STREPTOCOCCAL COLONIZATION OF HOST MUCOSAL SURFACES: A STUDY OF BIOFILM FORMATION AND DISPERSAL

BY

AMITY LEANN ROBERTS

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

In Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Microbiology and Immunology

December 2010

Winston-Salem, NC

Approved By:

Sean D. Reid, Ph.D., Advisor

Examining Committee:

Al Claiborne, Ph.D., Chair

Rajendar Deora, Ph.D.

Katherine A. Poehling, M.D., M.P.H

Elizabeth Hiltbold Schwartz, Ph.D.

W. Edward Swords, Ph.D.

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ACKNOWLEDGEMENTS

I would like to thank my family for their encouragement and support throughout the

years; I would not have made it to this point without them.

I would like to thank my mentor and advisor Sean D. Reid, Ph.D. for his thoughtful

guidance and direction on my project/s. He was instrumental in training me to

think outside the box and in helping me to become an independent scientist. His

passion for understanding infectious disease processes has provided me with the

opportunity to examine human samples and importantly has enabled me to take a

project from the research bench into animals and finally into a survey of the human

condition.

I would like to thank Chris Holder and Kristie Connolly for their help with the

chinchilla projects. Holder, thank you for being a molecular biology guru; your

cloning abilities have served us well. Kristie, thank you for being a great friend

and I will miss our lunch outings. Thank you both for being willing to listen to my

ramblings and for being wonderful sounding boards for project ideas. I would like

to thank Steve Richardson, Ph.D. for providing a wealth of experience and

knowledge to the lab and of course for comic relief, you know how to make us

laugh even on the dreariest days. I would also like to thank Chris Doern, Ph.D. for

setting the ground work for my project by examining the molecular aspects of Srv

and also for being a great colleague.

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I would like to thank the graduate school for the wonderful opportunity to receive

graduate training at Wake Forest University Health Sciences. I would like to thank

my graduate committee for their help and encouragement throughout the years; a

special thanks goes out to Al Claiborne, Ph.D. for agreeing to become my

dissertation defense committee chairman on such short notice. I would like to

thank the tonsil study group (Dan Kirse, MD; Adele Evans, M.D.; Kathy Poehling,

M.D., M.P.H.; and Tim Peters, M.D.) for helping with the IRB, obtaining the tonsils,

and for providing a clinical perspective to the project. I would like to thank Mark

Willingham, M.D. and Ken Grant for microscopy training and help with

immunofluorescence protocols. I would like to thank the Swords’ laboratory for

assisting with training me on how to handle the chinchillas, how to operate the

digital otoscope, and with the occasional extra set of hands.

Lastly, I would like to thank Michael Cutler for his encouragement and for helping

me to stay focused on the goal.

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TABLE OF CONTENTS

PAGE ACKNOWLEDGEMENTS................................................................................. ii LIST OF TABLES AND FIGURES................................................................... vi LIST OF ABBREVIATIONS ............................................................................. ix ABSTRACT ..................................................................................................... xi CHAPTER

I. INTRODUCTION ................................................................................... 1

II. BIOFILM FORMATION BY GROUP A STREPTOCOCCUS: A ROLE FOR THE STREPTOCOCCAL REGULATOR OF VIRULENCE (Srv) AND STREPTOCOCCAL CYSTEINE PROTEASE (SpeB) ................... 9

III. ALLELIC REPLACEMENT OF THE STREPTOCOCCAL CYSTEINE

PROTEASE SpeB IN A Δsrv MUTANT BACKGROUND RESTORES

BIOFILM FORMATION........................................................................ 32

IV. LOSS OF THE GROUP A STREPTOCOCCUS REGULATOR Srv

DECREASES BIOFILM FORMATION IN VIVO IN AN OTITIS MEDIA

MODEL OF INFECTION...................................................................... 50

V. STREPTOCOCCUS PYOGENES BIOFILMS DETECTED

WITHIN THE CRYPTS OF TONSILS FROM PEDIATRIC

CASES OF ADENOTONSILLAR HYPERTROPHY OR

RECURRENT S. PYOGENES INFECTION ........................................ 82

VI. DISCUSSION .................................................................................... 106 REFERENCES............................................................................................. 110

v

APPENDIX ................................................................................................... 133 CURRICULUM VITAE .................................................................................. 136 COPYRIGHT INFORMATION ...................................................................... 139

vi

LIST OF TABLES AND FIGURES

CHAPTER II PAGE Figure 1. Enzymatic inhibition or disruption of GAS biofilm formation 19 Figure 2. The transcriptional regulator Srv is required for maximal biofilm formation by MGAS5005 21 Figure 3. Casein agar assay for SpeB proteolytic activity 22 Figure 4. Inhibition of SpeB activity restores GAS biofilm formation 23 Figure 5. Detection of SpeB in GAS biofilms 25 Figure 6. Continuous-flow system for study of GAS biofilm formation 27 CHAPTER III Table 1. Primers and probes used in this study 40 Figure 1. Construction of MGAS5005ΔsrvΔspeB 39 Figure 2. Static crystal violet assays for the measurement of in vitro biofilm formation 44 Figure 3. MGAS5005ΔsrvΔspeB biofilm formation under continuous flow conditions 46 Figure 4. Addition of purified active SpeB inhibits biofilm formation 47 Figure 5. Hypothetical model of Srv/SpeB mediated GAS biofilm Formation 49 CHAPTER IV Table 1. Frequency of macroscopic structure formation over time following infection with GAS 62 Figure 1. Digital otoscopy of representative MGAS5005 and MGAS5005Δsrv at time 0 (uninfected) and 2, 4, and 7 dpi 60 Figure 2. GAS infection leads to the development of macroscopically visible surface attached structures in the chinchilla middle ear 61

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Figure 3. Increased clearance of MGAS5005, but not MGAS5005Δsrv, was observed over the course of experimental chinchilla otitis media within effusion samples 66 Figure 4. SEM reveals GAS biofilms embedded within the Macroscopic structures recovered from MGAS5005 infected middle ears 68 Figure 5. LIVE/DEAD viability staining of material recovered from the chinchilla middle ear following GAS infection 70 Figure 6. H&E (A, C, E, G) and Gram-staining (B, D, F, H) of macroscopic structures removed 7 dpi from the chinchilla middle ear cavity 72 Figure 7. SEM and Gram-staining reveals GAS biofilms embedded within the macroscopic structures recovered from MGAS5005ΔsrvΔspeB infected middle ear 75 Figure 8. MGAS5005ΔsrvΔspeB was maintained within middle ear effusion and macroscopic structures over the course of Infection 76 CHAPTER V Table I. Characteristics of study population undergoing Tonsillectomy 91 Figure 1. Fluorescent antibody staining of Cytokeratin 8 &18 lining the tonsillar crypts 92 Figure 2. Fluorescent antibody staining of S. pyogenes within the crypts of pediatric tonsils removed due to recurrent S. pyogenes infection 95 Figure 3. Fluorescent antibody staining of S. pyogenes within the crypts of pediatric tonsils removed due to ATH 97 Figure 4. SEM showing chains of cocci organized into biofilms attached to the tonsillar surface 99 Figure 5. Gram-stain showing the presence of Gram-positive bacteria in a biofilm within tonsil sections 102

viii

APPENDIX Figure 1. Biofilm formation of clinical GAS strains representing 32 distinct M-serotypes. 135

ix

ABBREVIATIONS

GAS Group A Streptococcus, Streptococcus pyogenes SEM Scanning Electron Microscopy CV Crystal Violet ARF Acute Rheumatic Fever RHD Rheumatic Heart Disease MBEC Minimal Biofilm Eradication Concentration MIC Minimal Inhibitory Concentration RRs ‘stand alone’ Response Regulator/s TCS Two-Component Signal Transduction System h hour ATH Adenotonsillar Hypertrophy SpeB Streptococcal Pyrogenic Exotoxin B/Cysteine Protease Srv Streptococcal Regulator of Virulence PrfA Pleiotropic Virulence Factor WHO World Health Organization CDC Centers for Disease Control and Prevention CLSM Confocal Laser Scanning Microscopy EMSA Electrophoretic Mobility Shift Assay i.p. Intraperitoneal HTH Helix-Turn-Helix cAMP cyclic-AMP

x

Crp/Fnr cAMP Receptor Protein/Fumarate and Nitrate Reduction Regulatory Family THY Todd Hewett Broth with 0.2% Yeast Extract Min Minute/s A Absorbance PBS Phosphate Buffered Saline BSA Bovine Serum Albumin HasA Hyaluronic Acid Capsule CovR/S Control of Virulence Operon Sda1 GAS Extracellular DNase NETs Neutrophil Extracellular Traps CFU Colony Forming Units g gram ml milliliter PCR Polymerase Chain Reaction RT-PCR Reverse Transcriptase PCR mRNA Messenger RNA ATH Adenotonsillar Hypertrophy OM Otitis Media/Middle Ear Infection dpi Days Post Infection CK Cytokeratin

xi

ABSTRACT

Group A Streptococcus (GAS) is a Gram-positive pathogen that is the

causative agent of a variety of human diseases. Infections include pharyngitis,

otitis media, sepsis, a toxic-shock syndrome, necrotizing fasciitis, and the post-

infectious sequelae acute rheumatic fever and acute rheumatic heart disease.

Thus, rather than exploit a singular niche, GAS has evolved to colonize and

disseminate within several physiologically distinct anatomical sites of the human

host. Such versatility requires the ability to coordinately regulate the expression

and production of numerous factors in rapid response to host and environmental

signals in order to facilitate attachment, replication, and eventual dispersal

(dissemination). Experimental evidence suggests that GAS forms biofilms during

the colonization of a surface. A biofilm is a bacterial sessile community encased in

an extracellular matrix and attached to a substratum or interface. Biofilms are

inherently tolerant to host defenses and antibiotic therapies and often involved in

chronic illness due to impaired clearance. It is estimated that upwards of 60% of

all bacterial infections involve biofilms. Presently, little is known about GAS biofilm

matrix composition, or the regulation of biofilm formation and dispersal.

Our evidence indicated that allelic replacement of the GAS transcriptional

regulator srv resulted in a significant reduction in GAS biofilm formation. Thus, we

hypothesized that srv is required for GAS biofilm formation, and that biofilms are

required for colonization of the host mucosa. Our research revealed that

complementation of srv in the srv mutant background restored biofilm formation

xii

indicating that srv is required for biofilm formation. Furthermore, we discovered

that the biofilm null phenotype of the srv mutant was due to the constitutive

production of a GAS cysteine protease (SpeB). Allelic replacement of speB in the

srv mutant background or chemical inhibition of SpeB restored GAS biofilm

formation. We further demonstrated that GAS formed biofilms in vivo in both

chinchillas and human patients. Finally, we demonstrated that biofilms may not be

required for infection of the host, but that biofilm dispersal may lead to more

severe disease. Under this model, Srv regulation of SpeB mediates biofilm

formation/dispersal.

1

CHAPTER I

INTRODUCTION

Impact of Group A Streptococcal Infections

Group A Streptococcus (GAS), also designated Streptococcus pyogenes,

is a Gram-positive β-hemolytic human pathogen that is capable of causing a wide

range of diverse human infections, including pharyngitis, otitis media, impetigo,

cellulitis, sepsis, streptococcal toxic-shock syndrome, and necrotizing fasciitis (13,

32, 36, 56, 91, 92, 113, 123, 127). Thus, the organism has evolved to colonize a

number of physiologically distinct host sites and has a significant impact on human

health. For example, GAS is the most common bacterial causative agent of

tonsillopharyngitis resulting in 15% to 36% of cases in children and 5% to 10% of

cases in adults (10, 101). The World Health Organization (WHO) reports that GAS

results in an estimated 616 million cases of tonsillopharyngitis each year globally

with it being more prevalent in underdeveloped countries (152). Complications of

a normally mild GAS infection can lead to the development of a severe and even

life threatening forms of systemic infection (152). As reported by the Centers for

Disease Control, an estimated 12,000 cases of severe invasive GAS infections

occur each year within the United States (US) resulting in ~ 1600 deaths (21). It is

estimated that 1.78 million new cases of severe GAS infection occur each year

world-wide, leading to ~ 517,000 deaths (152). The health care costs associated

with GAS infections world-wide and within the US alone each year are

tremendous. For example, within the US there are ~ 1.2 million doctor visits per

year for GAS pharyngitis, resulting in an estimated $381 million dollars in

2

healthcare and non-healthcare related costs (34, 78, 101). Futhermore, untreated

cases of GAS correlate with the development of the post-infectious sequelae:

acute rheumatic fever (ARF), rheumatic heart disease (RHD), and acute

glomerulonephritis (32). RHD is more prevalent in underdeveloped countries due

to minimal access to healthcare with ~ 15.6 million existing cases worldwide (152).

This results in RHD leading the way as the most common cause of preventable

heart disease in children (131-133).

Numerous questions remain to be answered in regards to understanding

the interaction between GAS and the human host. It is of great importance to

learn how this pathogen is successful at distinct sites within the host, and also,

what factors are determining whether the course of disease will be mild or severe?

It is still unknown, though many theories exist, how GAS is persisting within the

host and additionally how it is then able to spread throughout the host to different

anatomical sites.

Group A Streptococcal Serotypes

GAS strains are differentiated into serotypes based upon the emm

sequence of the unique M protein that is displayed upon the surface of the cell

(32). Thus far, over 100 different M-serotypes for GAS have been identified (46).

Interestingly, since the 1980’s, there have been reports that GAS infections are

shifting to a more invasive nature and that certain serotypes are becoming more

commonly associated with invasive disease. According to the CDC these

3

serotypes are M1, M3, M11, M12, M28 (95). This increase in severe infections is

coupled with an increase in penicillin treatment failure, the preferred and most

effective antibiotic for eradication of GAS (103, 105). Penicillin resistance

cassettes have not been identified in GAS, leading to several probable

explanations for penicillin failure: re-exposure to the organism post-treatment

through contact with peers or family members, the development of penicillin

tolerance by GAS, degradation of penicillin by beta-lactamase which can be

produced by co-colonizing beta-lactamase positive organisms, and intracellular

persistence of GAS within host cells (17, 71, 96, 97, 104, 105). However, these

studies have not provided conclusive evidence that these hypotheses are the

definitive reason for penicillin treatment failure. Another area of study that may

explain why penicillin treatment failure rates have increased in cases of GAS

infections is the formation of bacterial biofilms. Bacterial biofilms have been

shown to protect the bacterial agent against clearance by both the host immune

response and antimicrobial agents (49, 50).

Bacterial Biofilms

A bacterial biofilm is often defined as a grouping/community of either single

or multispecies bacteria encased in a matrix of extracellular polymeric substances

and attached to a biological or non-biological substratum or interface (40, 41, 52).

The composition of this matrix may differ between bacterial species but evidence

suggests the biofilm provides protection from the innate host response and

antimicrobial agents and is often associated with chronic infections due to an

4

inability of the host to completely eradicate the organism (31, 48-50, 77, 128, 146).

It is estimated that upwards of 60% of all bacterial infections involve biofilms

including dental caries, periodontitis, otitis media, endocarditis, and necrotizing

fasciitis (34, 39, 41, 48, 52). Additionally, this community may display an altered

phenotype with respect to growth rate and gene transcription when compared to

bacterial growth in the planktonic state (40, 52).

It is proposed that there are three stages of the biofilm growth cycle:

attachment, growth/maturation, and detachment/dispersal (75). Attachment

involves bacterial adherence to a surface, for instance the epithelial cells of the

nasal septum. Initial colonizers assemble into microcolonies, clusters of bacterial

cells, through bacterial division and recruitment of additional microbes of the same

or of a different species; the microcolony then matures into a matrix encased

biofilm. The presence of organized microcolonies is indicative of biofilm formation.

Dispersal occurs when individual or clusters of sessile bacteria leave the mature

biofilm and colonize new sites, forming progeny biofilm (31, 49, 52, 76, 134). The

mechanism by which dispersion occurs appears to be bacterial species specific

and is not well understood for most organisms. Some propose that biofilms are

highly organized and complex communities, the dynamics of which are constantly

changing due to the cyclic nature of the biofilm state. The biofilm cycle can be a

continuous process with controlled release/detachment of viable organisms as

single cells or as dense clusters which can then actively colonize other areas of

the host or facilitate spread to a new host, allowing the biofilm cycle to repeat (53).

