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. 2010 Jul 12;107(30):13473–13478. doi: 10.1073/pnas.1001815107

Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus α-hemolysin–mediated cellular injury

Georgia A Wilke a, Juliane Bubeck Wardenburg a,b,1
PMCID: PMC2922128  PMID: 20624979

Abstract

Staphylococcus aureus α-hemolysin (Hla), a potent cytotoxin, plays an important role in the pathogenesis of staphylococcal diseases, including those caused by methicillin-resistant epidemic strains. Hla is secreted as a water-soluble monomer that undergoes a series of conformational changes to generate a heptameric, β-barrel structure in host membranes. Structural maturation of Hla depends on its interaction with a previously unknown proteinaceous receptor in the context of the cell membrane. It is reported here that a disintegrin and metalloprotease 10 (ADAM10) interacts with Hla and is required to initiate the sequence of events whereby the toxin is transformed into a cytolytic pore. Hla binding to the eukaryotic cell requires ADAM10 expression. Further, ADAM10 is required for Hla-mediated cytotoxicity, most notably when the toxin is present at low concentrations. These data thus implicate ADAM10 as the probable high-affinity toxin receptor. Upon Hla binding, ADAM10 relocalizes to caveolin 1-enriched lipid rafts that serve as a platform for the clustering of signaling molecules. It is demonstrated that the Hla–ADAM10 complex initiates intracellular signaling events that culminate in the disruption of focal adhesions.

Keywords: pore-forming cytotoxin, cellular receptor

The Gram-positive extracellular pathogen Staphylococcus aureus is one of the leading causes of human bacterial infection. As a commensal of the skin, S. aureus is well positioned to cause infection of the skin and soft tissues, the bloodstream, and the lower respiratory tract, which are the principal sites of clinically relevant infection (1, 2). To facilitate entry and spread through the host tissue, S. aureus encodes a number of virulence factors that allow the organism to breach structural and immunological barriers to infection. One of the most prominent and well-characterized virulence factors produced by S. aureus is α-hemolysin (Hla), a pore-forming cytotoxin implicated in the pathogenesis of sepsis, pneumonia, and severe skin infection (3–6). Pore formation on susceptible host cell membranes triggers alterations in ion gradients, loss of membrane integrity, activation of stress-signaling pathways, and cell death (3, 7). Hla binds to most eukaryotic cells, often by a nonspecific adsorptive mechanism requiring micromolar concentrations of toxin (8). However, a high-affinity interaction of the toxin with a proteinaceous eukaryotic receptor has been suggested because rabbit erythrocytes are significantly more sensitive to Hla than human erythrocytes, correlating with the identification of 1,200–5,000 toxin-binding sites per rabbit cell (8, 9). Binding is saturable and time dependent, consistent with a ligand–receptor interaction (8, 10). In addition to these data, membrane lipids seem to be central to the interaction of the toxin with the eukaryotic cell. Membrane cholesterol or sphingomyelin depletion abrogates toxin binding and cytotoxicity, and the addition of exogenous phosphocholine disrupts toxin binding and impairs rabbit red cell hemolysis (11). These data have led to the hypothesis that clustered phosphocholine head groups serve as the high-affinity binding site for Hla. Despite intense investigation of this toxin, a proteinaceous cellular receptor has not yet been identified, and it remains unclear how Hla binding exhibits both species specificity and a requirement for particular membrane lipids.

Results

ADAM10 Mediates S. aureus α-Hemolysin Binding to Eukaryotic Cells.

We used a biochemical approach to purify a putative Hla receptor, taking advantage of species-specific receptor expression. Rabbit and human erythrocyte ghosts were incubated in the absence of detergent with GST or GST-HlaH35L, a Hla mutant that precludes pore formation while preserving membrane binding (12). After toxin treatment, ghosts were solubilized with Triton X-100, and GST or GST-HlaH35L precipitated proteins were analyzed by SDS/PAGE and silver staining. An ≈65-kDa protein bound GST-HlaH35L from rabbit erythrocytes (Fig. 1A, arrow). Mass spectroscopic analysis of this protein yielded peptides corresponding to ADAM10 (a disintegrin and metalloprotease 10), a zinc-dependent metalloprotease that is expressed on the surface of many distinct cell types as a type I transmembrane protein (Fig. S1, peptides highlighted in bold). GST or GST-HlaH35L bound proteins were then examined by immunoblotting with anti-human ADAM10 (hADAM10) antisera, leading to the detection of rabbit ADAM10 (rADAM10) specifically in GST-HlaH35L precipitates (Fig. 1B, Upper). Control immunoblots (Fig. 1B, Lower) demonstrating the presence of GST or GST-HlaH35L were prepared from samples processed in parallel or probed after removal of the upper section of the nitrocellulose containing ADAM10. Flow cytometric analysis revealed expression of ADAM10 on rabbit erythrocytes and toxin-susceptible human A549 alveolar epithelial cells (Fig. 1C, blue and green solid lines, respectively). Human erythrocytes were devoid of ADAM10 (Fig. 1C, red solid line), in agreement with the species-specific expression pattern predicted for the Hla receptor.

