RT-PCR)

HPTS142, HPTS65 and HPTS36 HPTS142, HPTS65 and HPTS36 are lysozyme-positive strains.are lysozyme-positive strains.

Cell wall lytic activity in Cell wall lytic activity in H. pyloriH. pylori protein protein extracts ( Zymogram gel analysis )extracts ( Zymogram gel analysis )

Protein extracts electrophoresis in SDS-PAGE gels Protein extracts electrophoresis in SDS-PAGE gels containing containing M. lysodeikticus M. lysodeikticus cells as a substratecells as a substrate

soaked in Milli-Q water then incubated in soaked in Milli-Q water then incubated in renaturrenaturation bufferation buffer with gentle agitation. with gentle agitation.

Staining with 0.1% Staining with 0.1% methylene bluemethylene blue in 0.01% KOH. in 0.01% KOH.

Zymogram gel analysisZymogram gel analysis

Lanes 1 and 2Lanes 1 and 2--crude protein extracts from --crude protein extracts from lysozyme-negative strainslysozyme-negative strains HPTS12 and HPTS90 HPTS12 and HPTS90

Lanes 3, 4, 5, and 6Lanes 3, 4, 5, and 6--crude protein extracts from --crude protein extracts from lysozyme-positive strainslysozyme-positive strains HPTS142, HPTS65,HPTS61,and HPTS36 HPTS142, HPTS65,HPTS61,and HPTS36

Lane 7Lane 7--bovine serum albumin and ovalbumin--bovine serum albumin and ovalbumin

13.5 kDa13.5 kDa

Cloning, over-expression, and Cloning, over-expression, and purification of the Lys proteinpurification of the Lys protein

E. coli BL21

thrombin cleavage site

HTPS142

pGEX4T-1

EcoRI XhoI

glutathioneS-

transferase

SDS-PAGE analysis of GST-SDS-PAGE analysis of GST-LysLys pro protein expressiontein expression

Lane 1Lane 1-- Marker -- Marker Lane 2Lane 2--crude extract from --crude extract from nontnontransformed cellsransformed cellsLane 3Lane 3--crude extract cells containing --crude extract cells containing

nonrecombinant pGEX nonrecombinant pGEX Lane 4Lane 4--crude extract from cells --crude extract from cells

containing the pGEX-containing the pGEX-lyslysLane 5Lane 5--solubilized pellet from --solubilized pellet from nontnontransformed cells ransformed cells Lane 6Lane 6--solubilized pellet from cells --solubilized pellet from cells

transformed with the transformed with the nonnonrecombinant pGEX recombinant pGEX Lane 7Lane 7--solubilized inclusion bodies --solubilized inclusion bodies

from cells containing the from cells containing the pGEX-pGEX-lys lys

GST-Lys protein purified by GST-Lys protein purified by affinity chromatographyaffinity chromatography

Lane 1Lane 1--markers--markersLane 2Lane 2--total proteins before affinity --total proteins before affinity

purification purification Lane 3Lane 3--total proteins after loading --total proteins after loading

on affinity resin on affinity resin Lane 4Lane 4--eluate from glutathione---eluate from glutathione-

Sepharose 4B resinSepharose 4B resinLane 5Lane 5--flowthrough following --flowthrough following thrthrombin digestionombin digestion of GST- of GST- Lys fusion Lys fusion protein bound to protein bound to glutathione-glutathione-Sepharose 4BSepharose 4BLane 6Lane 6--thrombin digest of eluate --thrombin digest of eluate

from glutathione-Sepharose from glutathione-Sepharose 4B resin4B resin

Zymogram analysis of hydrolytic activity of Zymogram analysis of hydrolytic activity of lys lys gene gene against gram-positive and gram-negative bacteriaagainst gram-positive and gram-negative bacteria

M. lysodeikticus M. lysodeikticus cells (Gcells (G++))

E. coli E. coli murein murein sacculi (G-)sacculi (G-)

H. pylori H. pylori sacculisacculi

controcontroll

LyLyss

Cell Cell extractextractLyLy

ss

LyLyss

Hydrolytic activity of purified recombinant HP0339 protein

M. lysodeikticus M. lysodeikticus cellscells

lys HP0339

H. pylori H. pylori murein sacculimurein sacculi

controlcontrol HP0339

Molecular modeling Molecular modeling HP0339HP0339 and and lys lys productsproducts

3D-pssm server3D-pssm server ：：Classified as a member of the phage T4 Classified as a member of the phage T4

lysozyme family (Proteins family d119l) lysozyme family (Proteins family d119l)

Expectation value of the match is >Expectation value of the match is >95%95%..

Two domains Two domains ：： one one αα, , one one ββ domain domain

Model Model ：： Swiss Model, Ramachandran plot checkSwiss Model, Ramachandran plot check

Refine Refine ：： loop library of Swiss-Pdb Viewerloop library of Swiss-Pdb Viewer

Tertiary structures of HP0339Tertiary structures of HP0339

one one αα domain domain ：： residues 38 to 111residues 38 to 111one one ββ domain domain ：： residues 3 to 27 residues 3 to 27

Tertiary structures of Tertiary structures of lyslys

one one αα domain domain ：： residues 56 to 119residues 56 to 119 one one ββ domain domain ：： residues 3 to 27 residues 3 to 27 insertinsert ：： residues 46 to 54 residues 46 to 54

Phage Phage T4 T4 lysozymlysozymee

HP0339 HP0339 productproduct lys lys productproduct

8 insertion8 insertion ：： LKENHRAL LKENHRAL

Conclusion Conclusion HP0339HP0339 gene product is an enzyme with lyso gene product is an enzyme with lyso

zyme activity.zyme activity.

LysLys gene is 96% identical to HP0339, except t gene is 96% identical to HP0339, except the he lyslys gene contained an insertion of 24 extra gene contained an insertion of 24 extra nucleotides.nucleotides.

There’s no apparent activity difference betwThere’s no apparent activity difference between een lyslys product and product and HP0339 HP0339 productproduct..

DiscussionDiscussion

The presence of an autolytic lysozyme in The presence of an autolytic lysozyme in H. H. pylori pylori is consistent with the hypothesis concis consistent with the hypothesis concerning altruistic behavior.erning altruistic behavior.

The activity of the Lys lysozyme could be mThe activity of the Lys lysozyme could be modulated by interactions with a larger compodulated by interactions with a larger complex of other enzymes.lex of other enzymes.

Structural modifications of the cell wall Structural modifications of the cell wall should play a role in should play a role in H. pylori H. pylori pathogenesis if pathogenesis if cell wall fragments are released.cell wall fragments are released.

One possible effect due to the insertion could One possible effect due to the insertion could be modulation of the domain motions and, of be modulation of the domain motions and, of the shape of the active cleft.the shape of the active cleft.

DiscussionDiscussion

Further functional characterization, such Further functional characterization, such

as in vivo localization and crystallographias in vivo localization and crystallographi

c structure analyses, will be performed.c structure analyses, will be performed.

Future WorkFuture Work

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Thank You For your AttentionThank You For your Attention ！！

lys

T4 lys