5

Group A Streptococcus Biofilms

There is a growing understanding of the importance of biofilm formation to

GAS (1, 7, 23, 28, 38, 76, 85, 88). One of the first observations of GAS biofilms

was reported by Akiyama et al. in an in vivo study examining one form of GAS skin

infection: impetigo (1). Confocal laser scanning microscopy (CLSM) of skin

lesions revealed the presence of GAS microcolonies that appeared to be

surrounded by a glycocalyx (1). Biofilm glycocalyx, or matrix material, can be

composed of a variety of substances, often species specific, including DNA,

protein, polysaccharides and other exogenous host components (42). As of yet,

the composition of the GAS matrix is not well understood. Lembke et al., utilized

GAS clinical isolates to show that the stages of GAS biofilm formation are

comparable to what is seen by other organisms: adhesion,

proliferation/maturation/matrix development, and finally detachment (76). Studies

have since shown that GAS clinical isolates from a variety of body sites can form

biofilm, however not every isolate forms biofilm and the amount of biofilm formed

can vary greatly between isolates (28, 76, 138).

In support of a biofilm hypothesis for antimicrobial treatment failure, one

study indicated that GAS clinical isolates which are susceptible to antibiotic

treatment with erythromycin tended to form thicker biofilms than strains carrying

erythromycin resistance cassettes (7). Moreover, the Calgary biofilm device was

utilized to determine the susceptibility of biofilm grown clinical isolates to penicillin

(28). The minimal biofilm eradication concentration (MBEC) values predicted a ~

6

60% likelihood of treatment failure whereas minimal inhibitory concentration (MIC),

the standard assay used in clinical laboratories to determine antimicrobial

susceptibility, indicated zero likelihood of failure. Isolates were obtained before

and after treatment, allowing verification of antibiotic failure cases (28).

As discussed, biofilm formation by GAS has been observed both in vitro

and in vivo however there is still much to learn about the about the makeup of the

GAS biofilm, how it is regulated, and how it contributes to virulence at

physiologically distinct host sites. Moreover, the detachment stage of GAS

biofilms is not well understood. Little is known about the composition of the GAS

matrix. In an attempt to determine the level of variability in biofilm forming ability

between serotypes of GAS, we tested 219 different clinical isolates representing

32 different M-serotypes. Of these isolates an invasive M1T1 serotype strain

MGAS5005, representative of M1T1 strains associated with invasive infections

worldwide, formed robust biofilm based on a commonly used crystal violet staining

assay (see Appendix Figure 1) (92, 113). Thus, MGAS5005 was selected for

our studies of GAS biofilm formation.

Streptococcal Regulator of Virulence (Srv)

In an attempt to understand how GAS regulates biofilm formation, we

examined the GAS regulator Srv (streptococcal regulator of virulence). We

hypothesized that the inactivation of srv would alter the GAS biofilm phenotype.

Srv is a highly conserved, transcriptional ‘stand alone’ response regulator encoded

7

by the gene srv (SPy1857 in the GAS SF370 genome bank, Spy1576 in the GAS

MGAS5005 genome bank) (111, 115). Stand alone response regulators (RRs)

are proteins that have a regulatory function and yet do not have an identified

sensor kinase or other sensory unit (90) as is common in two-component signal

transduction systems. Srv was identified through a GAS genome-wide search for

potential virulence factors and was selected due to its gene homology (27%

identical and 53% similar) to the pleiotropic virulence factor (prfA) gene; PrfA is a

virulence gene regulator in Listeria monocytogenes (74, 111, 115). Srv belongs to

the cyclic AMP (cAMP) receptor protein/fumarate and nitrate reduction regulator

(Crp/Fnr) family of positive transcriptional regulators (37, 115). This family of

regulators contains β-roll structures (short β-sheets separated by glycine residues)

at the N-terminal region of the protein which potentially bind allosteric effector

molecules and a helix-turn-helix (HTH) motif at the C-terminal region thought to

bind DNA at symmetrical consensus sequences near the regulated gene

promoters (115). Recently, Srv was shown to shift specific DNA targets in

electrophoretic mobility shift assays (EMSA). Point mutations of specific amino

acids, analogous to those shown to be critical for PrfA DNA binding (43, 55, 120,

124, 125, 144), in the C-terminal HTH region of Srv disrupted binding to these

specific DNA targets, indicating that the HTH motif is required for Srv to function in

DNA binding (37).

To further study the function of Srv, the gene srv was allelically replaced in

the clinical isolate MGAS5005 with a spectinomycin resistance cassette (spc) to

create MGAS5005Δsrv (115). Studies indicated that there were no defects in

8

mutant growth compared to wild-type and that in-frame insertion of spc did not

result in downstream polar effects (80, 115, 116). MicroArray genome analysis of

wild-type (MGAS5005) compared to its isogenic mutant of srv (MGAS5005Δsrv)

indicated a significant decrease in gene transcription across the entire GAS

genome and in particular expression of genes encoding for secreted extracellular

proteins was decreased (111). A study of MGAS5005Δsrv has shown that the srv

knockout displays a decreased level of virulence in a murine intraperitoneal (i.p.)

model of infection (115). The srv mutant was shown to constitutively produce a

streptococcal cysteine protease (SpeB) (37, 111). To confirm that the phenotypes

exhibited by MGAS5005Δsrv were due to loss of the gene srv, MGAS5005Δsrv

was complemented in trans with srv. The complemented strain showed that both

i.p. virulence and also SpeB levels were restored to those of wild-type (37).

Taken together, our data lead us to hypothesize that Srv may be involved in

the regulation of GAS biofilm formation. We hypothesized that allelic replacement

of srv would lead to decreased biofilm formation and that this phenotype was due

to the constitutive production of SpeB. We further sought to determine if these

hypotheses were valid in an in vivo model of GAS infection, and to determine if

there is evidence of GAS biofilms in humans.

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CHAPTER II

BIOFILM FORMATION BY GROUP A STREPTOCOCCUS: A ROLE FOR THE

STREPTOCOCCAL REGULATOR OF VIRULENCE (SRV) AND

STREPTOCOCCAL CYSTEINE PROTEASE (SPEB)

C. D. Doern†, A. L. Roberts†, W. Hong, J. Nelson, S. Lukomski, W. E. Swords, and Sean D. Reid

Department of Microbiology and Immunology

Wake Forest University School of Medicine

The following manuscript was published in Microbiology, Volume 155, pages 46-

52, 2009, and is reprinted with permission from the Society for General

Microbiology. Stylistic variations are due to the requirements for publication in the

journal. † C. D. Doern and A. L. Roberts are co-first authors and contributed

equally to the experiments and preparation of the manuscript. Dr. Sean Reid acted

in an advisory and editorial capacity. Copyright © Society for General Microbiology

2010.

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SUMMARY

Recently, biofilms have become a topic of interest in the study of the human

pathogen group A Streptococcus (GAS). In this study, we sought to learn more

about the make-up of these structures and gain insight into biofilm regulation.

Enzymatic studies indicated that biofilm formation by GAS strain MGAS5005

required an extracellular protein and DNA component(s). Previous results

indicated that inactivation of the transcriptional regulator Srv in MGAS5005

resulted in a significant decrease in virulence. Here, inactivation of Srv also

resulted in a significant decrease in biofilm formation under static and flow

conditions. Given that production of the extracellular cysteine protease SpeB is

increased in the srv mutant, we tested the hypothesis that increased levels of

active SpeB may be responsible for the reduction in biofilm formation. Western

immunoblot analysis indicated that SpeB was absent from MGAS5005 biofilms.

Complementation of MGAS5005∆srv restored the biofilm phenotype and

eliminated the over production of active SpeB. Inhibition of SpeB with E64 also

restored the MGAS5005∆srv biofilm to wild-type levels.

11

INTRODUCTION

Group A Streptococcus (GAS) is responsible for a wide range of human infections,

including pharyngitis and tonsillitis, skin infections, sepsis, a toxic-shock

syndrome, and necrotizing fasciitis (32, 92, 113). Until recently, there was little

association of GAS and biofilms in the published literature. One of the first

observations was reported by Akiyama et al. (2003) in a study examining GAS

in skin infections (1). Confocal laser scanning microscopy (CLSM) of skin

lesions revealed the presence of GAS microcolonies that appeared to be

surrounded by a glycocalyx (1). A limited number of subsequent studies have

demonstrated GAS biofilm formation in vitro and in vivo and microarray analyses

have shown that gene transcription varies markedly in biofilms compared to

planktonically grown (7, 23, 28, 76, 88, 118, 138). However, little is known about

whether the ability to form biofilms is a characteristic of all GAS strains, what the

biofilm matrix is composed of, or how this biofilm state is regulated.

In the present work, we demonstrate that a globally disseminated M1T1

clone associated with invasive disease produced one of the largest biofilms

observed. In addition, we demonstrate that DNA and protein are critical to GAS

biofilm formation. Furthermore, in an effort to gain insight into the regulation of the

biofilm process, we examined the effects of a loss of the virulence regulator srv on

GAS biofilm formation. Srv has been shown to be required for GAS virulence and

influence GAS gene expression (111, 115). Here we show that Srv is required for

12

biofilm formation and learn that increased activity of the cysteine protease SpeB in

the absence of Srv contributes to the loss of the biofilm phenotype.

METHODS

Bacterial strains. MGAS5005 is a representative M1T1 serotype strain (speA+)

which was isolated from a case of invasive GAS disease and has been the focus

of numerous studies (reviewed by (92, 113)). The MGAS5005 isogenic srv mutant

strain (MGAS5005∆srv) was generated by allelic replacement (115).

MGAS5005∆srv was complemented in trans (MGAS5005∆srv(pIAß8-srv)) as

previously described (37).

Adherence Assay. Overnight cultures of GAS grown at 37˚C (5% CO2) in Todd

Hewett Broth (TH) (Difco Laboratories, Sparks, MD) supplemented with 0.2%

yeast extract (THY) (Fisher Scientific, Fair Lawn, NJ) were harvested and used to

inoculate fresh THY. Cultures were then grown to an optical density (OD600nm) of

0.5. Tissue culture treated polystyrene 6-well cell culture plates (Corning Inc.,

Corning, NY) were seeded with 3-ml of culture per well. Plates were incubated 24

h at 37˚C, 5% CO2. Media was removed without disturbing the biofilm and wells

were washed 3x with dH2O. 1-ml aliquots of 0.1% crystal violet (CV) (Sigma-

Aldrich, Inc., St. Louis, MO) dissolved in dH2O were dispensed to each well.

Surface-attached bacteria were allowed to stain for 30 min at room temperature

and then washed three times with dH2O, after which 1 ml ethanol was added to

13

each well to solubilize the CV. An A600 reading (measuring the colour intensity of

the solubilised CV) was recorded for each sample. If staining was too dark for

spectrophotometric analysis, the sample was diluted with additional ethanol and

the dilution factor applied to the resulting reading (in these instances, the value is

referred to at the A600 extrapolated, or A600e).

Enzymatic inhibition/disruption of biofilms. Methods were adapted from Wang

et al. (149). To test the ability to inhibit biofilm formation, 2 ml aliquots of

exponentially growing GAS cultures (OD600 = 0.5) were mixed with 2 ml of one of

the following reagents (final conc.): 40 mM NaIO4 (metaperiodate), 200 μg/ml

DNaseI, or 1 mg/ml Proteinase K. Enzyme-treated cultures were added to six-well

tissue culture plates and incubated 24 h at 37˚C, 5% CO2. Wells were washed and

stained with CV as described. To test the ability to disrupt a pre-formed biofilm,

each of the reagents described above was added to a 24-h-old biofilm and allowed

to incubate for an additional 1 h. Wells were washed and stained as before.

Analysis of biofilm formation under continuous-flow conditions. Using a 25

gauge 1½ Precision Glide Needle (Becton Dickinson and Co., Franklinlakes, NJ),

a 10 ml aliquot of exponentially growing GAS culture (OD600 = 0.5) was inoculated

into a convertible flow-cell chamber (Stovall Life Sciences, Inc., USA). Each

inoculum was incubated 3 h at 37˚C without flow to allow initial attachment within

the chamber. Sterile THY media was connected to the flow chamber through the

pump head via Masterflex silicon tubing (peroxide treated) L/STM14 (Cole-Parmer

14

Instruments Co., Vernon Hills, IL), and flow discharge was collected in a waste

container downstream of the flow cell chamber. Flow was initiated using a

Masterflex Easy-Load II peristaltic pump (Cole-Parmer Instruments Co.) with a

flow rate of 0.7 ml/min and continued for 24 h at 37˚C.

Confocal laser scanning microscopy of flow cell biofilms. To visually

examine the health of the biofilm, samples were subjected to Live/Dead staining.

Samples contained within flow cell chambers were washed with PBS. A mixture of

SYTO 9 and propidium iodide (LIVE/DEAD BacLight Bacterial viability kit L7007,

Molecular Probes, Eugene, OR) contained in 10 ml of PBS buffer was added to

the chamber. The sample was allowed to incubate at room temperature for 1 h

with the LIVE/DEAD stain and then washed with PBS. Samples were visualized

using a Zeiss LSM510 confocal scanning laser microscope.

Scanning electron microscopy of flow-cell biofilms. Continuous flow-cell

samples were fixed within the flow-cell chamber with 2.5% glutaraldehyde in PBS.

After fixation for a minimum of 1 h, samples were washed twice and subjected to

dehydration, fixation, and critical-point drying for scanning electron microscopy.

Flow-cell chamber slides were then trimmed, mounted, and coated with palladium.

Biofilms were viewed on a Phillips SEM-515 scanning electron microscope.

15

RESULTS

Protein and DNA are significant components of the GAS biofilm.

We first determined the level of biofilm formed by MGAS5005, a representative

serotype M1T1 strain recently isolated from a case of invasive GAS disease.

Population genetic analysis has indicated that M1T1 strains are among the most

common causes of invasive GAS infections worldwide in most studies (92).

Dilution of the solubilised CV was necessary as the original CV staining was too

dark for spectrophotometric analysis.

We next sought to learn more about the biofilm structure by examining the

components present. We hypothesized that if proteins, DNA, or polysaccharides

are required, then biofilms should be inhibited or disrupted by proteinase K, DNase

I, or metaperiodate, respectively. Addition of proteinase K or DNase I at the time

six-well plates were seeded significantly inhibited biofilm formation (Fig. 1a). In

addition, a 1 h incubation with either proteinase K or DNase I disrupted an existing

24 h old biofilm (Fig. 1b). Treatment with metaperiodate, a compound capable of

oxidizing polysaccharides (86, 87, 149), led to a reduction in both the formation

and the stability of the biofilm, but measurable biofilm was still present (Fig. 1a-b).

As a control, we performed replicate plating to test the viability of the bacteria

following treatment. CFU numbers recovered following treatment with proteinase

K or DNase I were equivalent to those following treatment with PBS (~109

CFU/ml). However, we recovered between 106 – 107 CFU/ml from metaperiodate-

16

treated samples. This suggested that the decrease in biofilm observed may be

due to the effects of metaperiodate on bacterial viability. Thus, the GAS biofilm

requires a protein and a DNA component(s) for formation and stability. GAS

polysaccharides may increase the mass of the biofilm when present, but periodate

sensitive polysaccharides do not appear to be required for GAS biofilm formation.

Inactivation of the transcriptional regulator Srv results in a significant

reduction in GAS biofilm formation.

The observation that extracellular protein is important in the formation of the GAS

biofilm led us to consider the possible role of the transcriptional regulator Srv. Our

previous studies indicated that inactivation of srv resulted in a significant reduction

in GAS virulence (115) and differential transcription of 51 genes encoding proven

or putative extracellular proteins (111). The reduced expression of genes

encoding extracellular proteins in the srv mutant strain led us to hypothesize that

Srv may contribute to biofilm formation. Comparison of MGAS5005 and the srv

isogenic mutant derivative MGAS5005∆srv indicated that MGAS5005∆srv

produced significantly less biofilm (Fig. 2a). Complementation of srv in trans

(MGAS5005∆srv(pIAß8-srv)) restored biofilm production to near wild-type levels

(Fig. 2a).

To determine if the results observed were due to an impaired ability of

MGAS5005∆srv to form biofilms or an early disruption of the biofilm, we followed

formation of the structure over time. MGAS5005 and MGAS5005∆srv(pIAß8-srv)

17

produced biofilm at approximately the same rate and to the same level (Fig. 2b).

Maximal biofilm formation was observed at 24 h, with levels tapering off thereafter.

In contrast, CV staining of surface attached MGAS5005∆srv was significantly less

at all time points measured (Fig. 2b.).

Inhibition of SpeB in MGAS5005∆srv restores biofilm formation.

