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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: J Infect Dis. 2010 Jul 1;202(1):11–19. doi: 10.1086/653124

A Genetic Switch to Hypervirulence Reduces Colonization Phenotypes of the Globally Disseminated Group A Streptococcus M1T1 Clone

Andrew Hollands 1,2, Morgan A Pence 2, Anjuli M Timmer 2, Sarah R Osvath 4, Lynne Turnbull 4, Cynthia B Whitchurch 4, Mark J Walker 1, Victor Nizet 2,3,5
PMCID: PMC2880657  NIHMSID: NIHMS192950  PMID: 20507231

Abstract

Background

The recent resurgence of invasive group A streptococcal disease has been paralleled by the emergence of the M1T1 clone. Recently, invasive disease initiation to has been linked to mutations in the covR/S two-compnent regulator. Here we investigate if a fitness cost is associated with covS mutation that counterbalances hypervirulence.

Methods

Wild-type M1T1 GAS and an isogenic covS mutant derived from animal passage were compared for adherence to human laryngeal epithelial cells, keratinocytes or fibronectin, biofilm formation, and binding to intact mouse skin. Targeted mutagenesis of capsule expression from both strains was performed for analysis of its unique contribution to the observed phenotypes.

Results

The covS mutant bacteria showed reduced capacity to bind to epithelial cell layers as a consequence of increased capsule expression. The covS mutant strain also had reduced capacity to bind fibronectin and to form biofilms on plastic and epithelial cell layers. A defect in skin adherence of the covS mutant strain was demonstrated in a murine model.

Conclusions

Reduced colonization capacity provides a potential explanation as to why the covS mutation conferring hypervirulence has not become fixed in the globally-disseminated M1T1 GAS clone, but rather may arise anew under innate immune selection in individual patients.

Streptococcus pyogenes (group A Streptococcus, GAS) is a Gram-positive, human-specific pathogen responsible for over 500,000 deaths each year worldwide [1]. A resurgence of severe GAS disease in recent decades has been paralleled by the emergence of a globally disseminated GAS clone belonging to serotype M1T1 [2–4]. M1T1 GAS are the most common cause of streptococcal pharyngitis in developed countries, and are strongly overrepresented in cases of severe infection such as necrotizing fasciitis (NF) and toxic shock syndrome (TSS) [5–7].

An inverse relationship exists between expression of a broad-spectrum secreted cysteine protease, SpeB, and clinical disease severity in M1T1 clinical isolates [8]. Recently, mutations in the two-component regulatory system covR/S characteristic of bloodstream isolates of M1T1 GAS compared to pharyngeal isolates have been identified [9]. These mutations arise in vivo in the murine model of GAS M1T1 NF, leading to loss of SpeB expression and increased virulence [9–12]. SpeB is initially produced as a 40 kDa zymogen which is then converted to the 28 kDa active form by autocatalytic processing [13]. The role of SpeB in GAS infection is complex, multifaceted, and incompletely understood. SpeB has been shown to cleave a broad range of host proteins, including components of the extracellular matrix (ECM), cytokine precursors, immunoglobulins and antimicrobial peptides [14–16], which could promote tissue damage or impair host immune functions. However, SpeB also cleaves multiple of the bacterium’s own protein virulence factors, such as the fibrinogen-binding M1 protein [12, 17–19], various superantigens[20, 21], the secreted plasminogen activator streptokinase [22], and the DNase Sda1 [20], thus attenuating key aspects of GAS pathogenicity.

CovR/S is an important global gene regulator, responsible for regulating approximately 10% of the GAS genome [4, 9]. It has been found that CovR acts mainly as a negative regulator, even in the absence of CovS, and that CovS inactivates the function of CovR [23]. Specific point mutations in covR/S and truncation mutations of covS result in significant down-regulation of SpeB expression, but concurrent up-regulation of many virulence factor genes including those encoding Sda1, IL-8 protease SpyCEP, streptolysin O (SLO), streptococcal inhibitor of complement (SIC) and the hyaluronic acid capsule synthesis operon [9–12]. It is hypothesized that increased expression of these virulence determinants, along with sparing them from SpeB degradation, promotes the proliferation and invasive spread of the covR/S mutant [9, 10, 24]. Increased resistance to innate immune clearance, and in particular killing by neutrophils, may represent the major selection pressure favoring the covR/S mutation in vivo [10].