Fig. 1.

Fig. 1.

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Hla interacts with ADAM10 expressed on rabbit red blood cells. (A) Rabbit or human erythrocyte ghost preparations were precipitated with GST control or GST-HlaH35L and visualized by silver stain analysis. **Purified GST protein; *purified GST-HlaH35L fusion. (B) GST or GST-HlaH35L precipitates were immunoblotted for human ADAM10 or GST. (C) Flow cytometric analysis of ADAM10 on rabbit and human erythrocytes and human A549 cells.

To evaluate the Hla–ADAM10 interaction in intact cells, A549 cells were treated with HlaH35L or active Hla. Cells were washed and then lysed in Triton X-100 or SDS/DOC-containing RIPA buffer, and Hla immunoprecipiates were examined for hADAM10. Both forms of Hla precipitated ADAM10 (Fig. 2A), with an increased amount of ADAM10 detectable in RIPA lysates as compared with Triton X-100 lysates, suggesting localization of the complex to detergent-resistant lipid rafts. Hla treatment of A549 cells induced the aggregation of ADAM10 in discrete punctae (Fig. 2B, Middle Left, arrows). Previous studies have demonstrated an association of Hla with caveolin 1, a structural protein of specialized caveolar lipid rafts. The association of Hla with caveolin 1 is necessary for toxin oligomerization (13, 14). Upon Hla treatment of A549 cells, ADAM10-containing punctae colocalize with caveolin 1 (Fig. 2B, Middle Right; Manders’ coefficient = 0.15 ± 0.08, PBS; 0.28 ± 0.13, Hla; P < 0.0005). Punctae are not evident when cells are treated with a form of Hla that cannot form stable oligomers (HlaH35L; Fig. 2B, Bottom). These data together reveal that Hla interacts with ADAM10 in the context of the cell membrane, establishing an association that both parallels the known species-selectivity of the toxin evident in erythrocytes and forms a complex that seems to localize to caveolin 1-enriched lipid rafts.

Fig. 2.

Fig. 2.

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ADAM10 interacts with Hla and mediates toxin binding to human epithelial cells. (A) A549 alveolar epithelial cells were treated with nontoxigenic HlaH35L or active Hla, then Hla immunoprecipitates from cell lysates prepared in Triton X-100 or RIPA lysis buffer were immunoblotted for ADAM10. (B) Hla induces the relocalization of ADAM10 to discrete sites on the cell membrane that colocalize with caveolin 1. Quantification of the number of ADAM10 punctae ≥0.05 μm in diameter averaged on a per-cell basis revealed 0.08 ± 0.14, PBS; 1.63 ± 0.48, Hla; 0.13 ± 0.14, HlaH35L, with error values representing the SD. P = 8.7 × 10−6 PBS vs. Hla, P = 1.3 × 10−5 Hla vs. HlaH35L. A minimum of 45 cells was counted for each treatment condition. Quantification of ADAM10–caveolin 1 colocalization was determined by the Manders’ coefficient assessed on 20 distinct images (0.15 ± 0.08, PBS; 0.28 ± 0.13, Hla; P = 0.0005) (Scale bars, 10 μm.) (C) Binding of [35S]-methionine–labeled active Hla examined as a function of ADAM10 surface expression, quantified as the mean fluorescence intensity (MFI) shift by flow cytometric analysis in a panel of human epithelial cells. (D) Binding of [35S]-methionine–labeled active Hla to A549 cells treated with irrelevant or ADAM10-specific siRNAs (designated ADAM10.1 and ADAM10.2). *P < 2 × 10−6. (E) Flow cytometric detection of surface expressed ADAM10 in A549 cells.