Our data suggested that Srv-mediated gene transcription was required for proper

biofilm formation. This may be due to reduced transcription of genes encoding

extracellular proteins required for attachment and/or aggregation, or to the effects

of some other product that is directly or indirectly affected by Srv. Previously, we

discovered that inactivation of srv led to increased production of the GAS cysteine

protease SpeB (111). We can visualize the increased activity of SpeB in a casein

agar assay, in which strains are stab inoculated into casein agar and allowed to

grow at 37°C under microaerophilic conditions. The resulting zone of translucence

surrounding the stab site is indicative of SpeB caseinolytic activity. This assay

demonstrated that complementation with srv in trans restored SpeB activity to

wild-type levels (Fig. 3).

To test the hypothesis that an increase in SpeB may be responsible for the

MGAS5005∆srv biofilm defect, we examined biofilm formation in the presence of

an inhibitor of SpeB activity, E64. E64 is a potent and highly selective cysteine

protease inhibitor that irreversibly binds to an active thiol group to form a thioether

linkage. Previous groups have used concentrations of ~30µM to 10mM E64 (66,

145). However, these studies have added the inhibitor to assays using GAS

18

supernatants or purified proteins. Since we sought to add E64 to living cells and

allow the culture to incubate for 24 h, we elected to use a final concentration of

333 µM. Addition of E64 at the time the plates were seeded restored the ability of

MGAS5005∆srv to form biofilms (Fig. 4a). To determine that the observed results

were due to the inhibition of SpeB and not another protease, we examined the

biofilm formation of a speB isogenic mutant (79) with and without the addition of

E64 (Fig. 4a). No difference was observed in the ability of the ΔspeB strain to

form biofilms. Examination of the attached bacteria prior to solubilization with

ethanol revealed that the CV stain was not as uniform in E64 treated

MGAS5005∆srv as in the treated wild-type, but there was a clear increase in

attached bacteria compared to untreated MGAS5005∆srv (Fig. 4b).

Extracellular SpeB is absent from 24 h GAS biofilms.

Previously, we demonstrated that SpeB was detected by Western immunoblotting

in culture supernatants of MGAS5005 following 8 h of planktonic growth (111). By

contrast, SpeB was detectable in MGAS5005∆srv supernatants after just 2 h of

growth (111). For the experiments presented above, cultures were allowed to

grow planktonically for 3 h prior to seeding of the six-well plates. Biofilms were

then allowed to develop for 24 h. If SpeB inhibits or disperses the GAS biofilm,

then it must be present at very low levels if at all in the biofilm. To test this

hypothesis, GAS biofilms and their supernatants were collected, treated as a

single sample (biofilm and supernatant together), and probed by Western

19

Figure 1. Enzymatic inhibition or disruption of GAS biofilm formation. (a) To test

the ability of specific enzymes to inhibit biofilm formation, proteinase K, DNase I,

or metaperiodate was added when the six-well plates were initially seeded with

MGAS5005 in the exponential phase (OD600 = 0.5). Following 24 h growth, strains

incubated with proteinase K or DNase I produced significantly less, if any, biofilm.

While metaperiodate curtailed biofilm formation, it did not abolish this ability. Each

reported value is the mean ± SD of at least 6 replicates and is adjusted by the

dilution factor required to obtain a spectrophotometric reading (A600 extrapolated or

A600e ). (b) To test the ability of the same enzymes to disrupt an established GAS

biofilm, proteinase K, DNase I, or metaperiodate was added to cultures grown in

six-well plates for 24 h. CV assays were completed following 1 h incubation with

enzyme. Treatment with proteinase K or DNase I disrupted the biofilm and yielded

a significantly reduced A600e. Treatment with metaperiodate reduced the amount

of biofilm present, but was unable to completely disrupt the biofilm.

20

Figure 2. The transcriptional regulator Srv is required for maximal biofilm

formation by MGAS5005. Each reported value for the CV assay is an average of

at least six replicates and is adjusted by the dilution factor required to obtain a

spectrophotometric reading (A600e). (a) A comparison of the wild-type

(MGAS5005), the isogenic mutant lacking srv (∆srv), and the complemented strain

(MGAS5005(pIAβ8-srv)). The ∆srv mutant is significantly reduced in its ability to

form biofilms. Complementation of srv in trans restores biofilm formation. An

average A600 (extrapolation unnecessary) for wells containing THY alone is

provided as a negative control. (b) The kinetics of GAS biofilm formation. The

A600e values of solubilized CV from surface-attached cells grown in six-well

polystyrene plates were assayed over time.

21

22

Fig. 3. Casein agar assay for SpeB proteolytic activity. MGAS5005, Δsrv, and

MGAS5005Δsrv (pIAβ8-srv) were stab inoculated into agar plates containing skim

milk and incubated for 18 h microaerophilically. Proteinase activity is manifest as

a zone of translucence surrounding the stab sites. There is a marked increase in

the amount of SpeB proteolysis exhibited by Δsrv compared to MGAS5005 or

MGAS5005Δsrv (pIAβ8-srv).

23

Figure 4. Inhibition of SpeB activity restores GAS biofilm formation. (a)

Graphical representation of crystal violet assays of biofilm-forming ability in the

presence and absence of the cysteine protease inhibitor E64. While addition of

E64 may reduce the biofilm capacity of the wild-type slightly, biofilm formation of

the Δsrv strain is restored to comparable levels. E64 did not alter biofilm formation

by the ΔspeB isogenic mutant strain, suggesting the alteration in biofilm phenotype

is due to SpeB activity. (b) Single-well images of CV assays prior to solubilization

in ethanol. After image capture, the CV from surface-attached cells was

solubilized and the A600e values are shown. Note the increase in stained material

in the Δsrv well in the presence of E64.

24

immunoblot analysis. Abundant SpeB was detected in the sample from the biofilm

deficient MGAS5005∆srv (Fig. 5). However, SpeB was undetectable in

MGAS5005 samples, and only trace amounts of SpeB-zymogen (inactive) were

detected in MGAS5005∆srv(pIAß8-srv) samples (Fig. 5). As a negative control,

samples from MGAS5005∆speB were probed (Fig. 5) (79).

Analysis of GAS biofilm formation using continuous flow.

Research has indicated that the growth and structures of bacterial biofilms are

affected by hydrodynamic shear that may be generated by the flow of fluids over

epithelial surfaces in the host (75, 109, 134). While our data indicate that GAS is

capable of forming dense, surface-attached communities under static conditions,

we sought to determine the ability of GAS to form biofilms under continuous flow.

The convertible flow-cell chamber was inoculated with 10 ml of either the wild-

type, srv mutant, or complemented strain harvested at OD600 0.5, and allowed to

attach for 3 h at 37°C. Flow of fresh THY media was then initiated at a rate of

~0.7 ml/min, and the chamber was incubated for an additional 24 h. Following 24

h of growth, the MGAS5005 and the MGAS5005∆srv(pIAß8-srv) flow chambers

were completely filled with a viscous material (Fig 6a). Plating of a sample of this

material revealed GAS and no contaminants (data not shown). As observed under

static conditions, MGAS5005∆srv was largely unable to form a biofilm (Fig. 6a).

Small pockets of attachment were observed for MGAS5005∆srv, but these

pockets were washed away when the chamber was flushed with PBS in

preparation for microscopic analyses. Taken together, this data and our analysis

25

Figure 5. Detection of SpeB in GAS biofilms. Western immunoblot analysis failed

to detect the presence of SpeB in supernatants collected from MGAS5005 and

MGAS5005∆speB following growth for 24 h in a biofilm state. Elevated levels of

SpeB were detected in supernatants collected from MGAS5005∆srv. Low levels

of the inactive form of SpeB (zymogen-SpeB) were detected in supernatants

collected from the srv-complemented strain MGAS5005∆srv (pIAβ8-srv).

26

of static biofilms over time (Fig. 2b), suggested that MGAS5005∆srv was deficient

in attachment.

To determine the viability of cells within the biofilm, the MGAS5005 within the flow-

cell was stained with a commercial reagent (LIVE/DEAD, Molecular Probes) and

visualized by confocal microscopy (Fig. 6b). By this method, living cells with intact

membranes are stained green with SYTO 9 while dead cells are stained red with

propidium iodide. Colocalization of both signals gives a yellow colour. After 24 h,

the majority of cells in the biofilm were alive. Fifty consecutive micrographs of an

X-Y plane indicated the biofilm is approximately 80 μm thick. Vertical stacked Z

images (margins of Fig. 6b) showed living cells dispersed throughout the

macroscopic structure.

Electron microscopy revealed a densely packed population of MGAS5005 that

appear encased in an extracellular matrix (Fig. 6c-d). An angled image of the

biofilm removed from the corner of the flow chamber revealed the three-

dimensional nature of the structure (Fig. 6c). Higher magnification allowed the

visualization of chains of bacteria which appear coated in matrix (Fig. 6d). Fibrous

strands could be seen running within the matrix (Fig. 6d, white arrows). Some of

these appear to be sheared, probably during the fixation process.

27

Figure 6. Continuous-flow system for study of GAS biofilm formation. (a)

Inactivation of srv eliminates the ability of GAS to form biofilms under continuous-

flow conditions. Complementation of srv in trans (MGAS5005∆srv (pIAβ8-srv))

restores the biofilm phenotype. (b) Confocal microscopy of 24 h continuous-flow

biofilm. Image is an X-Y micrograph of MGAS5005 biofilm stained with

LIVE/DEAD commercial reagent. Cells with intact membranes (living) are stained

green while cells with disrupted membranes are stained red. Areas of overlap

appear yellow. Fifty X-Y plane micrographs indicate the biofilm is approximately

80 4m thick. Vertical stacked Z images (margins) show living cells dispersed

throughout the macroscopic structure. (c, d) Scanning electron microscopy of a 24

h continuous-flow biofilm; (c) shows the thickness of the biofilm formed within the

chamber. At high magnification (d), chains of GAS can be clearly seen. Note the

matrix material which appears to cover the bacterial chains. White arrows indicate

the presence of fibrous strands.

28

DISCUSSION

In a recent study, Baldassarri et al. (2006) examined a total of 289 GAS isolates

from different clinical sources and demonstrated that 90% of those strains were

capable of forming biofilms (7). In this investigation, we sought to learn more

about the make-up of these structures and gain insight into biofilm regulation.

Enzymatic treatment of GAS biofilms demonstrated a requirement for DNA and

protein in the structure, but suggested only a passive role for carbohydrate. This

is in contrast to numerous systems, Pseudomonas aeruginosa for example, for

which several genetically encoded polysaccharides have been shown to be

required (39, 41, 52, 122). Cho & Caparon (2005) found that the GAS hyaluronic

acid capsule (HasA) was not required for biofilm formation in a static system (CV

assay), but that following initial attachment, the hasA mutant was unable to

aggregate under flow conditions (23). Given the volume of enzyme that would be

required, we were unable to test inhibition or disruption under flow conditions.

Thus, hyaluronic acid does not appear necessary for initial biofilm attachment, but

may be needed for aggregation and biofilm maturation. Alternatively, differences

between the serotype M1 strain used here and the Cho M5 strain may account for

the observed differences. Further assessment in vivo will address these

possibilities.

Increasing evidence suggests that not only is DNA a major component of bacterial

biofilms, but that genetically encoded systems controlling programmed cell death

29

provide a mechanism for the release of bacterial DNA (reviewed in (8)).

Therefore, it is not surprising that we demonstrated a role for DNA in GAS biofilms.

However, the process by which GAS releases DNA is not yet known. GAS does

not appear to encode cid/lrg homologs which control programmed cell death in

other bacterial species, although the presence of additional or diverged genes

encoding holins cannot be ruled out.

DNA, or more appropriately the lack of extracellular DNA, has been tied to severe

GAS infection. Evidence has been presented that MGAS5005 has a mutation

within the GAS control of virulence operon (covRS/csrRS) which limits or

abolishes speB expression. This is hypothesized to allow the production of a GAS

extracellular DNase (Sda1), a protein that would normally be degraded by SpeB

(18, 136, 147). According to this model, Sda1 facilitates the escape from

neutrophil extracellular traps (NETs) by degrading the associated neutrophil DNA

(14, 147). The infecting strain is then free to disseminate and potentially cause

severe invasive disease (136, 147). Our data indicate that DNA is required for

MGAS5005 biofilm formation in vitro. This suggests that production of Sda1 is not

solely dependent on the absence of SpeB, but that it requires a signal

encountered in vivo. This idea is in concert with the data of Sumby et al., (2006),

who found naturally occurring examples of the covS mutation following passage in

a mouse model (136).

30

It is of note that our data also indicate that inactivation of srv in MGAS5005

compensates for the covS mutation and restores SpeB production in this strain. In

addition, our data indicate that there is a necessary protein component for initial

adherence and/or aggregation leading to biofilm maturation. We hypothesized

that the high levels of SpeB may be responsible for the biofilm- deficient

phenotype of the srv mutant. Addition of the cysteine protease inhibitor E64

restored the ability of MGAS5005∆srv to form biofilms suggesting SpeB degrades

GAS proteins needed for establishment of the biofilm (E64-treated

MGAS5005ΔspeB produced wild-type levels of biofilm). This leads to the

hypothesis that the timed production of SpeB might contribute to GAS biofilm

dispersion. Under this model, GAS begins in a biofilm state. In reaction to an

unknown stimulus, an alteration in Srv-mediated control occurs and results in the

direct or indirect increase of SpeB production and/or secretion. SpeB degrades

GAS and host proteins integral to the biofilm. This alone may disperse the biofilm

or, as put forth by others, the increased activity of and resulting damage from

SpeB is perceived as a signal (in addition to host signals) which drives selection

for mutations in covS (136, 147). Inactive CovS leads to the repression of speB,

induction of sdaI, and the degradation of host and bacterial DNA (which may be

more accessible due to the prior cleavage of proteins by SpeB). GAS is now free

to disperse and potentially adopt an invasive phenotype (136, 147). While this

model is preliminary, we believe it is testable. Clearly, further study of GAS biofilm

formation will provide new insights into the pathogenesis of GAS.

31

ACKNOWLEDGEMENTS

This project was supported by Public Health Service Grant R01AI063453 from the

National Institutes of Health to S.D.R. We thank D.J. Wozniak for critical reading

of the manuscript as well as Robert C. Holder for technical assistance.

32

CHAPTER III

ALLELIC REPLACEMENT OF THE STREPTOCOCCAL CYSTEINE PROTEASE

SpeB IN A Δsrv MUTANT BACKGROUND RESTORES BIOFILM FORMATION

A. L. ROBERTS, R. C. HOLDER, AND S. D. REID

Department of Microbiology and Immunology

Wake Forest University School of Medicine

The following manuscript was published in BMC Research Notes, Volume 3, page

281, November 2010 and is reprinted with permission from the BioMed Central Ltd

Journal Department. Stylistic variations are due to the requirements for

publication in the journal. The experimentation, writing, and interpretation in this

paper were performed primarily by Amity L. Roberts. RCH assisted designed and

created the MGAS5005ΔsrvΔspeB mutant. Dr. Sean Reid acted in an advisory

and editorial capacity.

33

Abstract

Background. Group A Streptococcus (67) is a Gram-positive human pathogen

that is capable of causing a wide spectrum of human disease. Thus, the organism

has evolved to colonize a number of physiologically distinct host sites. One such

mechanism to aid colonization is the formation of a biofilm. We have recently

shown that inactivation of the streptococcal regulator of virulence (Srv), results in a

mutant strain exhibiting a significant reduction in biofilm formation. Unlike the

parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is

constitutively produced by the srv mutant (MGAS5005Δsrv) suggesting Srv

contributes to the control of SpeB production. Given that SpeB is a potent

protease, we hypothesized that the biofilm deficient phenotype of the srv mutant

was due to the constitutive production of SpeB. In support of this hypothesis, we

have previously demonstrated that treating cultures with E64, a commercially

available chemical inhibitor of cysteine proteases, restored the ability of

MGAS5005Δsrv to form biofilms. Still, it was unclear if the loss of biofilm formation

by MGAS5005Δsrv was due only to the constitutive production of SpeB or to other

changes inherent in the srv mutant strain. To address this question, we

constructed a ΔsrvΔspeB double mutant through allelic replacement

(MGAS5005ΔsrvΔspeB) and tested its ability to form biofilms in vitro.

Findings. Allelic replacement of speB in the srv mutant background restored the

ability of this strain to form biofilms under static and continuous flow conditions.

Furthermore, addition of purified SpeB to actively growing wild-type cultures

significantly inhibited biofilm formation.

34

Conclusions. The constitutive production of SpeB by the srv mutant strain is

responsible for the significant reduction of biofilm formation previously observed.

The double mutant supports a model by which Srv contributes to biofilm formation

and/or dispersal through regulation of speB/SpeB.