The routine occurrence of covR/S mutation in M1T1 GAS and the dramatically increased animal virulence following this event raise the question of why this particular mutation has not become fixed during GAS evolution. We hypothesized that a counter-balancing selection pressure acts to maintain the wild-type genotype, and thus sought to elucidate fitness cost(s) of covR/S mutation in the GAS M1T1 population.

METHODS

Bacterial strains, media and growth conditions

Well-characterized M1T1 clinical isolate 5448 and its mouse-passaged covS mutant derivative 5448AP were used [10]. Correction of the covS mutation in 5448AP restores WT phenotypes [25]. GAS strains were propagated using Todd-Hewitt broth (THB) or agar (THA). Escherichia coli were grown using Luria-Bertani broth or agar. Erythromycin (Erm) selection was used at 5 μg/ml (GAS) and 500 μg/ml (E. coli).

Growth curves and chain length assays

Overnight cultures of GAS bacteria were diluted into fresh THB and OD600 followed every 30 min for growth curves. For chain length assay, GAS were centrifuged for 5 min at 3,200 x g to create a monolayer, viewed at 10−3 dilution using a Zeiss Axiovert 100 inverted microscope, and chain length calculated using one random field of view from three separate wells. Statistical significance was determined by one way ANOVA with Tukey’s post-hoc test.

Plasmid integrational mutagenesis

An intragenic fragment of hasA was amplified using forward primer hasAint-F-BamHI (5′-gcaggatccttggaacatcaactgtagg-3′) and reverse primer hasAint-R-XbaI (5′-gcatctagattaattcaaatgtcctgttgcagc-3′), then cloned by BamHI/XbaI digestion into conditional vector pHY304. The resultant plasmid was transformed into 5448 and 5448AP by electroporation and Ermr transformants were grown at the permissive temperature for plasmid replication (30°C). Single-crossover chromosomal insertions were selected by shifting to the nonpermissive temperature (37°C), maintaining Erm selection [26]. Integrational knockouts were confirmed unambiguously by PCR, and designated 5448ΔhasA and 5448APΔhasA.

Epithelial cell adherence assays

Assays were performed using HEp-2 (human laryngeal epithelial cells) and HaCaT (human keratinocyte cells) as described [27]. Cells were plated at 2 × 105 cells/well and grown overnight at 37°C in 5% CO2. Mid-logarthmic growth phase GAS were resuspended in RPMI + 2% fetal calf serum (FCS) and added at multiplicity of infection 10:1. Plates were spun for 10 min at 500 x g and incubated for 30 min at 37°C in 5% CO2, then washed 5 times with PBS to remove non-adherent bacteria. 100 μl of trypsin was added to release cells, which were lysed with 0.02% Triton X-100. Bacteria were serially diluted and plated on THA for enumeration. Adherence was determined as a percentage of initial inoculum. By trypan blue staining, > 95% cell viability of Hep2 and HaCat cells was documented in all assays. Bacterial strains grew equally in RPMI + 2% FCS for the 30 min duration of the experiment. Significance was determined by one-way ANOVA with Tukey’s post-hoc test.

Competition adherence assays were performed identically, but adding 2 × 106 cfu/well of an equal mix of 5448 and 5448AP, or 5448ΔhasA and 5448APΔhasA, to cell monolayers. After serial dilutions were plated on THA overnight, 50 individual colonies from each condition (5448/5448AP or 5448ΔhasA/5448APΔhasA) were picked. The proportion of 5448 vs. 5448AP was determined by the proportion of SpeB positive vs. SpeB negative colonies, respectively, by published methods [28]. Assays were performed in triplicate and statistical significance determined using an unpaired T-test.

Hyaluronic acid capsule assay

Bacterial cultures were grown to mid-log phase in THB. 5 ml of OD600=0.4 culture was spun down and resuspended in 500 μl H2O. Serial dilutions of bacterial suspension were plated to confirm equivalent cfu. 400 μl of the bacterial suspension was placed in a 2 ml screw cap tube with 1 ml chloroform. Tubes were shaken for 5 min in a mini-beadbeater-8 (Biospec Products), then spun at ~13,000 x g for 10 min. Hyaluronic acid in aqueous phase was determined using a Hyaluronic Acid Test Kit (Corgenix) per manufacturer’s instructions.