To further examine the role of ADAM10 as a potential Hla receptor, we quantified the binding of radiolabeled, active Hla to a panel of human epithelial lines expressing varying amounts of ADAM10. A linear correlation was evident between ADAM10 expression and Hla binding (Fig. 2C). In contrast, an inverse correlation was derived when toxin binding was compared with expression of the related metalloproteases ADAM9 and ADAM17 (Fig. S2A), demonstrating the specificity of binding to ADAM10. We assessed binding of radiolabeled Hla to A549 cells treated with irrelevant siRNA or two siRNAs targeting ADAM10 (ADAM10.1 and ADAM10.2; Table S1). Hla bound cells treated with irrevelant siRNA (Fig. 2D). This interaction was diminished upon siRNA-mediated knockdown of ADAM10, with residual toxin binding to knockdown cells likely reflecting incomplete knockdown, because ADAM10 remains detectable on the cell surface after siRNA treatment, as assessed by flow cytometric analysis (Fig. 2E). Binding was unaltered in ADAM9 and ADAM17 siRNA-treated cells (Fig. S2 B and C).

ADAM10 Is Required for α-Hemolysin–Mediated Cytotoxicity.

Hla-deficient S. aureus strains are impaired in causing cytotoxicity in an A549 coculture model with live staphylococci and are severely attenuated in a mouse model of S. aureus pneumonia (4). To examine the requirement for ADAM10 in Hla-mediated cytotoxicity, A549 cells were treated with irrelevant or ADAM10 siRNAs, then infected with WT S. aureus Newman, an isogenic Hla− mutant of S. aureus Newman, or the S. aureus Hla− strain complemented with plasmid-encoded Hla (Hla− phla). ADAM10 knockdown afforded protection from S. aureus–induced injury as compared with irrelevant siRNA-transfected cells (Fig. 3A, WT, Hla− phla). This injury was dependent on Hla expression (Fig. 3A, Hla-). ADAM10 was also required for cytotoxicity mediated by purified Hla (Fig. 3B). The requirement for ADAM10 in Hla-mediated injury was most evident at concentrations of toxin in the nanomolar range, corresponding to the addition of 30 or 60 hemolytic units (HU)/mL of toxin (0.375 μM and 0.750 μM, respectively). Incubation of cells with 1.25 μM toxin (120 HU/mL) resulted in a smaller, yet significant difference in cytotoxicity between irrelevant and ADAM10 siRNA treated cells, whereas a toxin concentration of 2.5 μM (180 HU/mL) led to no observable difference upon ADAM10 knockdown. This observation is highly consistent with previous work that has described a dual mechanism of toxin binding to Hla-targeted cells—a high-affinity, receptor-dependent binding interaction that prevails at low toxin concentrations, and a nonspecific binding event that occurs at higher toxin concentrations (8). Physiologic lytic injury occurring at toxin concentrations of ≈50 HU/mL have been suggested to be associated with target cell expression of high-affinity binding sites (3), in good agreement with the data presented herein.

Fig. 3.

Fig. 3.

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ADAM10 is required for Hla-mediated cytotoxicity. (A) Irrelevant or ADAM10 siRNA transfected A549 cells were cocultured with WT S. aureus, S. aureus deficient in production of Hla (Hla−), or S. aureus Hla− complemented with plasmid-encoded Hla (Hla− phla), and cell injury measured by lactate dehydrogenase (LDH) release. *P < 0.008 vs. irrelevant siRNA treated cells. Error bars delineate the SEM examined in six replicates from two independent experiments. (B) LDH release from A549 cells treated with the indicated concentrations of purified, active Hla. *P < 0.0002 vs. irrelevant siRNA treated cells. Error bars delineate the SD of six replicates. (C) Detection of radiolabeled, oligomeric Hla (Hla7) bound to irrelevant or ADAM10 siRNA transfected A549 cells. (D) LDH release in a panel of epithelial cell lines cultured with purified active Hla examined as a function of ADAM10 surface expression, quantified as the mean fluorescence intensity (MFI) shift by flow cytometric analysis.