35

Findings

Group A Streptococcus (GAS) is a Gram-positive human pathogen that is

capable of causing a wide spectrum of human disease (13, 32, 36). Thus, the

organism has evolved to colonize a number of physiologically distinct host sites.

One such mechanism to aid colonization is the formation of a biofilm (29, 30, 42).

As put forth by Donlan and Costerton, a biofilm is a community of bacteria

encased in an extracellular matrix (41). The structure of this matrix may differ by

bacterial species but evidence suggests the biofilm provides protection against the

innate host response and therapeutic agents (31, 48, 77, 128). In a study of the

biofilm forming ability of 219 clinical GAS isolates representing 32 serotypes, we

observed considerable strain to strain variation in biofilm formation based on a

crystal violet staining assay (unpublished). This variation has also been observed

by others (7). In our study, one strain named MGAS5005 formed amongst the

largest biofilms we observed (38). MGAS5005 is representative of a M1T1 clone

that is globally disseminated and a leading cause of invasive infections world-wide

(92, 113, 136). This strain has been shown to have a mutation in the histidine

kinase encoded by covS, part of the two component regulatory system CovRS

(CsrRS) (135). This mutation results in CovR repression of the cysteine protease

speB (54, 143). Repression of SpeB is thought to contribute to the invasive

phenotype of this clone (58, 135, 147). We have recently shown that inactivation

of the streptococcal regulator of virulence (Srv), a proposed transcriptional

regulator with homology to the Listeria monocytogenes regulator PrfA, results in a

36

mutant strain exhibiting a significant reduction in biofilm formation (38, 115).

Unlike in the wild-type parental strain, the streptococcal cysteine protease (SpeB)

is constitutively produced by the srv mutant suggesting Srv contributes to the

control of SpeB production (111). SpeB is capable of cleaving both host

(vitronectin, fibronectin, C3b) and self (M-protein, C5a peptidase, Fba, Sda1)

extracellular proteins (9, 62, 66, 69, 110, 142, 147, 151). Previous studies have

shown that SpeB production leads to localized tissue damage and dissemination

from the sight of infection in several murine models (73, 79, 83, 139). Given these

previous observations, we hypothesized that the biofilm deficient phenotype of the

srv mutant was due to the constitutive production of SpeB. In support of this

hypothesis, we demonstrated that treating cultures with E64, a commercially

available chemical inhibitor of cysteine proteases, restored the ability of the srv

mutant to form biofilms (38). Furthermore, mature SpeB was undetected in wild-

type in vitro biofilms by western immunoblot analysis (38). Still, it was unclear if

the loss of biofilm formation by MGAS5005Δsrv was due only to the constitutive

production of SpeB or to other changes inherent in the srv mutant strain. To

address this question, we constructed a ΔsrvΔspeB double mutant through allelic

replacement (Figure 1). If our hypothesis is correct, biofilm formation would be

restored in the MGAS5005ΔsrvΔspeB strain. Furthermore, one would expect that

the addition of exogenous SpeB to a growing culture of the wild-type strain would

significantly decrease biofilm formation.

The sequence located upstream of the speB ORF was amplified from

MGAS5005 genomic DNA using speBsrv UP FWD (Table 1) and speBsrv UP REV

37

(Table 1), generating an ~1.1kb DNA fragment. The fragment was cloned into the

BsrGI-XhoI site of pFW14 (82, 108, 115), forming plasmid pFW14ΔspeB-UP.

Sequence located downstream of the speB ORF was amplified from MGAS5005

genomic DNA using speBsrv DOWN FWD (Table 1) and speBsrv DOWN REV

(Table 1), generating an ~1.1 kb DNA fragment. The fragment was cloned into the

XmaI-AgeI site of pFW14ΔspeB-UP. The resulting plasmid (pFW14ΔspeB) was

transformed into NovaBlue competent cells (Novagen). Electrocompetent

MGAS5005Δsrv cells (200 μL) were incubated with pFW14ΔspeB (2 μg, 10 μL) for

10 minutes on ice. The competent cells and DNA were placed in a pre-chilled 0.2

cm cuvette and electroporated (2.5 kV, 25 μF, 200 Ω). Electroporated cells were

incubated for 10 minutes on ice. Cells were allowed to outgrow at 37°C with 5%

CO2 for 3.5 h in Todd Hewitt broth supplemented with 2% Yeast extract (THY)

(Becton, Dickson, Company). Selection for MGAS5005ΔsrvΔspeB occurred on

THY agar supplemented with chloramphenicol (5 μg/mL) (Sigma) and incubated at

37°C with 5% CO2 for 48 hours. The speB deletion was verified in

chloramphenicol resistant transformants using PCR and restriction digestion. A

PCR utilizing internal srv and internal speB primers (Table 1) was performed on

genomic DNA of MGAS5005 wild-type (I), MGAS5005Δsrv (II), MGAS5005ΔspeB

(III) and MGAS5005ΔsrvΔspeB (IV) (Figure 1B) to validate deletion of either srv or

speB or both within the indicated mutants.

To verify that speB mRNA was not produced by MGAS5005ΔsrvΔspeB,

total RNA was isolated from MGAS5005 (control) and MGAS5005ΔsrvΔspeB and

38

Figure 1. Construction of MGAS5005ΔsrvΔspeB.

(A) speB flanking sequences were cloned upstream and downstream of the

chloramphenicol resistance cassette cat (Cmr) in pFW14. The resulting plasmid

was transformed into MGAS5005Δsrv, and allelic replacement yielded

MGAS5005ΔsrvΔspeB. (B) PCR of (I) MGAS5005, (II) MGAS5005Δsrv, (III)

MGAS5005ΔspeB and (IV) MGAS5005ΔsrvΔspeB using primers srv internal

FWD/REV (347 bp fragment) and internal speB FWD/REV (80 bp fragment) to

verify deletion of the genes srv and speB within the MGAS5005 mutants. Ladder

(L) is a 1 kB ladder.

39

40

Table 1. Primers and probes used in this study

Primer or probe

Sequence

speB internal FWD speB internal REV internal srv FWD internal srv REV prsA 309AA FWD prsA 309AA REV prsA 309AA Probe speBsrv UP FWD speBsrv UP REV speBsrv DOWN FWD speBsrv DOWN REV gyrA FWD gyrA REV gyrA Probe

5’-TCAACATGCAGCTACAGGATGTG-3’ 5’-TCAACCCTTTGTTAGGGTAATTATGATA-3’ 5’-GCATTGTGAAACAGAGTGTTCTTTCAAAATATGG-3’ 5’-TAGTTCTTCGCCAAATAGGGTCATTAAGTC-3’ 5’-GCGACAGTCGTGACCTTATCAG-3’ 5’-CTGACAGTGATGGTGTCTCCTTTC-3 5’-CATCACACAACAACACCAAACTCGTC-3’ 5’ ATATATATTGTACACGATAATAGGTTTGCCTAGTGAG-3’ 5’-ATATATATCTCGAGGCTAAAAGACTTAATAATCTGACACC-3’ 5’-ATATATATCCCGGGCAGTATACTACCAAGGTGTCGG-3’ 5’-ATATATATACCGGTCGCCAGCGTTACCACTC-3’ 5’-CGACTTGTCTGAACGCCAAA-3’ 5’-TTATCACGTTCCAAACCAGTCAA-3’ 5’-CGACGCAAACGCATATCCAAAATAGCTTGE-3’

41

subjected to TaqMan real-time reverse transcriptase PCR (RT-PCR) analysis (22,

112). Results indicated that transcript was not produced for either srv or speB

(data not shown) in the MGAS5005ΔsrvΔspeB strain. Transcript of prsA, a gene

located immediately downstream of speB, was ~ 3 fold higher in

MGAS5005ΔsrvΔspeB than MGAS5005, indicating that transcription of

downstream genes was not disrupted. It should be noted that MGAS5005Δsrv

(115) and MGAS5005ΔspeB have previously been shown to be free of detectable

polar effects (79, 81, 83). Also, Srv and SpeB have both been shown to be

produced by MGAS5005 (111, 115).

To examine biofilm formation, MGAS5005, MGAS5005Δsrv,

MGAS5005ΔspeB (79, 81, 83) and MGAS5005ΔsrvΔspeB cultures were grown

under static conditions (0.5 h - 48 h); biofilm production was measured through

crystal violet (CV) staining as previously described (38) (Figure 2). Inactivation of

speB in the srv mutant background restored biofilm formation to near wild-type

levels after 24 h (Figure 2A). Inactivation of speB in the MGAS5005 wild-type

background does not alter biofilm formation (Figure 2A). MGAS5005ΔsrvΔspeB

formed significantly more biofilm than that of MGAS5005Δsrv (P < 0.001, unpaired

student’s t-test). Over time, biofilm formation of MGAS5005ΔsrvΔspeB closely

resembled what we have previously reported for MGAS5005 with maximal

formation occurring between 24 h and 30 h with a subtle decline in CV staining

thereafter (Figure 2B) (38). Planktonic growth of MGAS5005, MGAS5005Δsrv,

MGAS5005ΔspeB, and MGAS5005ΔsrvΔspeB indicated that there was no growth

defect of the mutant strains compared to the wild-type as equivalent bacterial

42

loads were recovered over time (e.g. AVG 8.32 ± 0.72 Log10 CFU/mL 7 h post-

growth initiation).

Studies have shown that hydrodynamic shear forces are often needed for

biofilm formation as these conditions are comparable to that of the host

environment (75, 109, 134). MGAS5005Δsrv was unable to form a biofilm under

continuous flow conditions (38). To verify that the restored biofilm phenotype

observed for MGAS5005ΔsrvΔspeB was maintained under continuous flow,

MGAS5005ΔsrvΔspeB was grown in a flow cell chamber under a flow rate of ~ 0.7

mL/min for 24 h as previously described (38). After 24 h, the flow chamber was

completely filled with a viscous substance (Figure 3A) comparable to flow chamber

grown wild-type MGAS5005 (Figure 3B). Once again, MGAS5005Δsrv failed to

attach and form a biofilm under these conditions (Figure 3C). Electron microscopy

revealed a dense population of MGAS5005ΔsrvΔspeB organized in a three-

dimensional structure (Figure 3E-G). Visually, this structure is equivalent to the

MGAS5005 biofilms we have observed (Figure 3D) (38). Higher magnification

revealed chains of MGAS5005ΔsrvΔspeB (Figure 3G) which appeared to be

coated in a matrix material comparable to what has been seen in MGAS5005

biofilms (Figure 3D) (38). Thus, MGAS5005ΔsrvΔspeB can form a biofilm under

continuous flow conditions. To prove that SpeB alone is capable of disrupting GAS

biofilm formation, we added purified active SpeB (Toxin Technology, Inc.,

Sarasota, FL)(final concentration 1 μg/mL) 3 times over the course of static biofilm

development (0, 6 h, and 12 h). CV staining was performed on treated and

untreated samples at 18 h post-seeding (Figure 4). SpeB addition resulted in a

43

Figure 2. Static crystal violet assays for the measurement of in vitro biofilm

formation. MGAS5005, MGAS5005Δsrv, MGAS5005ΔspeB and

MGAS5005ΔsrvΔspeB were grown in 6-well tissue culture treated polystyrene

plates for 24 h (A), stained with crystal violet, and solubilized with ethanol. Each

reported value for the CV assay is an average of at least 6 replicates and is

adjusted by the dilution factor required to obtain a spectrophometric reading

(A600nm) (P < 0.0001, unpaired t-test). (B) Biofilm formation for each strain over

time is shown out to 48 h.

44

45

significant decrease in measurable biofilm of all treated strains to levels

comparable to MGAS5005Δsrv (Figure 4).

Taken together, the data indicate that the biofilm deficient phenotype

of MGAS5005Δsrv is due to the constitutive production of mature SpeB.

Inactivation of speB in the MGAS5005Δsrv background restored biofilm formation

to wild-type levels. Complementation of MGAS5005ΔsrvΔspeB through the

addition of exogenous SpeB significantly reduced biofilm formation to

MGAS5005Δsrv levels. These results support a model in which the Srv mediated

control of SpeB production regulates GAS biofilm formation (Figure 5). Following

initial exposure and attachment, our model would predict Srv-based negative

regulation of SpeB production. This state would allow biofilm formation and

colonization. Likewise, an opposite state would be predicted in which SpeB

production is upregulated allowing biofilm dispersal and

dissemination/transmission of GAS. We hypothesize an equilibrium exists

between these two states such that controlled levels of SpeB may be produced to

facilitate transmission while preventing complete biofilm disruption. For clarity, it is

important to point out that our work was done in the MGAS5005 background, a

background which contains a mutation in covS, which has been shown to be

involved in invasive disease and is characterized by an invasive transcriptome

profile (92, 136). Recently, Hollands et al. have shown in a separate M1T1 strain

(5448) that mutation of covS (obtained following passage through an animal

model) resulted in a strain with decreased biofilm formation due to increased

capsule production (58). They show that 5448 formed more biofilm than the 5448

46

Figure 3. MGAS5005ΔsrvΔspeB biofilm formation under continuous flow

conditions.

(A-C) Representative flow cell chambers containing 24 h grown cultures under a

flow rate of ~ 0.7 mL/min of MGAS5005ΔsrvΔspeB, MGAS5005, and

MGAS5005Δsrv, respectively. (A and B) Chambers inoculated with (A)

MGAS5005ΔsrvΔspeB or (B) MGAS5005 were filled with dense viscous material

indicative of GAS biofilms. (C) MGAS5005Δsrv was unable to form biofilms under

flow conditions. Scanning electron microscopy of a 24 h (D) MGAS5005 and (E-

G) a MGAS5005ΔsrvΔspeB continuous flow biofilm clearly depicts chains of cocci

organized into a 3-dimensional structure encased in a matrix-like material.

47

Figure 4. Addition of purified active SpeB inhibits biofilm formation.

MGAS5005, MGAS5005ΔspeB and MGAS5005ΔsrvΔspeB were either untreated

or treated with 1 μg/mL of purified SpeB (Toxin Technology, Inc., Sarasota, FL) 3

times at time 0, 6 h, and 12 h. Biofilm was measured at 18h using CV staining as

previously discussed. The level of reduction in biofilm formation was statistically

significant ((***) P < 0.0001) compared to the untreated samples.

MGAS5005Δsrv, with constitutive production of SpeB, is presented for

comparison.

48

covS mutant (58). Thus, our future work is directed at studying the effects of

mutation of srv in a covS+ M1T1 background (as well as in other serotypes) to

understand the role of Srv in biofilm formation and GAS disease.

Acknowledgements

This work was supported by Public Health Service Grant R01A1063453 from the

National Institutes of Health to SDR. We would like to thank S. Lukomski for

graciously providing us with the MGAS5005ΔspeB single mutant.

49

Figure 5. Hypothetical model of Srv/SpeB mediated GAS biofilm formation

and dispersal. Following GAS exposure, Srv-mediated negative regulation of

SpeB production would allow biofilm formation and colonization. As of yet

unidentified environmental signals may reverse this control, promoting SpeB

production and subsequent biofilm dispersal in order to facilitate

dissemination/transmission of the organism. We hypothesize that this cycle is

likely held in equilibrium such that controlled amounts of SpeB may be produced to

allow dissemination without complete disruption of the GAS biofilm.

50

CHAPTER IV

LOSS OF THE GROUP A STREPTOCOCCUS REGULATOR Srv DECREASES

BIOFILM FORMATION IN VIVO IN AN OTITIS MEDIA MODEL OF INFECTION

Amity L. Roberts1, Kristie L. Connolly1, Christopher D. Doern2, Robert C.

Holder1, and Sean D. Reid1*.

The following manuscript was published in Infection and Immunity, Volume 78,

Issue 11, pages 4800-4808, November 2010 and is reprinted with permission from

the American Society for Microbiology Journals Department (Doi: 1128/IAI.00255-

10. License # 2550860578434). Stylistic variations are due to the requirements for

publication in the journal. The experimentation, writing, and interpretation in this

paper were performed primarily by Amity L. Roberts. KLC and RCH assisted with

handling of the chinchillas and assisted in editing of this manuscript. Dr. Sean

Reid acted in an advisory and editorial capacity.