Fibronectin-binding assays

Fibronectin was bound to 96-well plates (Costar) as described [29]. Bacteria were grown to mid-logarithmic phase, washed in PBS and resuspended to 2 × 108 cfu/ml. Plates were washed three times with sterile PBS, 100 μl (2 × 107 cfu) of bacterial solution added to each well, plates centrifuged at 500 x g for 10 min, incubated at 37°C for 1 h, then washed five times with PBS to remove non-adherent bacteria. Adherent bacteria were released by using 100 μl of 0.25% trypsin/1mM EDTA (Gibco) for 10 min at 37°C. Bacteria were serially diluted in PBS and plated onto THA for cfu enumeration. Adherence was determined as a percentage of the initial inoculum. Assays were performed in triplicate and significance determined using a one-way ANOVA with Tukey’s post-hoc test.

Fluorescence microscopy

Glass coverslips were coated with fibronectin by incubating overnight at 4°C in a 50 μg/ml solution in PBS. Slides were then washed with PBS and blocked overnight at 4°C with PBS + 1% BSA. Bacteria were grown to mid-log phase (OD600 = 0.4), spun down and resuspended in PBS to OD600 = 1.0. FITC was added to a final concentration of 100 μg/ml and incubated on ice for 30 min. FITC- labeled bacteria were pelleted, washed twice with sterile PBS, then resuspended to OD600 = 0.4 in PBS. 500 μl of FITC-labeled bacterial solution was added to glass coverslips in the bottom of 24-well tissue culture plates. Plates were spun for 10 min at 500 x g, then incubated for 1 h at 37°C. Coverslips were washed five times with PBS then fixed with 4% PFA overnight at 4°C, washed again and mounted on microscopes slides with ProLong Gold (Invitrogen). Slides were visualized using a DeltaVision RT Deconvolution microscope (UCSD Neuroscience Microscopy Shared Facility).

Static biofilm formation on polystyrene

Determination of biofilm formation on polystyrene was performed using a modified O’Toole and Kolter crystal violet (CV) stain assay [30]. Briefly, 8 individual wells of a black-sided, clear-bottomed 96-well microtiter plate (Greiner CELLSTAR, Cat# 655090) were inoculated with 150 μL of overnight culture diluted 1:100 in THB supplemented with 1% (w/v) yeast extract for each strain studied. Plates were sealed with Aeraseal breathable film (Excel Scientific) and incubated for 24h at 37°C. Plates were washed and cells fixed with 4% paraformaldehyde. 6 wells were stained with 0.2% CV, extracted in acetone/ethanol and CV absorbance assayed at A595nm for biofilm biomass quantification. The remaining two wells for each strain were stained with SYTO 9 nucleic acid stain (Invitrogen) and biofilms visualized using a Nikon A1 laser scanning confocal microscope (LSCM). Images were reconstructed from Z-sections and rendered for 3D visualization using NIS Elements software (Nikon). Assay was performed 4 times and statistical significance determined using a one-way ANOVA with Tukey’s post-hoc test.

Static biofilm formation on epithelial cells

Visualisation of biofilm formation by GAS strains on epithelial cells was modified from method of Manetti et al 2007 [31]. HaCaT keratinocytes were seeded in RPMI into 35 mm tissue culture dishes with a 10 mm diameter glass-coverslip insert (Fluorodish, World Precision Instruments) and cultured for 48 h. HaCaT cells were then washed and 200 μL of 1:10 dilutions of overnight cultures of GAS strains in RPMI were added to each dish. After 15 min, HaCaT cells were washed to remove unattached bacteria. Pilot experiments revealed 8h incubation was sufficient for WT GAS (5448) to form a robust biofilm on HaCaT cells without extensive cell loss/death. At 8h, dishes were fixed with 4% paraformaldehyde, blocked and blotted with mouse-anti-GAS-M1 polyclonal antisera (provided by Anna Henningham, University of Wollongong) and Alexa Fluor 488-conjugated goat anti-mouse (Invitrogen) as a secondary antibody to allow fluorescent visualisation. The HaCaT cells were stained with DAPI (nucleic acid) and Alexa Flour 647-conjugated phalloidin (actin). The cells were mounted in glycerol, imaged using a Nikon A1 LSCM and 3D-rendered as above.