Examination of toxin oligomerization on the surface of A549 cells revealed that ADAM10 is required for the formation of the toxin heptamer, because this product is not detected on the membrane of cells treated with ADAM10 siRNAs (Fig. 3C). A direct correlation between the level of surface expression of ADAM10 and susceptibility to intoxication was seen in a panel of human epithelial cells (Fig. 3D), with the relatively toxin-insensitive line 1299 displaying a 3.6-fold reduction in ADAM10 expression compared with A549 cells. Expression of ADAM9 and ADAM17 did not markedly affect sensitivity to Hla (Fig. S2 D and E).

α-Hemolysin–ADAM10 Complex Alters Integrin-Mediated Cell Signaling Events.

ADAM family proteins regulate cell migration and adhesion through the combined actions of the metalloprotease and disintegrin domains (Fig. S1) (15, 16). The N-terminal enzymatic domain mediates proteolysis of cell adhesion molecules, disrupting lateral cell junctions to facilitate cell mobility and epithelial barrier remodeling. The adjacent disintegrin domain of many ADAM metalloproteases harbors a conserved aspartate-rich motif that binds cellular integrins; such ADAM–integrin associations also occur in the absence of this specific motif, exerting both pro- and antiintegrin function (16). Although ADAM10 lacks this specific motif, a reported association of Hla with β1-integrin (17) raised the interesting possibility that the Hla–ADAM10 complex may alter integrin-mediated signaling. Integrin heterodimers bind extracellular matrix proteins, coupling signals from the extracellular environment to intracellular responses at focal adhesion sites (18). Integrin activation leads to the tyrosine phosphorylation and activation of the FAK and Src tyrosine kinases (19). FAK and Src in turn phosphorylate the adaptor proteins p130Cas and paxillin, allowing these proteins to function as molecular scaffolds that link integrin receptors to Rho family GTPases and the actin cytoskeleton (20, 21). This phosphorylation-regulated signal transduction cascade permits the dynamic assembly and turnover of focal adhesions. To determine whether Hla alters focal adhesion complex signaling, lysates from A549 cells transfected with irrelevant or ADAM10 siRNAs were examined for the presence of tyrosine-phosphorylated FAK, Src, p130Cas, and paxillin (Fig. 4 A–D). Treatment of irrelevant transfectants with Hla led to the dephosphorylation of all four proteins; this response was abrogated upon ADAM10 knockdown (Fig. 4 A–D). This activity requires toxin oligomerization, because the HlaH35L mutant does not induce tyrosine dephosphorylation of cellular proteins (Fig. S3). Knockdown of β1-integrin expression by specific siRNAs (Table S1) did not alter Hla binding (Fig. S4A), cellular injury (Fig. S4B), or p130Cas dephosphorylation (Fig. S4C). Further, β1-integrin expression by a panel of epithelial cell lines did not correlate strongly with susceptibility to the cytotoxic effects of Hla (Fig. S4D), together suggesting that Hla binding and cytotoxicity are not directly dependent on β1-integrin.

Fig. 4.

Fig. 4.

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ADAM10 is required for Hla-mediated focal adhesion disruption. (A) Cell lysates from irrelevant or ADAM10 siRNA transfected A549 cells treated with active Hla were probed with an antibody specific for phospho-Tyr397 FAK (Upper) or anti-FAK control (Lower). (B) Lysates prepared as described in A were probed with an antibody specific for phospho-Tyr-416 Src (Upper) or anti-Src control (Lower). (C) Lysates prepared as described in A were probed with an antibody specific for phospho-Tyr-165 p130Cas (Upper) or anti-p130Cas control (Lower). (D) Lysates prepared as described in A were probed with an antibody specific for phospho-Tyr-118 paxillin (Upper) or antipaxillin control (Lower). (E) Irrelevant (Top) or ADAM10 (Middle, Bottom) siRNA transfected A549 cells were treated with PBS or active Hla and stained to detect vinculin (red) and filamentous actin (phalloidin, green). (Scale bars, 10 μm.)

Focal Adhesions Are Disrupted by the α-Hemolysin–ADAM10 Complex.