51

ABSTRACT

Group A Streptococcus (GAS) is a common causative agent of pharyngitis, but the

role of GAS in otitis media is underappreciated. In this study, we sought to test the

hypothesis that GAS colonizes the middle ear and establishes itself in localized, 3-

dimensional communities representative of biofilms. To test this hypothesis, the

middle ears of chinchillas were infected with either a strain of GAS capable of

forming biofilms in vitro (MGAS5005), or a strain deficient in biofilm formation due

to the lack of the transcriptional regulator Srv (MGAS5005Δsrv). Infection resulted

in the formation of large, macroscopic structures within the middle ears of

MGAS5005 and MGAS5005Δsrv infected animals. Plate counts, scanning

electron microscopy, LIVE/DEAD staining, and Gram-staining revealed a

difference in the distribution of MGAS5005 vs. MGAS5005Δsrv in the infected

samples. High CFUs of MGAS5005Δsrv were isolated from the middle ear

effusion and MGAS5005Δsrv was found randomly distributed throughout the

excised macroscopic structure. Conversely, MGAS5005 was found in densely

packed microcolonies indicative of biofilms within the excised material from the

middle ear. CFU levels of MGAS5005 from the effusion were significantly lower

than that of MGAS5005Δsrv early during the course of infection. Allelic

replacement of the chromosomally encoded streptococcal cysteine protease

(speB) in the MGAS5005Δsrv background restored biofilm formation in vivo.

Interestingly, our results suggest that GAS naturally forms a biofilm during otitis

52

media, but biofilm formation is not required to establish infection following

transbullar inoculation of chinchillas.

53

INTRODUCTION

Group A Streptococcus (GAS) is a Gram-positive pathogen that is capable

of causing a variety of infections in the human host. These infections vary from

mild illnesses such as pharyngitis and impetigo, to more severe diseases including

necrotizing fasciitis and streptococcal toxic shock syndrome (32, 56, 91, 123, 127).

Thus, the organism has evolved to colonize a number of physiologically distinct

host sites. The role of GAS in otitis media (OM) is often underappreciated due to

the effectiveness of beta-lactam antibiotics to eliminate GAS; however, there have

been studies showing GAS to be one of the top 4 causative agents of OM infection

and more importantly in the development of OM complications such as acute

mastoiditis (56, 102, 127). Mastoiditis is a severe bacterial infection of the mastoid

bone and air cell system often requiring surgical intervention and aggressive

antibiotic treatment (121). Various studies have reported GAS to be one of the

leading causes of severe mastoiditis (84, 121, 126), and an increase in the

number of GAS mastoiditis cases has occurred since administration of the

heptavalent pneumococcal conjugate vaccine in the years 2001 to 2005 (121). A

five year study of 11,311 acute OM episodes found that 3.1% were positive for

GAS and 4 of the 346 GAS acute OM cases developed into acute mastoiditis

(123). The mechanism by which GAS transitions from colonizing the middle ear to

colonizing the mastoid process is unclear.

One mechanism which may contribute to GAS colonization of the middle

ear is the ability to form biofilms. As hypothesized by Donlan and Costerton, a

biofilm is a bacterial sessile community encased in a matrix of extracellular

54

polymeric substances and attached to a substratum or interface (41). Biofilms are

believed to be inherently tolerant to host defenses and antibiotic therapies and

often linked to chronic illness due to impaired clearance (49, 50). There are some

estimates that suggest upwards of 60% of all bacterial infections involve biofilms

including dental caries, periodontitis, otitis media, chronic tonsillitis, endocarditis,

necrotizing fasciitis and others (34, 41).

There is a growing understanding of the importance of biofilm formation to

GAS (1, 7, 23, 28, 38, 76, 85, 88). Recently, we sought insight into how GAS

regulates biofilm formation by testing the hypothesis that the inactivation of the

streptococcal regulator of virulence (srv) would alter the GAS biofilm phenotype

(38). We found that loss of Srv resulted in a significant reduction in the ability of

an M1T1 serotype invasive clone of GAS, MGAS5005, to form biofilms under

static and continuous flow conditions in vitro (38). One of the phenotypes of the

srv mutant strain, MGAS5005∆srv, is the constitutive production of an active

cysteine protease, SpeB (37, 38, 111). Chemical inhibition of SpeB restored the

biofilm phenotype of MGAS5005∆srv to wild-type levels in vitro (38). Furthermore,

MGAS5005ΔsrvΔspeB formed biofilms at wild-type levels (Roberts, A. L.,

submitted for publication). Taken together, these data led us to hypothesize that

Srv-regulated production of SpeB may be one mechanism by which GAS can

actively disperse the biofilm.

We still have much to learn about the makeup of the GAS biofilm, how it is

regulated, and how it contributes to virulence at physiologically distinct host sites.

In this study, we sought to examine the role of GAS biofilm formation in vivo in a

55

chinchilla model of otitis media. Based on our previous findings, we hypothesized

that MGAS5005∆srv would not form biofilms in vivo. Furthermore, we

hypothesized that the lack of biofilm formation would lead to enhanced clearance

of the infection. The majority of animals inoculated with either MGAS5005 or

MGAS5005∆srv developed an infection which resulted in inflammation and was

accompanied by effusion and the formation of a dense macroscopic structure

within the middle ear chamber. Counter to our hypothesis, MGAS5005∆srv

infected animals had a significantly higher bacterial load present in the effusion

early during the course of the infection. Microscopic analysis identified

MGAS5005 present in microcolonies reminiscent of biofilms within the

macroscopic structure, while MGAS5005∆srv was found to be dispersed

throughout the infected samples. Interestingly, we found that allelic replacement

of the chromosomally encoded cysteine protease (speB) in the MGAS5005∆srv

background (MGAS5005ΔsrvΔspeB) restored biofilm formation in vivo. Taken

together, our results suggest that GAS naturally forms a biofilm during otitis media,

but biofilm formation is not required to establish infection following transbullar

inoculation of chinchillas. Furthermore, these data provide additional support for a

role for Srv in the establishment of GAS biofilms in vivo and suggest that SpeB is

involved in biofilm dispersal.

56

MATERIALS AND METHODS

Bacterial strains and growth conditions. MGAS5005, a clinical group A

Streptococcus M1T1 isolate, obtained from an invasive form of infection was

utilized in this study (92, 113). MGAS5005Δsrv, the isogenic srv mutant strain was

generated by allelic replacement (115). MGAS5005ΔsrvΔspeB was generated by

allelic replacement of speB in the MGAS5005Δsrv background (Roberts, A. L.,

submitted for publication). Cultures were grown in Todd Hewitt broth (Becton-

Dickinson) supplemented with 2% yeast extract (THY)(Fisher Scientific) at 37˚C,

5% CO2 overnight and diluted in pyrogen-free phosphate-buffered saline (PBS)

prior to infection. Serial dilutions were plated onto THY agar plates.

Chinchilla infections. Studies were approved by the Animal Care and

Use Committee of Wake Forest University Health Sciences. Healthy adult

chinchillas (Chinchilla lanigera) were purchased from Rauscher’s Chinchilla Ranch

and allowed to acclimate to the animal holding facility for 5 or 6 days prior to

infection. None of the animals had any signs of otitis media or any other overt

disease. The infectious dose was verified through bacterial plate counts.

Chinchillas were anesthetized with isoflurane and inoculated via transbullar

injection with 0.2 mL of bacterial suspension containing a total range of 5 × 104 to

2.5 × 105 CFU.

57

Otoscopy images were captured by digital imaging prior to infection and at

2, 4, and 7 days post-infection (dpi). Images were assessed for indicators of

inflammation including vessel dilation, fluid accumulation and opacity of the

tympanic membrane (61). Groups of animals were euthanized at 2, 4, or 7 dpi and

the superior bullae were opened to expose the middle ear cavity as previously

described (61). The presence of surface attached material was assessed by

visual inspection and photographed. When present, this material was removed

and analyzed as described below. A subset of the macroscopic structures were

weighed, suspended in 1 mL of sterile PBS and homogenized with FastPrep-24

lysing matrix D (MP Biomedicals) for 40 seconds, speed 6 in a FastPrep FP-120

(Thermo Electron Corp.). The homogenized suspension was serially diluted and

plated. Effusion was removed from the middle ear cavity and the middle ear cavity

was then washed with 0.5 mL of sterile PBS. Wash and effusion fluids were

combined and equalized to a total volume of 1 mL and serially diluted. Bacterial

load (CFU/ml) was determined by plate counts.

Scanning electron microscopy (84). The excised surface attached

communities were fixed for 1 h with 2.5% glutaraldehyde-PBS and then rinsed

twice (10 min/wash) in PBS prior to dehydration in a graded ethanol series. The

samples were then subjected to critical point drying, mounted onto stubs, and

sputter coated with palladium prior to viewing with a Philips SEM-515 scanning

electron microscope.

58

LIVE/DEAD staining. The material isolated from the middle ear was

analyzed for viability with LIVE/DEAD stain at 7 dpi (Molecular Probes). The

surface attached communities were incubated in 0.5 ml of PBS containing an

equal mixture of SYTO 9 and propidium iodide for 25 min and rinsed in PBS 3-

times. Samples were visualized using a Zeiss LSM 510 confocal laser-scanning

microscope (CLSM) and Zeiss LSM Image Browser.

Hematoxylin & eosin staining and Gram-staining. Fresh macroscopic

structures were snap frozen in OCT resin (Sakura Finetek) and stored at -80˚C.

Samples were acclimated to -20˚C, cut into 10 μm sections with a cryotome and

placed onto positively charged microscope slides (Fisher Scientific). The sections

were stored at -20˚C post-sectioning until processed with a standard hematoxylin

& eosin (H&E) protocol, utilizing Harris’s hematoxylin formula and eosin-phloxine.

Gram-stain processing was conducted utilizing the Taylor’s Brown-Brenn modified

Gram-stain. Samples were analyzed with a Nikon Eclipse TE300 Light

Microscope (Nikon) and were blind scored. Images were taken using a QImaging

Retiga-EXi camera (AES) and stored through ImageJ.

59

RESULTS

Defining the GAS biofilm. To assist in the interpretation of the results to

follow we feel it necessary to briefly discuss what may constitute a GAS biofilm.

We consider a GAS biofilm to be a 3-dimensional clustering of the bacteria into

microcolonies that are non-randomly distributed at the infected host site. While

there is undoubtedly a matrix of bacterial components and possibly host derived

components necessary for the formation of these microcolonies, the microcolonies

themselves may be present in a larger macroscopic structure that consists of

primarily host derived products such as neutrophils, macrophages, fibrin,

fibronectin, etc. For the purposes of this study, we consider the microcolony to be

evidence of GAS biofilm formation. As our understanding of the GAS biofilm

evolves, our GAS biofilm definition will expand.

GAS infection of the chinchilla middle ear resulted in inflammation,

effusion, and the formation of visible macroscopic structures. To begin to

study GAS biofilm formation in an in vivo model of OM, cohorts of chinchillas were

infected with MGAS5005 or MGAS5005Δsrv. A subset of 20 animals was

assessed by digital otoscopy prior to infection and at 2, 4, and 7 dpi. Prior to

infection, each of the animals was observed to be free of middle ear disease. At

each of the time points post-infection the majority of ears showed visible signs of

tympanic membrane and inner ear inflammation (Fig. 1). Removal of the superior

60

Figure 1. Digital otoscopy of representative MGAS5005 and MGAS5005Δsrv at

time 0 (uninfected) and 2, 4, and 7 dpi. MGAS5005 infected ears showed an

inflammatory response accompanied by inflammation by 2 dpi. Redness and

swelling remained over the course of the infection with effusion developing over the

course of the infection. By 2 dpi MGAS5005Δsrv infected ears showed signs of

redness and swelling accompanied by effusion and an air-fluid level at the tympanic

membrane. Otorrhoea was visible at 7 dpi.

61

Figure 2. GAS infection leads to the development of macroscopically visible surface

attached structures in the chinchilla middle ear. Images show representative middle

ear chambers of euthanized animals. (A) PBS mock infected middle ear, (B)

MGAS5005, and (C) MGAS5005Δsrv. Arrows indicate the visible structures for (B)

and (C).

62

TABLE 1. Frequency of macroscopic structure formation over time following

infection with GAS

No. (%) of infected ears showing formation of visible structure/total no. of

infected ears

dpia

MGAS5005

MGAS5005Δsrv

2 12/20 (60%)c 16/17 (94%)c 4 20/24 (83%) c 21/22 (95%)c 7 22/33 (66%) c 20/25 (80%)c

aDays post-infection

63

bulla revealed that this inflammation coincided with the presence of macroscopic

structures within the middle ear cavity (Fig. 2, Table 1). In, addition, the presence

of serous middle ear fluid, or effusion, was observed. In all cases, the

macroscopic structures were dense enough to permit physical removal with

forceps. Structures were removed, the effusion collected, and the middle ears

washed with PBS. The effusion and wash, referred to as the effusion hereafter,

were combined, brought up to 1 mL total volume and plated. A statistically higher

number of CFU/mL (P < 0.05) was recovered from the effusion of MGAS5005∆srv

infected ears 2 and 4 dpi (Fig. 3A). The average CFU recovered from the effusion

by 7 dpi (~ 1 × 108) was roughly equivalent for both MGAS5005 and

MGAS5005∆srv infected animals (Fig. 3A). However, more animals had cleared

the MGAS5005 from the effusion by 7 dpi (Fig. 3A). Homogenization and plating

of excised macroscopic structures revealed a trend in bacterial counts similar to

that observed for the CFU recovered from the effusion although the bacterial load

decreased over time (Fig. 3B). While not statistically significant, survival analysis

indicated a larger number of MGAS5005∆srv infected animals had succumbed to

infection (Fig. 3C).

GAS and host components are detected within macroscopic

structures. As an initial step toward understanding the composition of the

material observed, scanning electron microscopy was used to analyze the

macroscopic structures excised from the middle ear. As a comparison, we also

analyzed a 24 h in vitro MGAS5005 biofilm grown under continuous flow (38). The

64

in vitro biofilm consisted of densely packed chains of GAS, with each cell

measuring approximately 0.5 to 1.0 μm in diameter (Fig. 4A). Close examination

revealed the chains were coated with a matrix-like material of varying thickness

(Fig. 4A). In the MGAS5005 in vivo samples, we also observed three-dimensional

communities or groupings of chains of cocci that morphologically resembled GAS

microcolonies (Fig. 4B, C). These MGAS5005 communities were reminiscent of

the biofilm observed in vitro. In contrast, we were only able to identify individual

chains or pairs of cocci in the MGAS5005∆srv samples (Fig. 4D, E). We take this

as evidence of MGAS5005 biofilms embedded within the macroscopic structures.

Viability staining revealed a difference in the distribution of

MGAS5005 and MGAS5005Δsrv. To further examine the distribution of GAS

within the macroscopic structures, unfixed material was recovered 7 dpi and

stained with the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes). In

this method, live cells with intact membranes were stained green with SYTO 9,

while dead cells, nuclei, and DNA were stained red with propidium iodide.

Colocalization of both signals appeared yellow. In both MGAS5005 and

MGAS5005∆srv infected samples, dead cells and associated material were

distributed throughout the field of view and stained at varying intensities (Fig. 5A,

D). However, living cells in the MGAS5005 sample were non-randomly distributed

and localized to a distinct community resembling an intact biofilm. Vertical stacked

Z-slice images (Fig. 5C, margins) revealed that this community had a three-

65

Figure 3. Increased clearance of MGAS5005, but not MGAS5005Δsrv, was

observed over the course of experimental chinchilla otitis media within effusion

samples. (A) Higher concentrations (CFU/ml) of MGAS5005Δsrv were recovered

from middle ear effusions/washes. Conversely, more middle ear effusion samples

were cleared of MGAS5005 infection over time. *P < 0.005. Statistics were

determined by using an unpaired student’s t-test. (B) Bacterial concentrations

(CFU/g) recovered from middle ear macroscopic structures. Higher

concentrations of MGAS5005Δsrv were recovered from homogenized

macroscopic structures at 2 and 4 dpi. At 7 dpi, macroscopic structures recovered

from all 8 MGAS5005 infected ears were positive for GAS. Only 2 structures

recovered from MGAS5005Δsrv infected ears were positive for GAS at 7 dpi. Of

note, 2 MGAS5005Δsrv infected animals had to be euthanized before the 7 day

time point was reached. *P < 0.05 at 4 dpi. (C) Kaplan-Meier survival curves

showing the relative rates of chinchilla mortality caused by MGAS5005 (solid line)

and MGAS5005Δsrv (dashed line). Two groups of 21 (MGAS5005) and 21

(MGAS5005Δsrv) animals received ~ 2 × 105 CFU/mL by transbullar inoculation.

While not significant (Wilcoxon; P value < 0.0755), more animals succumbed to

MGAS5005Δsrv infection. All remaining animals were euthanized at day 7.