Murine skin adherence assay

Bacterial cultures were grown to mid-logarithmic phase (OD600=0.4) and washed with sterile PBS. Bacteria were diluted to 2 × 107 cfu/ml. 10 μl of bacterial solution (2 × 105 cfu) was spotted onto prewarmed THA plates. Once the droplets had dried, agar discs containing the bacteria were excised using an 8 mm biopsy punch. Shaved CD1 mice were anaesthetized with Ketamine/Xylazine and bacterial agar discs affixed with Tegaderm transparent wound dressing (3M). A total of four mice, each with two discs of 5448 bacteria on their left flank and two discs of 5448AP bacteria on their right flank, were used. After 1 h, the mice were euthanized with isoflurane. The skin under the bacterial discs was excised and placed into 2 ml screw cap tubes containing 1ml PBS. The tubes were shaken in a mini-beadbeater-8 (Biospec Products) on mix setting for 2 min to remove non-adherent bacteria. Skin was then transferred to a fresh screw cap tube containing PBS, shaken for 2 min, and transferred to a fresh 2 ml tube containing 1 ml PBS and 1 mm silica/zirconia beads (Biospec Products). Tissue was homogenized by shaking twice with the mini-beadbeater-8 at full for speed for 1 min, placing on ice in between. The homogenate was serially diluted in sterile PBS and plated on THA for enumeration. Adherence was calculated as a percentage of the initial inoculum. Statistical significance was determined using an unpaired T-test.

Histology

The in vivo adherence assay was performed as described above using 2 × 107 cfu/spot. Excised skin was placed in Formalde-fresh solution (Fisher) overnight, then sectioned and Gram-stained by the UCSD Histopathology Core Facility (Nissi Varki, Director).

LL-37 resistance assays

Bacteria were grown to mid-logarithmic phase and resuspended in PBS + 20% THB at 1 × 105 cfu/ml. 90 μl of bacteria was then added to 10 μl of varying concentrations of human cathelicidin LL-37 or the murine cathelicidin CRAMP in a 96-well plate. At 24 h, 5 μl from each well was plated on THA and incubated overnight. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of LL-37 or CRAMP yielding no detectable bacterial growth when the sample was plated on THA. Where there was variance in the MIC between replicates, the values are shown as a range.

Ethics approvals

Permission to obtain human blood and undertake animal experiments was obtained from University of California, San Diego and University of Wollongong human and animal subject protection committees. Human volunteers provided informed consent before blood samples were obtained.

RESULTS

Adherence to epithelial cells is reduced in hypervirulent covS mutant M1T1 GAS

The well-characterized M1T1 clinical isolate 5448 and the isogenic covS mutant derivative 5448AP were used in this comparative analysis. To investigate the ability of WT and covS mutant bacteria to adhere to epithelial cells, we utilized two human cell lines: HEp-2 laryngeal cells and HaCaT keratinocytes, representative of the throat and skin focal points of GAS colonization and mucosal infection. 5448AP had a marked decrease in adherence compared to WT 5448 (P < 0.001) in both HEp-2 and HaCaT cells (figure 1A). In a competition binding assay, WT 5448 outperformed 5448AP in adherence to both HEp-2 and HaCaT cells (figure 1B). The observation that the adherence of 5448AP was not rescued by co-infection with 5448 suggests that a cell surface factor and not a secreted factor, was responsible for this defect in epithelial cell binding.

Figure 1.

Figure 1

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CovS mutation results in increased capsule expression and reduced capacity to adhere to epithelial cells. (A) Adherence to Hep-2 cells (human pharyngeal epithelial cells) and HaCat cells (human keratinocytes). (B) Competitive adherence to Hep-2 and HaCat cells. (C) Hyaluronic acid capsule levels. (D) Adherence to Hep-2 and HaCat cells using capsule deficient mutants 5448ΔhasA and 5448APΔhasA. Values shown in all panels are mean ± sd.

Up-regulation of capsule expression has previously been shown in covS mutant GAS [9, 11]. We hypothesized that this up-regulation of capsule in the 5448AP was an important contributor to the phenotype of decreased epithelial cell binding. Excluding differences in growth characteristics that may affect subsequent assays, we showed that the capsule deficient mutant strains, 5448ΔhasA and 5448APΔhasA, exhibited similar growth to their respective parent strains in THB and formed similar length chains (data not shown). 5448AP was found to have significantly more hyaluronic acid capsule than 5448, while the mutant strains 5448ΔhasA and 5448APΔhasA were found to have no capsule (Fig 1C). Upon a genetically defined disruption of the capsule biosynthesis gene hasA, a difference in adherence between WT and the covS mutant strain to either epithelial cell line was no longer observed (figure 1D). While there was no difference observed between 5448ΔhasA and 5448APΔhasA, both of these strains displayed less binding than WT 5448. This supports a model in which low level capsule expression can contribute to epithelial cell adherence, in particular through hyaluronic acid binding to host CD44 receptors [32, 33], while marked hyperencapsulation impairs adherence, likely through cloaking of higher affinity GAS adhesins and extracellular matrix binding proteins. It is important to note that the M1T1 genome sequence [34] lacks most of the well-characterized GAS surface proteins known to bind fibronectin or other extracellular matrix proteins, including Sfb1, PrtF, PrtFII, SOF, SfbX. The two known GAS fibronectin binding proteins encoded in the GAS genome sequence (FBP54 and FbaA) are not differentially regulated upon the in vivo selection of covS mutation [9]. Thus the relative contribution of capsule to adherence in the covRS intact WT M1T1 strain may be greater than in other serotype backgrounds. Nevertheless, our data suggest that up-regulation of capsule expression following covS mutation is the principal reason for reduced binding to epithelial cells observed in the 5448AP strain.