The data just described demonstrate that the Hla–ADAM10 complex rapidly alters signaling cascades that contribute to the stability of focal adhesions. In this regard, ADAM10 may function in a manner analogous to other ADAM metalloproteases, creating a functional disruption of integrin signaling. Several bacterial virulence factors target the host cytoskeleton through the direct modification of the phosphorylation state of focal adhesion proteins. The Yersiniae YopH tyrosine phosphatase dephosphorylates FAK and p130Cas (22), uncoupling integrin-mediated signals from intracellular signaling machinery and promoting cytoskeletal derangements that permit evasion of phagocytosis. The Clostridium difficile toxin A disrupts focal adhesion signaling through Src inactivation, with the resultant dephosphorylation of FAK and paxillin leading to colonic epithelial cell detachment (23). To evaluate the cytoskeletal effects of Hla, siRNA transfected A549 cells were treated with PBS or Hla, then stained with an antivinculin antibody (red) and phalloidin (green). Actin stress fibers originating from vinculin-containing focal adhesions are apparent in PBS-treated cells (Fig. 4E, Left). Hla treatment of irrelevant siRNA transfected cells induces loss of focal adhesions and actin fibers (Fig. 4E, Top Right), which is prevented by ADAM10 knockdown (Fig. 4E, Middle/Bottom Right). Toxin treatment of irrelevant siRNA treated cells results in an F/G actin ratio of 1.26, decreased from 1.92 in PBS-treated cells (P = 7.2 × 10−10). The F/G actin ratio after toxin treatment of ADAM10 siRNA treated cells is 1.7, reduced from 2.1 (P = 0.004). The absolute change in this ratio in ADAM10 siRNA treated cells is blunted, consistent with preservation of the F actin cytoskeleton, as viewed microscopically in Fig. 4E.

Discussion

Several features of the Hla–ADAM10 interaction identify ADAM10 as the likely proteinaceous cellular receptor for the toxin. First, toxin binding to eukaryotic cells requires ADAM10 expression. Second, Hla physically interacts with ADAM10 in vivo. Third, ADAM10 is required for Hla-mediated cytotoxicity. The requirement for ADAM10 is most evident at low toxin concentrations in which a high-affinity receptor-driven binding event would be essential. Importantly, an examination of cell lines that naturally express differing amounts of ADAM10 confirms the strong correlation between ADAM10 expression and the ability of Hla to bind to the membrane and induce cytotoxic injury. These data shed light on the mechanism by which the toxin exhibits species-specificity.

Hla pore formation is a complex series of events, initiated by monomeric toxin binding to the surface of the eukaryotic cell. Multiple lines of evidence confirm the importance of the membrane lipid environment in Hla-induced injury, because the membrane-opposed region of the toxin interacts with phosphatidylcholine (24), and cholesterol/sphingomyelin-rich membrane domains are required for cytotoxicity (11). On the basis of these observations, clustered phosphocholine head groups have been hypothesized to be the toxin receptor (11). Although the requirement for membrane lipids in toxin biology has been well described, a model proposing these as the sole cell-binding moiety fails to explain several important observations. Most notably, the species specificity of the toxin implies a proteinaceous receptor, because liposomes mirroring rabbit and human red blood cells do not parallel the sensitivities of these cells (10). Further, protease treatment of highly sensitive rabbit red blood cells abrogates toxin binding (10). The identification of ADAM10 as an Hla-interacting protein that is required for initial toxin binding and multiple cellular cytotoxic insults provides important insight into how Hla engages the host cell. Our investigations have revealed that the strong in vivo interaction of Hla with ADAM10 must occur in the context of an intact cell membrane, because solubilization of the membrane before toxin binding precludes the association of these proteins. It thus seems that Hla demonstrates a dual requirement for both ADAM10 and membrane lipids to establish a high-affinity interaction with the eukaryotic cell. This model thereby draws together seemingly disparate observations in the field, solving the paradox of how Hla can simultaneously exhibit cell specificity, which can only be conferred by a proteinaceous receptor, and yet also require membrane cholesterol and sphingomyelin.

At present, it has not been possible to dissociate ADAM10 from the membrane environment to demonstrate a direct in vitro interaction with Hla. Crystallographic studies of a portion of native ADAM10 and the related vascular apoptosis-inducing protein-1 reveal a highly conserved, multidomain C-shaped structure containing 17 disulfide bonds and several calcium-binding sites (25, 26). The structural complexity of ADAM10, in concert with the clear requirement for the membrane context, will likely preclude in vitro interaction studies of these proteins until such time that ADAM10 can be incorporated in its native form into an artificial lipid matrix. It remains possible that ADAM10 requires a membrane-localized coreceptor to bind Hla. The association of the ADAM10–Hla complex in SDS-containing buffer suggests a direct interaction of these proteins, because multimolecular complexes are readily dissociated under these conditions. Similarly, we did not observe other proteins to be precipitated from rabbit erythrocytes with GST-HlaH35L.