66

67

FIG. 4. SEM reveals GAS biofilms embedded within the macroscopic structures

recovered from MGAS5005 infected middle ears. (A) GAS biofilm grown in an in

vitro continuous flow cell. Chains of cocci coated in a matrix material are visible.

(B,C) White arrows demonstrate chains of cocci that are consistent in size and

length with GAS. Note the seemingly complex matrix, 3-dimensional community

structure, and other cellular features present. Images taken at 2 dpi (B) and 7 dpi

(C). (D,E) Only isolated chains of cocci were identified in MGAS5005Δsrv infected

samples 2 (D) and 7 (E) dpi. Scale bars are 10 μm.

68

69

dimensional structure with living cells present throughout. In the MGAS5005∆srv

infected sample, a dense structure of cells was also observed (Fig. 5E), but it was

not as organized. Overlap of the two fluorescent signals indicated the structure

was composed of more dead cells than living (Fig. 5F). However, a large number

of living cells are randomly distributed throughout the field of view in the

MGAS5005∆srv infected sample (Fig. 5F). Images are representative of what

was observed.

Gram-staining revealed differences in microcolony formation between

MGAS5005 and MGAS5005Δsrv infected samples. To further investigate the

apparent difference between the structure of the MGAS5005 and MGAS5005∆srv

populations in vivo, H&E-staining and Gram-staining were used. H&E-staining

revealed abundant polymorphonuclear leukocytes (blue) and fibrin (pink) within

sections of the macroscopic material removed from the chinchilla middle ear (Fig.

6A, C, E, G). Blind analysis revealed that the degree of cellularity and fibrin

composition was approximately equivalent between the MGAS5005 and

MGAS5005∆srv samples examined. However, Gram-staining revealed densely

packed and darkly stained microcolonies of bacteria within the MGAS5005

infected sample 7 dpi (Fig. 6B, D). Such microcolonies have been interpreted as

evidence of a biofilm (29, 30, 41, 150). In comparison, Gram-stained

MGAS5005∆srv was diffused throughout the samples examined (Fig. 6F, H).

While microcolony formation was not absent, fewer and less dense microcolonies

were observed consistent with the differences observed by SEM and LIVE/DEAD

staining.

70

Figure 5. LIVE/DEAD viability staining of material recovered from the chinchilla

middle ear following GAS infection. Material was recovered 7 dpi with MGAS5005

(A-C) or MGAS5005Δsrv (D-E), and stained with a two-color fluorescence assay of

bacterial viability (LIVE/DEAD BacLight, Molecular Probes). Non-viable cells with

damaged membranes are depicted in red (A and D), and cells with intact

membranes stain green (B and E). Overlap of both signals appears yellow (C and

F). A horizontal Z-slice image (center) and vertical stacked images (margins)

show living MGAS5005 in a concentrated community suggestive of a biofilm (C).

Alternatively, MGAS5005Δsrv was found dispersed throughout (F) the

macroscopic structure depicted in Figure 2. Zeiss LSM Image Browser Software

3-dimensional distance analysis predicted the thickness of the MGAS5005 (C)

community to be ~ 680 μm and the MGAS5005Δsrv (F) community to be ~ 696

μm, as indicated by the line.

71

FIG. 6. H&E (A, C, E, G) and Gram-staining (B, D, F, H) of macroscopic

structures removed 7 dpi from the chinchilla middle ear cavity. MGAS5005 is

represented in the top two rows and MGAS5005Δsrv is represented in the bottom

two rows. H&E allows detection of host fibrin and PMN infiltration whereas the

Gram-stain allows detection of the Gram-staining phenotype, shape, and

groupings of the bacteria. 20 × magnification. Note the abundant microcolonies

present in the MGAS5005 Gram-stained samples (B, D) whereas the bacteria

appear more dispersed in the MGAS5005Δsrv Gram-stained samples (F, H).

72

73

Allelic replacement of speB in the MGAS5005Δsrv background restored

biofilm formation. Previously, we have provided evidence that constitutive

production of SpeB by MGAS5005∆srv led to a decrease in biofilm formation in

vitro (38). More recently, we have shown that allelic replacement of speB in the

MGAS5005∆srv background restores biofilm formation in vitro (Roberts, A. L.,

submitted for publication). Given the observations described above, we sought to

directly address whether loss of speB would restore the ability to form biofilms by

MGAS5005∆srv. Macroscopic structure development and effusion was observed

in the middle ears of animals infected with MGAS5005ΔsrvΔspeB (Fig. 7A).

Similar to MGAS5005 infected samples, SEM revealed the presence of 3-

dimensional MGAS5005ΔsrvΔspeB microcolonies embedded within the

macroscopic structures (Fig. 7B). Gram-staining further confirmed the presence of

MGAS5005ΔsrvΔspeB microcolonies in the infected samples (Fig. 7C). Finally,

viable MGAS5005ΔsrvΔspeB was recovered from both the effusion and

macroscopic structures (Fig. 8).

74

FIG. 7. SEM and Gram-staining reveals GAS biofilms embedded within the

macroscopic structures recovered from MGAS5005ΔsrvΔspeB infected middle

ears. (A) Middle ear chamber of a euthanized chinchilla infected with

MGAS5005ΔsrvΔspeB filled with a visible macroscopic structure (black arrow).

(B) SEM of a representative 2 dpi macroscopic structure. Notice the clustering of

chains of cocci into microcolonies (white arrow). (C) H&E (left-hand column) and

Gram-stain (right-hand column) of sections from two representative 7 dpi

macroscopic structures. Notice the presence of host fibrin and numerous Gram-

positive microcolonies (white and black arrows). 20 × magnification.

75

76

FIG. 8. MGAS5005ΔsrvΔspeB was maintained within middle ear effusion and

macroscopic structures over the course of infection. Animal euthanasia reduced

the number of samples recovered 7 dpi.

77

DISCUSSION

In this study, we sought to test the hypothesis that GAS colonizes the

middle ear in a biofilm. The chinchilla animal model has been a useful tool for

studying in vivo biofilm formation (2, 6, 41, 61, 114). Recently, the chinchilla

model was used to investigate Streptococcus pneumoniae biofilm formation in

vivo (114). In that study, middle ear effusion and inflammation characteristic of

OM were observed along with macroscopic structures that were indistinguishable

by the naked eye from the structures observed in this study (114). Similar

effusion, inflammation, and structures were also observed following infection with

Haemophilus influenzae (61). Thus, the formation of these structures appears to

often accompany the onset of otitis media in this model; however, our data

indicates that the presence of these macroscopic structures does not determine

the distribution of the infecting organism within the structure. That is the pathogen

may be aggregated into microcolonies/biofilms or may be more randomly

distributed throughout the structure and the accompanying effusion.

In the S. pneumoniae study, biofilm formation was correlated with

resistance to clearance by the host immune system (114). Given that

MGAS5005∆srv does not form biofilms in vitro (38), we hypothesized we would

see decreased biofilm formation by MGAS5005Δsrv and an increased rate of

clearance in vivo. While we did note that biofilm formation was decreased to

absent, animals infected with MGAS5005Δsrv had higher bacterial loads in middle

ear effusions out to 7 dpi, and significantly higher bacterial loads at 2 and 4 dpi in

the effusion (Fig. 3A). Furthermore, animals infected with MGAS5005Δsrv had a

78

lower rate of clearance of the organism from the effusion compared to MGAS5005

(Fig. 3A). While not statistically significant, the rate of mortality for those animals

infected with MGAS5005Δsrv was also greater (Fig. 3C).

Conversely a different phenotype was observed in the MGAS5005 infected

samples. Plate counts of the homogenized macroscopic structures revealed the

presence of MGAS5005 in all 8 of the experimentally infected ears 7 dpi (Fig. 3B).

A combination of SEM, LIVE/DEAD-, and Gram-staining revealed the presence of

3-dimensional aggregates of cells/microcolonies indicative of biofilms within the

macroscopic structures removed from the middle ears of MGAS5005 infected

samples. For GAS, evidence of biofilm formation has been shown as dense

clusters of bacteria or microcolonies in zebrafish muscle tissue (23).

Microcolonies have also been presented as evidence of GAS biofilms on human

cells in vitro (88). These microcolonies were largely absent in the analyzed

MGAS5005Δsrv infected samples. Rather, the MGAS5005Δsrv organisms were

randomly distributed throughout the macroscopic structure and dispersed into the

effusion. This is perhaps best observed in the LIVE/DEAD stained samples (Fig.

5) in which living MGAS5005 cells were clustered into a densely packed 3-

dimensional core compared to the randomly distributed viable MGAS5005Δsrv

organisms. This is not to say that some clustering of MGAS5005Δsrv was not

observed, but it was found dispersed far more frequently than MGAS5005. Thus,

the MGAS5005Δsrv structures are less adherent, less organized, and less stable.

These results do not contrast with our in vitro data of MGAS5005Δsrv (38), rather

the in vivo data suggests that host components may partially rescue a biofilm

79

deficient mutant and likely add to overall biofilm structural stability of a biofilm

proficient strain(60, 61, 114). We conclude that the increased CFUs of

MGAS5005Δsrv found in the effusion is due to the decreased biofilm phenotype

exhibited by this strain. Furthermore the increase in CFUs of MGAS5005 found in

the effusion 7 dpi may reflect a population density cue signaling dispersal into the

effusion. We are continuing to investigate this hypothesis.

In our in vitro work examining GAS biofilm formation, we presented

evidence that the significant deficiency in the ability of MGAS5005Δsrv to form

biofilms was due to the constitutive production of SpeB. SpeB has been shown to

degrade GAS proteins such as M protein and DNase (9, 94, 147), as well as host

proteins including complement, antibodies, and extracellular matrix components

(27, 45, 70, 72). Further work supports a role for SpeB in dissemination, tissue

damage, and prevention of phagocytosis by polymorpholeukocytes (73, 79). In

addition, patients suffering from severe, invasive GAS disease have decreased

antibody levels to SpeB compared to healthy counterparts, suggesting that

individuals with low anti-SpeB antibody titers may be at a higher risk for

developing severe disease (59, 89). Thus, if our hypothesis is correct, allelic

replacement of speB in the MGAS5005Δsrv background should restore biofilm

formation. SEM and Gram-staining revealed the presence of biofilms within the

macroscopic structures recovered from MGAS5005ΔsrvΔspeB infected animals

(Fig. 7B,C).

Taken together, we believe this work is significant on three counts. First,

we have demonstrated a model for the study of GAS otitis media. While the

80

chinchilla model itself is not new, we are unaware of GAS being used in this model

previously. Transbullar infection with GAS resulted in inflammation including

vessel dilation, fluid accumulation and opacity of the tympanic membrane that is

associated with otitis media and is similar to what has been described for other

organisms (2, 6, 41, 61, 114). It will be important to continue to develop this model

to determine if intranasal infection can lead to GAS OM. Alternatively, we are

currently developing this model to study the advancement of GAS OM to the more

severe mastoiditis in an effort to understand how GAS biofilms or biofilm dispersal

may be involved in this process.

Second, we have provided evidence that GAS naturally forms biofilms

during otitis media infection, and that these biofilms may contribute to persistence

within the macroscopic structures that form as a result of the disease and immune

response. However, through the MGAS5005Δsrv data, we have also

demonstrated that biofilms are not required for infection or overt disease following

transbullar inoculation. Thus, it appears that GAS can exist at the site of infection

in either a biofilm or non-biofilm state. Longer time courses of infection will be

needed to determine if the GAS biofilm does provide a resistance to overall

clearance from the middle ear, but it appears the non-biofilm state, as seen in the

MGAS5005Δsrv infected animals is more virulent based upon increased bacterial

loads in the effusion, and increased mortality.

Finally, the ability of GAS to exist in a biofilm or non-biofilm state during an

active infection strongly suggests that there is a mechanism for the dispersal of the

biofilm. Our previous in vitro work and the genetic evidence presented here

81

support one hypothetical mechanism by which Srv mediated regulation of SpeB

contributes to biofilm stability or dispersal and dissemination (38, Roberts, A. L.,

submitted for publication). During colonization and biofilm maturation, Srv

mediated control, whether direct or indirect, of speB/SpeB is tightly regulated

allowing little to no production of SpeB. Upon the sensing of some environmental

signal(s), control is relaxed, SpeB is produced, and the biofilm is dispersed

allowing for dissemination and possible disease transmission. We are continuing

to explore this hypothesis.

ACKNOWLEDGEMENTS

This work was supported by Wake Forest Venture Funds and Public Health

Service grant R01AI063453 from the National Institutes of Health to S.D.R.

We thank the Swords laboratory for expert technical assistance. We thank

Rajendar Deora, Karen Haas, Steve Richardson, and Ed Swords (Wake Forest

University School of Medicine) for helpful discussions and a detailed critique of the

manuscript. A special thanks to Dan Wozniak (Ohio State University) for his

mentorship. We thank Nancy Kock for assistance with histopathology.

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CHAPTER V

STREPTOCOCCUS PYOGENES BIOFILMS DETECTED WITHIN THE CRYPTS

OF TONSILS FROM PEDIATRIC CASES OF ADENOTONSILLAR

HYPERTROPHY OR RECURRENT S. PYOGENES INFECTION

Amity L. Roberts, Daniel J. Kirse, Adele K. Evans, Katherine A. Poehling,

Timothy R. Peters, Sean D. Reid

To be submitted for publication, submitted October 2010.

83

ABSTRACT

Objective/Hypothesis. The objective of this study was to test the hypothesis that

Streptococcus pyogenes is present in biofilm communities within tonsil tissue at

the time of tonsillectomy.

Study Design. Blinded immunofluorescence and histological evaluation of

palatine tonsils from children undergoing routine tonsillectomy

Methods. Immunofluorescent and histological methods were employed to

evaluate palatine tonsils from children after tonsillectomy for adenotonsillar

hypertrophy or recurrent Streptococcus pyogenes infections.

Results. Immunofluorescence analysis using anti-S. pyogenes antibodies was

positive in 11/30 (37%) children who had tonsillectomy for adenotonsillar

hypertrophy and in 10/30 (33%) children who had tonsillectomy for recurrent S.

pyogenes pharyngitis. Fluorescent microscopy with anti-S. pyogenes and anti-

cytokeratin 8 & 18 antibodies revealed S. pyogenes was localized to the tonsillar

reticulated crypts. Scanning electron microscopy identified 3-dimensional

communities of cocci similar in size and morphology to S. pyogenes. The

characteristics of these communities are similar to S. pyogenes biofilms from in

vivo animal models.

Conclusions. Our study provides evidence that S. pyogenes may reside in

biofilms within the tonsillar reticulated crypts of approximately one-third of children

who undergo tonsillectomy for adenotonsillar hypertrophy or recurrent S.

pyogenes infections.

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INTRODUCTION

Streptococcus pyogenes, commonly known as group A Streptococcus, is a

β-hemolytic, Gram-positive human pathogen capable of causing a wide variety of

human disease. S. pyogenes is one of the predominant causes of acute bacterial

tonsillopharyngitis (11, 12, 19, 106, 107, 141). Tonsillopharyngitis is an acute

infection of the palatine tonsils and pharynx often presenting symptomatically with

a sore throat, fever and cervical lymphadenopathy (105). Patients diagnosed with

S. pyogenes tonsillopharyngitis are prescribed antibiotic therapy to avoid the

potential development of post-infectious sequelae such as acute rheumatic fever

and acute rheumatic heart disease (11, 12, 19, 106, 107, 141).

Prevention of rheumatic fever with antibacterial therapy can be life-saving,

so it is important to identify patients with S. pyogenes pharyngitis. Because

accurate clinical differentiation between viral and S. pyogenes pharyngitis is not

possible, laboratory confirmation of S. pyogenes pharyngitis is recommended for

children (98). A common clinical problem occurs when patients frequently present

with episodes of acute viral pharyngitis, but S. pyogenes is repeatedly detected by

throat culture or antigen detection methods because some of these children may

be chronic carriers of S. pyogenes. Approximately 20% of school-age children are

estimated to be chronic carriers of S. pyogenes, defined as prolonged persistence

of S. pyogenes without evidence of infection or an immune response (68).

Although chronic carriage is well known and widespread, it is poorly understood

and its clinical relevance is unclear.

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Antibacterial therapy sufficient to treat S. pyogenes pharyngitis and prevent

acute rheumatic fever is not effective in eradicating S. pyogenes carriage (33, 67).

There are a number of hypotheses proposed to explain chronic S. pyogenes

carriage. 1) Intracellular survival of S. pyogenes in tonsillar epilthelium has been

reported (96, 97). 2) Non-S. pyogenes organisms present in the pharynx that

produce beta-lactamases may confer antibacterial resistance to otherwise

susceptible S. pyogenes by proximity. 3) Carriage may occur due to an absence

of normal oral flora that inhibit S. pyogenes (140).