Hypervirulent covS mutant M1T1 GAS have reduced capacity to bind fibronectin

Fibronectin (Fn) is an important component of the extracellular matrix (ECM) that acts as a target for GAS adhesins; thus Fn binding represents an important initial step in the colonization process [35, 36]. The covS mutant strain 5448AP has significantly reduced capacity to bind Fn (P < 0.01) compared to the WT parental strain (figure 2A). The capsule-deficient strain 5448ΔhasA exhibited similar binding capacity to WT 5448, whereas 5448APΔhasA showed increased binding compared to 5448AP (figure 2A). Fluorescence microscopy of FITC labeled bacteria corroborated these findings (figure 2B). Reduced capacity to bind ECM components may affect the ability of covS mutant GAS to colonize the host. Moreover, increased binding by 5448APΔhasA compared to WT 5448 suggests that the dramatic capsule up-regulation effectively masks binding increases that would otherwise result from gene expression changes linked to covS mutation.

Figure 2.

Figure 2

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CovS mutation results in reduced binding to fibronectin. (A) Binding of 5448 and 5448AP and the capsule deficient mutants 5448ΔhasA and 5448APΔhasA to immobilized fibronectin. Values are mean ± sd. (B) Fluorescence microscopy of FITC labeled GAS to fibronectin-coated glass coverslips. Scale bars shown in white are 20 μm.

Biofilm formation is reduced in hypervirulent covS mutant M1T1 GAS

Biofilm formation has been proposed to play a role in GAS colonization as well as the persistence and recurrence of GAS infection [31, 37]. We found that covS mutant 5448AP exhibits significantly less biofilm formation than WT 5448 (figure 3A, P < 0.001). 5448ΔhasA exhibited similar biofilm formation to WT, whereas 5448APΔhasA produced greater biofilms than either WT or 5448ΔhasA. Together, these data illustrated that while further gene regulation differences may affect biofilms, capsule up-regulation in covS mutant GAS is the major factor limiting biofilm formation and effectively negates any potential positive contribution of up-regulated genes in the covS mutant strain. Confocal microscopy was used to visualize biofilm formation on both polystyrene (figure 3B) and epithelial cell layers (figure 3C). Impaired biofilm formation may contribute to a reduced ability of covS mutant M1T1 GAS to colonize and persist in new hosts.

Figure 3.

Figure 3

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CovS mutation reduces biofilm formation. (A) Quantification of biofilm biomass after 24 h growth in 96-well polystyrene microtitre plates. GAS biofilms were stained with crystal violet and extracted with acetone/ethanol. Values are mean ± sd of absorbance at A595nm. (B) Visualization of 24h biofilm formation on polystyrene. Biofilms were cultured in 96-well polystyrene microtitre plates and stained with SYTO 9 nucleic acid stain (green). Progressive Z-sections of the biofilms were imaged at 400x magnification by laser scanning confocal microscopy and rendered into 3D images using NIS Elements software. (C) Visualization of 8h biofilms on epithelial cells. GAS biofilms were cultured on HaCaT keratinocytes for 8h, stained and imaged by confocal microscopy. The HaCaT keratinocytes were stained with phalloidin to outline actin (red) and the DNA stain DAPI (blue) to show the nucleus. The GAS cells were immunostained with mouse anti-GAS-M1 polyclonal antisera and Alexa Fluor488-conjugated goat anti-mouse secondary antibody and are shown in green. Scale in μm is shown by white grids in each image.