These data provide a mechanistic view of the assembly of Hla, suggesting that its initial interaction with ADAM10 and the membrane directs the assembly of the Hla–ADAM10 complex in cholesterol/sphingolipid-rich caveolar rafts. This clustering likely increases the local concentration of Hla, permitting caveolin 1-directed oligomerization of the toxin and providing accessibility to caveolae-associated proteins FAK and Src that mediate the biologic effects of Hla (Fig. 5). Focal adhesion disruption by the Hla–ADAM10 complex provides a mechanism by which the toxin may perturb cellular barriers to cause invasive disease and facilitate superantigen permeation through impenetrable stratified cell layers (27). By placing ADAM10 as a proteinaceous Hla receptor, genetic mutation of ADAM10 should confer resistance to toxin-mediated disease. ADAM10 null mice display embryonic lethality (28), thus conditional knockouts will be essential to define the relevance of the Hla–ADAM10 interaction in S. aureus infection. In addition, inhibitors of zinc metalloproteases, including ADAM10, are being investigated for clinical potency in a wide array of inflammatory and malignant diseases and may have utility as tools to combat S. aureus infection.

Fig. 5.

Fig. 5.

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Model depicting the ADAM10-Hla complex localized to caveolin 1-enriched membrane domains, causing cytolysis and focal adhesion disruption in toxin-sensitive cells.

Methods

Bacterial strains, cell lines and culturing conditions, antibodies, recombinant proteins, buffers, and flow cytometry techniques are detailed in SI Methods.

Cellular Protein Precipitations and Analysis.

Rabbit or human red cell ghosts (5 × 109) were incubated 20 min at room temperature with either 2 μg GST or GST-HlaH35L, whereas intact A549 cells (1 × 107) were incubated with 2 μg untagged HlaH35L or HIS-Hla proteins for 20 min at room temperature for coprecipitation studies, which are fully described in SI Methods. Mass spectrometry was performed by the Taplin Mass Spectrometry Facility at Harvard Medical School.

siRNA Experiments.

siRNAs were purchased from Applied BioSciences (Table S1) and used as described previously (29), with minor modifications relevant to A549 cells as detailed in SI Methods.

Radiolabeled Hla Studies.

Binding of radiolabeled Hla prepared as described previously (30) was assessed after treatment of 1 × 106 cells/500 μL with 0.3 nM Hla in PBS over 5 min. Processing of cells and assessment of toxin oligomerization is detailed in SI Methods.

Immunofluorescence Microscopy.

Cells stained for microscopic analysis were treated for 60 min (ADAM10, caveolin 1 studies) or 2 h (focal adhesion studies) in F12K media with 30 nM recombinant active Hla or the corresponding HlaH35L mutant and stained with the indicated antibodies as detailed in SI Methods. Microscopy was performed using an Olympus DSU confocal microscope or a Nikon Eclipse TE2000U microscope. Image processing and analysis of immunofluorescence microscopy was performed with ImageJ software (http://rsbweb.nih.gov/ij/). The quantification of ADAM10 punctae was performed with ImageJ software by automated counting of particles with a lower size cutoff of 0.05 μM; quantification of colocalization as determined by Manders’ coefficient was performed using JACoP (http://rsb.info.nih.gov/ij/plugins/track/jacop.html). Quantification of the F/G actin ratio was performed on toxin-treated cells according to previously described methods (31), as detailed in SI Methods.

Statistical Analysis.

Statistical significance was calculated using the two-tailed Student's t test.

Supplementary Material

Supporting Information
supp_107_30_13473__index.html (1,002B, html)

Acknowledgments

We thank D. Missiakas, O. Schneewind, A. Schwartz, and R. Haselkorn for critical discussions and comments on the manuscript; B. Ragle for optimizing siRNA knockdown in A549 cells and early experiments on radiolabeled Hla binding; G. Randall and N. Heaton for assistance with siRNA experiments; and J. Kern, V. Bindokas, and C. Labno for microscopy support. This work was supported by the Deparments of Pediatrics and Microbiology at the University of Chicago. The authors acknowledge membership in and support from the Region V “Great Lakes” Regional Center of Excellence (National Institutes of Health Award 2-U54-AI-057153).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1001815107/-/DCSupplemental.

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