We have shown that S. pyogenes forms biofilms in vitro and in vivo

(38)[authors’ unpublished data]. As put forth by Donlan and Costerton, a biofilm is

a bacterial sessile community encased in a matrix of extracellular polymeric

substances and attached to a substratum or interface (41). Biofilms are inherently

tolerant to host defenses and antibiotic therapies and often involved in chronic or

recurrent illness due to impaired clearance (48, 50). It is estimated that upwards

of 60% of all bacterial infections involve biofilms including dental caries,

periodontitis, otitis media, chronic tonsillitis, endocarditis, necrotizing fasciitis and

others (39, 41, 48). Recently, bacterial biofilms have been shown on the tonsillar

surface although the colonizing organism(s) has not been identified (63).

We sought to test the hypothesis that S. pyogenes biofilms are present on

pediatric tonsil samples after tonsillectomy. This study involved examination of the

tonsillar reticulated crypt epithelium, which is a branching network throughout the

palatine tonsil that increases surface area and functions to allow more efficient

antigen sampling (25, 93, 100). We used immunofluorescence to demonstrate the

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presence of S. pyogenes biofilms within the reticulated crypts of tonsils recovered

from pediatric patients undergoing tonsillectomy for recurrent S. pyogenes

infection or adenotonsillar hypertrophy (ATH). Scanning electron microscopy (84)

and Gram-staining confirmed the presence of biofilms on the surface of and within

tonsils recovered from both pediatric populations (recurrent S. pyogenes infection

and ATH).

MATERIALS AND METHODS

This study was approved by the Wake Forest University Health Sciences

Institutional Review Board. We analyzed specimens of tonsils from children 2-18

years of age undergoing tonsillectomy for management of either adenotonsillar

hypertrophy (ATH) or recurrent S. pyogenes infections in 2009-2010. Upon

removal, tonsils were placed in sterile PBS and kept at 4˚C until processing. One

tonsil per child was prepared for immunofluorescence staining and three

immunofluorescence-positive samples underwent scanning electron microscopy

and tissue Gram-staining. Clinical information without personal identifiers was

collected on a standardized form.

Immunofluorescence.

Processing. One palatine tonsil per child was fresh frozen in OCT resin

(Sakura Finetek, Torrance, CA) within a peel-a-way disposable plastic tissue

embedding mold (Polysciences, Inc., Warrington, PA) and stored at -80˚C.

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Samples were acclimated to -20˚C, cut into 10 μm sections with a cryotome,

placed onto positively charged microscope slides (Fisher Scientific, Fair Lawn,

NJ), and stored at -20˚C until immunofluorescence staining. For

immunofluorescence staining, the slides were brought to room temperature, briefly

fixed with 4% paraformaldehyde-PBS (PFMA)(Sigma-Aldrich, St. Louis, MO), and

blocked for 30 min with 1% bovine serum albumin (BSA)(Amresco, Solon, OH) to

control for non-specific antibody staining prior to addition of primary antibodies at a

1 to 500 dilution.

Group A Streptococcus. Individual sections were stained with primary

rabbit anti-Streptococcus group A IgG (anti-S. pyogenes) (US Biological,

Swampscott, MA) in 1% BSA-PBS for 30 min in a 37˚C incubator. The S.

pyogenes antibody is derived from the cell wall of S. pyogenes and did not cross-

react with group B Streptococcus, viridans group Streptococcus nor Streptococcus

pneumoniae (Tigr4).

Tonsillar crypt epithelial. Cytokeratin 8 & 18 are co-expressed

specifically by tonsillar crypt epithelial cells (25, 99). Individual sections were

stained with primary mouse monoclonal anti-Cytokeratin 8 and Cytokeratin 18

(Thermo Scientific, Fremont, CA) in 1% BSA-PBS for 30 min in a 37˚C incubator.

Analysis. Individual sections were concurrently stained with antibody for S.

pyogenes and for cytokeratin 8 & 18 to identify the presence of S. pyogenes and

determine if it was localized to the tonsillar crypt epithelial cells. To control for

autofluorescence and non-specific antibody staining, adjacent slides were either

left unprobed with antibody or were probed with a rabbit anti-Borrelia burgdorferi

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IgG, (US Biological, Swampscott, MA) to control for IgG cross-reactivity and non-

specific binding. As a positive control, slides of S. pyogenes in vivo biofilm

sections collected from infected animals were stained with rabbit anti-S. pyogenes.

Secondary antibodies ((goat anti-rabbit IgG-Alexa 568) (Invitrogen Molecular

Probes, Eugene, OR) and goat anti-mouse-IgG-Alexa 488 (Invitrogen Molecular

Probes, Eugene, OR)) were applied and samples were incubated for 30 min in a

37˚C incubator. Samples were coated with ProLong Gold antifade reagent

(Invitrogen Molecular Probes, Eugene, OR). Specific identification of S. pyogenes

within the immunofluorescence stained tonsil material was primarily visualized

using a Nikon Eclipse TE300 Light Microscope equipped with an EXFO Xcite 120

Illumination System (Nikon Instruments Inc., Melville, NY) with a QImaging Retiga-

EXi camera (AES, Perth, Australia) and Image J software.

Gram-staining.

Three tonsils that were positive by immunofluorescence analysis also

underwent analysis by Gram-staining. Adjacent slides to those positive for

immunofluorescence were Gram-stained using the Taylor’s Brown-Brenn modified

Gram-stain procedure. Samples were analyzed with a Nikon Eclipse TE300 Light

Microscope (Nikon Instruments, Inc., Melville, NY). Images were taken using a

QImaging Retiga-EXi camera (AES, Perth, Australia) and stored through Image J

software.

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Scanning electron microscopy.

A portion of three immunofluorescence-positive tonsils from each group

were fixed for 1 hour with 2.5% glutaraldehyde-PBS and then rinsed twice for 10

minutes in PBS prior to dehydration in a graded ethanol series. The samples were

then subjected to critical point drying, mounted onto stubs, and sputter coated with

palladium prior to viewing with a Philips SEM-515 scanning electron microscope

(FEI, Hillsboro, OR). As a positive example of S. pyogenes biofilm formation on

the surface of tissue, excised skin epithelium from pigs colonized with S. pyogenes

was processed and viewed as described above.

Statistical Analysis.

Categorical variables were analyzed by chi-square or Fisher’s exact tests.

A P-value of <0.05 was considered statistically significant. Stata 8.1 (Stata

Corporation, College Station, TX) was used for all analyses.

RESULTS

Characteristics of study population undergoing tonsillectomy. The children

undergoing tonsillectomy ranged in age from 2 years to 18 years with over half the

children being 5-13 years of age. The age groups, gender, and race/ethnicity of

children undergoing tonsillectomy for recurrent S. pyogenes pharyngitis and for

ATH were similar (Table I). Children undergoing tonsillectomy for recurrent S.

pyogenes pharyngitis were more likely to have had a diagnosis of streptococcal

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pharyngitis in the prior year and history of ear tube placement than those with

ATH.

Prevalence of S. pyogenes in pediatric tonsils after tonsillectomy. Overall,

21 (35%) of 60 tonsils were positive for S. pyogenes by immunofluorescence. The

proportion of tonsil samples that had S. pyogenes detected by

immunofluorescence with or without acute symptoms of streptococcal pharyngitis

was similar for children undergoing surgery for ATH (11 (37%) of 30 samples) and

for those with recurrent S. pyogenes infection (10 (33%) of 30 samples, P = 0.79).

Detection of S. pyogenes within the tonsillar crypts. We hypothesized that the

detected S. pyogenes was localized in the tonsillar crypts. Given that the

reticulated crypt epithelium expresses unique cytokeratin markers 8 & 18, we

elected to use dual staining immunofluorescence in an effort to detect the

colocalization of crypt markers with S. pyogenes (25, 99). As proof of principle,

the branching network of the crypts was readily visible following staining with anti-

CK8 and anti-CK18 antibodies (Fig. 1).

Dual staining immunofluorescence revealed that S. pyogenes (RED)

consistently localized to the tonsillar reticulated crypt epithelium (GREEN) in both

samples from recurrent S. pyogenes infection (Fig. 2. row B, C) and ATH (Fig. 3.

row B, C, D) cases. Representative images of S. pyogenes localization are shown

(Fig. 2, Fig. 3). Positive controls for the detection of S. pyogenes consisted of in

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Table I. Characteristics of study population undergoing tonsillectomy

a Characteristic

Recurrent Streptococcus

pyogenes pharyngitis

Adenotonsillar hypertrophy

bP-value

Age group 0.67

< 5 yrs 8 (27%) 11 (38%)

5-13 yrs 19 (63%) 16 (55%)

> 13 yrs 3 (10%) 2 (7%)

Gender 0.51

% Female 14 (47%) 16 (55%)

% Male 16 (53%) 13 (45%)

Race/Ethnicity 0.10

White 22 (76%) 17 (55%)

Black 5 (17%) 8 (26%)

Hispanic 1 (3%) 6 (19%)

Black and

White

1 (3%) 0 (0%)

Streptococcus pyogenes pharyngitis diagnosed within the past 12 months <0.001

Yes 30 (100%) 5 (17%)

No 0 (0%) 24 (83%)

History of ear tubes 0.04

Yes 9 (31%) 2 (7%)

No 20 (69%) 27 (93%)

a There are 1 or 2 missing values for each category. b Statistical Analysis. Categorical variables were analyzed by chi-square or Fisher’s exact tests. A P- value of <0.05 was considered statistically significant. Stata 8.1 (Stata Corporation, College Station, TX) was used for all analyses.

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Fig. 1. Fluorescent antibody staining of Cytokeratin 8 & 18 lining the tonsillar

crypts. 10 μm sections of fresh frozen tonsils were stained with anti-Cytokeratin 8

& 18 (WHITE) to visualize the tonsillar crypt epithelium. Representative images of

tonsils removed due to ATH (left panel) or recurrent S. pyogenes infection (right

panel) are shown at 4× magnification.

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vivo biofilm samples collected from a chinchilla middle ear model of S. pyogenes

infection (Fig. 2 and Fig. 3. row A)[authors’ unpublished data].

SEM detection of S. pyogenes biofilms on the tonsillar surface. SEM was

utilized to identify the presence of 3-dimensional communities of S. pyogenes on

the tonsillar surface that are indicative of biofilms. As a positive control, excised

skin epithelium from pigs was incubated in the presence of S. pyogenes for a

period of 24 h and analyzed by SEM for the presence of biofilms. As expected for

in a positive control, 3-dimensional communities of chains of S. pyogenes cocci

(biofilms) were observed on the epithelium surface (Fig. 4A, Fig. 4B). Biofilms

were not evenly distributed across the skin surface. Close inspection revealed the

presence of extracellular matrix material within the makeup of the biofilm (Fig. 4A,

Fig. 4B). SEM revealed homologous biofilm structures on the surface of tonsils

that had previously tested positive for the presence of S. pyogenes by

immunofluorescence (Fig. 4C-F). As in our positive controls, biofilms were not

evenly distributed across the tonsillar surface but relegated to folds in the tonsil

epithelium. While the size of the biofilms differed from sample to sample, the

overall morphology of the structures observed were consistent among samples

from recurrent S. pyogenes infection (Fig. 4C, Fig. 4D) and ATH (Fig. 4E, Fig. 4F).

Close inspection revealed the presence of extracellular matrix associated with the

biofilms.

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Fig. 2. Fluorescent antibody staining of S. pyogenes within the crypts of pediatric

tonsils removed due to recurrent S. pyogenes infection. 10 μm sections of fresh

frozen tonsils were stained with anti-S. pyogenes (RED) and anti-Cytokeratin 8 &

18 (GREEN). Row A is representative images of positive controls for the detection

of S. pyogenes within in vivo biofilm samples collected from a chinchilla middle ear

model of S. pyogenes infection. Row B and C positive detection of S. pyogenes

(RED) localized within the tonsillar crypts (GREEN). Representative images are

shown at 20× and 40× magnification.

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Fig. 3. Fluorescent antibody staining of S. pyogenes within the crypts of pediatric

tonsils removed due to ATH. 10 μm sections of fresh frozen tonsils were stained

with anti-S. pyogenes (RED) and anti-Cytokeratin 8 & 18 (GREEN). (A)

Representative images of positive controls for the detection of S. pyogenes within

in vivo biofilm samples collected from a chinchilla middle ear model of S.

pyogenes infection. (B, C, and D) Shows positive detection of S. pyogenes (RED)

localized within the tonsillar crypts (GREEN). Representative images are shown

at 20× and 40× magnification.

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Fig. 4. SEM showing chains of cocci organized into biofilms attached to the

tonsillar surface. As a positive control for biofilm formation, excised skin

epithelium from pigs was incubated in the presence of S. pyogenes. The positive

control revealed 3-dimensional communities of chains of S. pyogenes cocci on the

epithelium surface (A and B). Low (A) and high (B) magnification are shown. (C

and D) Representative images of a tonsil removed due to recurrent S. pyogenes

infection. Low (C) and high (D) magnification are shown. Visible chains of cocci

organized into biofilm (b) are readily visible. Note the presence of extracellular

matrix material (e) associated with the biofilm. (E and F) Representative images

of a tonsil removed due to ATH. Two regions are shown. Once again, visible

chains of cocci organized into biofilm (b) associated with extracellular matrix (e)

are seen. These tonsil samples tested positive for the presence of S. pyogenes by

immunofluorescence.

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Gram-staining was utilized to provide further evidence of Gram-positive

biofilm formation. As a positive control for biofilm detection, specimens collected

from a chinchilla model of middle ear S. pyogenes infection were Gram-stained.

Biofilms of S. pyogenes were clearly evident in addition to dispersed S. pyogenes

(Fig. 5A). Homologous biofilms consisting of Gram-positive cocci were observed

in tonsil specimens collected from children presenting with either recurrent S.

pyogenes infection (Fig. 5B) or ATH (Fig. 5C, Fig. 5D).

DISCUSSION

In the present study, we sought to test the hypothesis that S. pyogenes was

present within or on pediatric palatine tonsils as a biofilm. To achieve this, we

analyzed surgically excised tonsils from 30 pediatric patients undergoing

tonsillectomy due to recurrent S. pyogenes infection. Originally, we planned to

examine a limited number of surgically excised tonsils from patients undergoing

tonsillectomy for ATH as these patients were asymptomatic for S. pyogenes

infection, and thus we thought these samples would provide a negative control for

the detection of S. pyogenes. However, we readily detected the presence of S.

pyogenes in these samples by immunofluorescence and SEM revealed the

presence of biofilms made up of chains of cocci which morphologically resembled

S. pyogenes. Given this result, we examined the tonsils excised from a total of 30

patients presenting with ATH in addition to the tonsils excised from 30 patients

presenting with recurrent S. pyogenes infection.

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Fig. 5. Gram-stain showing the presence of Gram-positive bacteria in biofilm

within tonsil sections. (A) Gram-stain showing the positive detection of S.

pyogenes biofilm (b) in a sample taken from a chinchilla middle ear infection

model. Dispersed S. pyogenes are also visible. (B) Detection of Gram-positive

bacteria in a tonsil removed due to recurrent S. pyogenes infection. Dispersed

bacteria and bacteria organized into biofilm (b) are visible. This tonsil sample

tested positive for the presence of S. pyogenes by immunofluorescence. (C and

D) Detection of Gram-positive bacteria in tonsils removed due to ATH. Dispersed

bacteria and bacteria organized into biofilm (b) are visible. These tonsil samples

tested positive for the presence of S. pyogenes by immunofluorescence.

Representative images are shown at 20× and 40× magnification.

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We discovered that a similar proportion of tonsils from children with either

ATH or recurrent S. pyogenes infection were positive for S. pyogenes by

immunofluorescence (~ 35% positive). Of note, our method of

immunofluorescence allowed the dual detection of both S. pyogenes and the

cellular markers of the reticulated crypt epithelium, cytokeratin 8 & 18. By this

method, we were able to show that the S. pyogenes detected had colonized

throughout the tonsillar crypts in both sets of patient tonsil samples.