Hypervirulent covS mutant M1T1 GAS have reduced capacity to bind murine skin

A mouse model of skin colonization was used to investigate the comparative capacity of 5448 and 5448AP to colonize the host at a relevant infection site. The covS mutant 5448AP was found to have significantly reduced (P < 0.001) ability to adhere to live mouse skin compared to 5448 (figure 4A), as further illustrated by the lack of adherent bacteria visible in Gram-stained skin sections (figure 4B). Reduced survival of GAS on live skin may relate to sensitivity to cathelidicin antimicrobial peptides [38, 39]. However, no significant difference between the WT 5448 and covS mutant 5448AP was noted in resistance to killing by the human cathelicidin LL-37 (MIC 14–16 μM for both strains) or the murine cathelicidin CRAMP (MIC 4 μM for both strains). Thus the colonization defect of the covS mutant is likely related to reduced adherence and/or biofilm phenotypes and not increased susceptibility to these cutaneous antimicrobials.

Figure 4.

Figure 4

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CovS mutation results in reduced colonization capacity. (A) Adherence of bacteria to live mouse flank after 1 h incubation. Values are mean ± sd. (B) Gram-stained sections of GAS colonized skin tissue. Red arrows indicate groups of Gram-stained bacteria. Scale bars shown in white are 50 μm.

DISCUSSION

M1T1 GAS is the most common cause of streptococcal infections in several western countries [1, 5, 6]. An inverse relationship between SpeB expression in M1T1 clinical isolates and disease severity indicates that inactivation of SpeB through mutation in the covR/S regulator facilitates invasive disease initiation [8–10]. While covS mutation in M1T1 GAS results in improved neutrophil resistance and propensity for bacterial dissemination [9, 10], here we identify potential counterbalancing fitness costs associated with covS mutation in the realm of GAS fibronectin binding, epithelial adherence and biofilm formation.

We recovered significantly more 5448 than 5448AP from the mouse skin adherence model, showing that covS mutation and associated phenotypes, in particular upregulation of capsule biosynthesis, confer a colonization defect in M1T1 GAS despite the dramatic increase in virulence at subsequent stages of infection. This data is supported by a recently published finding of an inverse correlation between ability to adhere to host cells and GAS virulence [40]. Recently, it was also shown that WT GAS outcompete covS mutants in human saliva [41], a finding that supports a model in which such mutations result in increased systemic virulence, but come at a fitness cost for other stages of the infection process.

Differential optimization of GAS phenotypic characteristics for survival at different stages of disease pathogenesis is evident in this study. Epithelial cell binding via the extracellular matrix and biofilm formation can promote GAS colonization of pharynx or skin in the face of competition from the normal resident microflora. However, such close interactions with host cells could be disadvantageous in systemic or bloodstream infection where phagocytes of the innate immune system seek to eradicate the pathogen, hence the selective pressure for covR/S mutation with upregulation of capsule and other neutrophil and serum resistance factors including SpyCEP [42], SLO [43], Sda1 [9, 10] and SIC [44]. Analogous patterns of in vivo evolution have been recently described in relation to persistent Pseudomonas aeruginosa infection in cystic fibrosis patients, with positive selection of mutations allowing for genetic changes advantageous to life within the host [45].

The global dissemination and persistence over decades of the M1T1 GAS clone as the prevalent disease-associated strain not only indicates robust colonization properties but also the propensity to mutate to an immune-resistant phenotype capable of systemic dissemination. Our data indicate that this phenotype does not become fixed because the cost of covS mutation, and in particular hyper-encapsulation, makes the mutant strain less capable of epithelial colonization than the parent phenotype. It is conceivable that similar paradigms exist for many leading human bacterial pathogens, where relatively uncommon invasive disease events occur in certain individuals against a much larger backdrop of asymptomatic colonization or self-limited mucosal infection.

Acknowledgments

Financial support: This work was funded by NIH grant AI077780 (VN, MW), National Health and Medical Research Council of Australia Grant 459103 (MW) and UCSD Neuroscience Microscopy Shared Facility Grant P30 NS047101. AH is the recipient of an Australian Postgraduate Award. MAP was supported by the UCSD Genetics Training Program. AMT is a recipient of the A.P. Giannini Foundation Postdoctoral Fellowship. CW is a National Health and Medical Research Council of Australia Senior Research Fellow.

The authors wish to thank Anna Cogen (University of California, San Diego) for experimental advice and Anna Henningham (University of Wollongong) for providing antisera.

Footnotes

Potential conflicts of interest: The authors have no conflicts of interest to report.

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