The finding that tonsillar tissue from children with ATH patients showed S.

pyogenes in such a high percentage was unexpected. However, bacterial carriage

by children with ATH is not unprecedented. Several previous studies of

adenotonsillar hypertrophy have provided evidence that increased numbers of

pathogenic bacteria can be recovered from homogenized hypertrophic tonsil cores

compared to swabs of those tonsils alone (15, 129). Indeed, it is proposed that

lymphoid hyperplasia (chronic enlargement) is correlated with increased bacterial

load and increased B- and T-lymphocyte proliferation (16, 20). This phenomenon

has been associated with a number of bacteria including Staphyloccocus aureus,

Haemophilus influenzae, S. pneumoniae, as well as S. pyogenes (15). However,

a review of the literature revealed that the percentage of hypertrophic tonsils

positive for S. pyogenes by Brodsky et al. (16%) and Stiernquist-Desatnik et al.

(20%) was lower than the 36.7% positive that we observed (15, 130). Our method

of detection is more sensitive (antibody based immunofluorescence vs. culture),

but this difference in frequency of detection may also be due to geographic

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location or the fact that our study occurred almost 20 years after the reports

referenced.

Our results support the hypothesis that S. pyogenes colonized pediatric

palatine tonsils as a biofilm. SEM clearly reveals the presence of 3-dimensional

communities of chains of cocci in tonsils that had tested positive for the presence

of S. pyogenes by immunofluorescence. These structures closely resemble the in

vivo S. pyogenes biofilms grown on pig epithelium. Furthermore, Gram-staining

reveals the presence of microcolonies of Gram-positive cocci indicative of biofilms

in samples that tested positive for the presence of S. pyogenes.

This study has important limitations. Detailed information regarding

antibiotic exposure prior to tonsillectomy was not collected. The finding that S.

pyogenes biofilms were detected in roughly equivalent percentages in these two

patient groups is consistent with the hypothesis that biofilms may be important in

carrier state antibacterial resistance, but this study is not designed to directly

address that hypothesis.

CONCLUSIONS

We find evidence for S. pyogenes biofilms in approximately 1/3 of children

undergoing tonsillectomy for management of either recurrent S. pyogenes

pharyngitis or ATH. Our findings support the hypothesis that biofilm formation may

be important in the persistence of S. pyogenes in the pharynx of children and that

biofilms may contribute to resistance to clearance by both the host immune system

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and antibiotic therapy. A determination of the role of biofilm formation in the S.

pyogenes carrier state will require further study.

ACKNOWLEDGEMENTS

We would like to thank Robert C. Holder, MS, for his assistance. We would like to

thank Elizabeth Palavecino, MD, of the WFUBMC Clinical Microbiology

Laboratories for supplying group B Streptococcus and viridans group

Streptococcus isolates for testing anti-S. pyogenes antibody cross-reactivity to

other Streptococcus species.

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CHAPTER VI

DISCUSSION As each of the preceding chapters include a discussion specific to each data set,

the focus of this section is to comment on how our work melds with existing GAS

research and how hypotheses are changed going forward. As such, our

discussion begins with the strain at the center of this work MGAS5005.

MGAS5005 is serotype M1T1 and representative of a clone that has spread world-

wide and is the most frequently recovered strain from cases of invasive/systemic

infection (57, 92). It has been hypothesized that the transition from mild to severe

invasive disease by M1T1 clones is due to a mutation in covS, the gene encoding

for the sensor kinase portion of the two-component signal transduction system

CovRS (5, 64, 135). Normally, CovS phosphorylates the DNA-binding cytoplasmic

protein CovR (24, 35, 44, 47, 51). Importantly, CovS has not only been shown to

activate CovR repression of target genes through phosphorylation, but also inhibit

CovR activity on certain subsets of target genes (143). For example, covS

mutants with a non-functional CovS have heightened repression of speB by CovR.

It is hypothesized that selection of the covS mutation occurs in nature to select

against SpeB production to prevent SpeB mediated degradation of GAS protein

products involved in immune evasion during systemic infection (4, 26, 64, 143,

147). Truncation of CovS leads to repression of speB, increased expression and

maintenance of sda1/Sda1 (GAS extracellular DNase) and ska (streptokinase)

among other important GAS virulence proteins (26, 65, 117, 137, 147, 148). For

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example, studies indicate that Sda1 is essential to invasive disease since it is

proposed to facilitate escape from neutrophil extracellular traps (NETs) by

degrading the associated neutrophil DNA (14, 18, 135, 147). Similarly,

streptokinase is a secreted GAS protein that activates human plasminogen

(proprotein for plasmin) leading to accumulation of plasmin (a host serine

protease) on the GAS surface and degradation of host fibrin clots, which are used

by the host to contain the organism (117, 137, 148). Studies of humanized

transgenic murine models of infection suggest that loss of SpeB activity leads to

accumulation of this surface plasmin activity thereby triggering systemic spread

(26, 148). It is proposed that GAS cocci possessing the covS mutation are then

able cause widespread systemic infection within the host (26, 117, 147). Naturally

occurring strains possessing the covS mutation have been recovered following

passage in an invasive mouse model, indicating that in vivo signals are important

to facilitate mutation of covS (3, 136, 147).

This present hypothesis concerning the arise of the M1T1 invasive clone is

inconsistent with our data on some levels. First, there have been no published

reports identifying naturally occurring covS mutations from upper respiratory tract

infections (143). However, given this association between covS mutation and a

successful invasive phenotype has only recently been reported it is unclear if a

sufficient number of upper respiratory tract isolates have been examined for the

presence of this mutation. Second, amongst the proteins that would be protected

in a SpeB null phenotype are Sda1 and Ska, a DNase and a plasmin activator,

- 108 -

respectively. Our data indicates MGAS5005 produces robust biofilms and DNA is

required for their formation. This suggests that there is still a mechanism in place

for the control of Sda1 to allow it to switch between inactive and active in a biofilm

versus systemic (planktonic) mode of growth. While it seems likely that

plasminogen might be incorporated in biofilm formation, we have not tested this or

looked at the activity of Ska in MGAS5005 biofilms.

Our data clearly show that a biofilm is not required for infection. Thus, it is likely

that GAS can switch from a biofilm mode of growth to a mode of growth that may

be more suitable for survival under systemic conditions. However, as suggested

by our tonsil study the biofilm provides a clear advantage for GAS in allowing it to

colonize tonsillar crypts asymptomatically. We hypothesize a model in which Srv

regulation of SpeB mediates GAS biofilm formation and dispersal. Under this

model, controlled amounts of SpeB could be produced over time to allow regulated

dispersal of the biofilm free planktonic cells. Once released, we hypothesize that

these planktonic cells undergo a number of regulatory changes. Srv

downregulation of SpeB would allow for the production of Sda1 and Ska through,

perhaps, another regulatory system. Thus, these cells would be best suited for

survival in the planktonic state. Upon encountering a susceptible host surface,

sda1 and ska are downregulated and biofilm formation and subsequent

colonization occur.

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Why has M1T1 spread globally? If our hypothesis is correct, the increased biofilm

phenotype exhibited by MGAS5005 would allow for increased colonization of a

higher proportion of susceptible hosts. Under this model, the increased rate of

recovery of M1T1 clones from cases of invasive disease is merely a side effect of

a larger proportion of the population being colonized by the M1T1 clone. It is

important to note that this model assumes the eventual isolation of this clone from

cases of upper respiratory tract infection.

In summary, our work has made several key observations. First, it is clear that

GAS can exist in both a biofilm or a planktonic state within the host. Furthermore,

our work suggests that while the biofilm may provide a means to persist in an

asymptomatic state the planktonic mode of growth appears to be more virulent.

This switch between biofilm and planktonic modes of growth may be regulated by

more than one mechanism. However, our data indicate that one mechanism is

Srv mediated control of speB/SpeB. Finally, our work further develops the

hypothesis of the evolution of the invasive M1T1 clone.

- 110 -

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APPENDIX

A globally disseminated serotype M1T1 isolate produced the largest biofilm

in an analysis of 219 strains.

To determine the degree of strain-to-strain variation in GAS biofilm formation, we

utilized a crystal violet assay to determine the ability of 219 strains isolated from

human patients to form biofilms (Figure 1). 218 of these strains represented 32 M

serotypes and were obtained across the country in a larger survey of 1885 isolates

from 45 U.S. medical centers (119). In total, 177 strains were isolated from the

throat, 9 from the lower respiratory tract, 8 from the blood, 4 from the middle ear,

and 20 from patients for which the site of isolation was not indicated. In addition,

we tested MGAS5005, a representative serotype M1T1 strain recently isolated

from a case of invasive GAS disease. A serotype M1T1 strain was chosen

because population genetic analysis has indicated that M1T1 strains are among

the most common causes of invasive GAS infections worldwide in most case

studies (92, 113). Each of the other M1 strains tested was isolated from a case of

non-invasive disease.

Spectrophotometric analysis of the solubilized crystal violet from each sample

revealed strain-to-strain variation in the ability to form biofilms (Figure 1). This

variation was exhibited within and between serotypes. MGAS5005 produced the

highest average biofilm (OD600 = 37.913 ± 5.813), a reading that was significantly

greater than any of the other serotype M1 strains examined. Moreover, this value

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represented a biofilm that was larger than any produced by 8 other invasive

isolates (Avg OD600 = 7.547 ± 5.667). Among the remaining strains, biofilm

formation varied considerably. At least one isolate in each of the serotypes 1, 12,

22, 28, 73, 83, and 114 were identified as weak or non-biofilm producers based on

a solubilized CV reading of ≤ 1.0 (A600). We did not find any association between

biofilm capacity and site of strain isolation. Given the biofilm formation exhibited

by MGAS5005, its biomedical relevance, and our previous work with this strain, it

was selected for further analysis.

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Figure 1. Biofilm formation of clinical GAS strains representing 32 distinct M serotypes. The number of strains (n) tested for each serotype is listed in italics. MGAS5005 is a representative serotype M1T1 strain recently isolated from a case of invasive GAS disease. M1T1 strains are among the most common causes of invasive GAS infections worldwide in most case studies. The remaining strains were isolated from cases of clinical infection from 45 medical centers in the United States. The A600 values of solubilized crystal violet from surface-attached cells grown in 6-well polystyrene plates for 24 h are presented. The values reported are averages for all of the strains in a given serotype. Error bars indicate the standard deviation for the overall average per serotype and is intended to provide an illustration of the degree of variation in biofilm formation among strains of a given serotype.

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CURRICULUM VITAE

Amity L. Roberts

ADDRESS: Department of Microbiology and Immunology Wake Forest University School of Medicine Medical Center Boulevard Winston-Salem, NC 27157 Telephone: (336) 716-7597 Email: amrobert@wfubmc.edu EDUCATION: 2010 Ph.D., Microbiology and Immunology Wake Forest University Graduate School of Arts & Sciences Winston-Salem, NC 2006 B.S. summa cum laude, Microbiology, Mississippi State University Starkville, Mississippi RESEARCH EXPERIENCE: 2006-2010 Graduate Research Student Wake Forest University School of Medicine Department of Microbiology and Immunology Advisor: Sean D. Reid, Ph.D. Dissertation title: Streptococcal colonization of host mucosal surfaces: a study of biofilm formation and dispersal. 2005-2006 Senior Thesis Research, Mississippi State University Department of Biological Sciences Major Professor: Franklin R. Champlin, Ph.D. Senior thesis title: Characterization of growth inhibition by a synergistic combination of triclosan and compound 48/80 in Psuedomonas aeruginosa.

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PUBLICATIONS: Roberts, A. L., D. J. Kirse, A. K. Evans, K. A. Poehling, T. R. Peters, S. D. Reid. 2010. Streptococcus pyogenes biofilms found in pediatric tonsillectomy specimens. Submitted. Roberts, A. L., R. C. Holder, S. D. Reid. 2010. Allelic replacement of the streptococcal cysteine protease SpeB in a Δsrv mutant background restores biofilm formation. BMC Research Notes. 3:281. Roberts, A. L., K. L. Connolly, C. D. Doern, R. C. Holder, S. D. Reid. 2010. Loss of the group A Streptococcus regulator Srv decreases biofilm formation in vivo in an otitis media model of infection. Infection and Immunity. 78(11):4800-4808. Doern†, C. D., A. L. Roberts†, W. Hong, J. Nelson, S. Lukomski, W. E. Swords, and S. D. Reid. 2009. Biofilm formation by group A Streptococcus: a role for the streptococcal regulator of virulence (Srv) and streptococcal cysteine protease (SpeB). Microbiol.155:46-62. † contributed

equally to this work

Ellison, M. L., A. L. Roberts, F. R. Champlin. 2007. Susceptibility of compound 48/80-sensitized Pseudomonas aeruginosa to the hydrophobic biocide triclosan. FEMS Microbiol Lett. 269:295-300. FORMAL PRESENTATIONS: Roberts, A. L., D. J. Kirse, A. K. Evans, K. A. Poehling, T. R. Peters, S. D. Reid. 2010. Streptococcus pyogenes biofilm communities are detected within the tonsillar crypts of tonsil from children with recurrent Streptococcus pyogenes tonsillopharyngitis and adenotonsillar hypertrophy. Oral Presentation. International Conference on Gram- Positive Pathogens. Omaha, NE, U.S.A. Kirse, D. J., A. L. Roberts, A. K. Evans, S. D. Reid. 2010. Streptococcus pyogenes biofilm communities are detected within the tonsillar crypts of pediatric patients who have undergone tonsillectomy for obstructive sleep apnea. Poster Abstract. Annual Meeting of the American Society of Pediatric Otolaryngology (ASPO), Las Vegas, NV, U.S.A. Roberts, A. L., K. L. Connolly, R. C. Holder, and S. D. Reid. 2009. Loss of biofilm formation by the group A Streptococcus leads to increased disease severity in otitis media. Poster Abstract. American Society for Microbiology Conference on Biofilms. Cancun, Mexico.

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Roberts, A. L., C. D. Doern, R. C. Holder, J. Nelson, W. Hong, W. E. Swords, and S. D. Reid. 2009. Biofilm formation by group A Streptococcus: a role for the streptococcal regulator of virulence (Srv) and streptococcal cysteine protease (SpeB). Presentation Abstract. Mid- Atlantic Microbial Pathogenesis Meeting. Wintergreen, VA, U.S.A. Roberts, A. L., C. D. Doern, R. C. Holder, J. Nelson, W. Hong, W. E. Swords, and S. D. Reid. 2008. Biofilm Formation by Group A Streptococcus: A Role for the Streptococcal Regulator of Virulence (Srv) and Streptococcal Cysteine Proteinase (SpeB). Poster Abstract. American Society for Microbiology General Meeting. Boston, MA, U.S.A. Ellison, M. L., A. L. Roberts, and F. R. Champlin. 2006. Compound 48/80 sensitization of Pseudomonas aeruginosa to low concentrations of triclosan. Abstract. American Society for Microbiology General Meeting. Orlando, FL, U.S.A. PROFESSIONAL AFFILIATIONS: 2005-present American Society for Microbiology 2005-2006 South Central Branch of the American Society for Microbiology 2007-2008 Mid-Atlantic Branch of the American Society for Microbiology 2006-2007 Phi Kappa Phi HONORS AND AWARDS: 2009 American Society for Microbiology Meeting on Biofilms. Student Travel Award. 2009 Mid-Atlantic Microbial Pathogenesis Meeting. Student Travel Award. 2005-2006 Northeast Mississippi Daily Journal Undergraduate Research Award, Northeast Mississippi Journal, Tupelo, MS. Awarded to A. L. Roberts for undergraduate senior research. 2003-2004 Drs. Karen and William Hulett Endowed Scholarship for Science Majors.

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COPYRIGHT INFORMATION

The work presented in Chapter II of this thesis was reprinted with permission from the Microbiology, 155, Doern, C.D., A. L. Roberts, W. Hong, J. Nelson, S. Lukomski, W. E. Swords, and S. D. Reid. 2009. Biofilm formation by group A Streptococcus: a role for the streptococcal regulator of virulence (Srv) and streptococcal cysteine protease (SpeB), pp. 46-52. Copyright © 2009, permission from Society for General Microbiology. The work presented in Chapter III of this thesis was reprinted with permission from BMC Research Notes, 3, Roberts, A.L., R. C. Holder, and S. D. Reid. 2010. Allelic replacement of the streptococcal cysteine protease SpeB in a deltasrv mutant background restores biofilm formation, pp. 281. Copyright © 2010, permission from BioMed Central Ltd. The work presented in Chapter IV of this thesis was reprinted with permission from Infection and Immunity, 78 (11), Roberts, A.L., K. L. Connolly, C. D. Doern, R. C. Holder, and S. D. Reid. 2010. Loss of the Group A Streptococcus Regulator Srv Decreases Biofilm Formation In Vivo in an Otitis Media Model of Infection, pp. 4800-4808. Copyright © 2010, permission from American Society for Microbiology (Doi: 1128/IAI.00255-10. License # 2550860578434).