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Infection and Immunity, November 2008, p. 5006-5015, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00300-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Shr Is a Broad-Spectrum Surface Receptor That Contributes to Adherence and Virulence in Group A Streptococcus{triangledown}

Morly Fisher,1,{dagger} Ya-Shu Huang,2,{dagger} Xueru Li,2 Kevin S. McIver,3 Chadia Toukoki,2 and Zehava Eichenbaum2*

Department of Infectious Diseases, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona 74100, Israel,1 Biology Department, Georgia State University, P.O. Box 4010, Atlanta, Georgia 30302-4010,2 University of Maryland Department of Cell Biology and Molecular Genetics and Maryland Pathogen Research Institute, College Park, Maryland 207423

Received 5 March 2008/ Returned for modification 10 April 2008/ Accepted 1 August 2008


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ABSTRACT
 
Group A streptococcus (GAS) is a common hemolytic pathogen that produces a range of suppurative infections and autoimmune sequelae in humans. Shr is an exported protein in GAS, which binds in vitro to hemoglobin, myoglobin, and the hemoglobin-haptoglobin complex. We previously reported that Shr is found in association with whole GAS cells and in culture supernatant. Here, we demonstrate that cell-associated Shr could not be released from the bacteria by the muralytic enzyme mutanolysin and was instead localized to the membrane. Shr was available, however, on the exterior of GAS, exposed to the extracellular environment. In vitro binding and competition assays demonstrated that in addition to hemoprotein binding, purified Shr specifically interacts with immobilized fibronectin and laminin. The absence of typical fibronectin-binding motifs indicates that a new protein pattern is involved in the binding of Shr to the extracellular matrix. Recombinant Lactococcus lactis cells expressing Shr on the bacterial surface gained the ability to bind to immobilized fibronectin, suggesting that Shr can function as an adhesin. The inactivation of shr resulted in a 40% reduction in the attachment to human epithelial cells in comparison to the parent strain. GAS infection elicited a high titer of Shr antibodies in sera from convalescent mice, demonstrating that Shr is expressed in vivo. The shr mutant was attenuated for virulence in an intramuscular zebrafish model system. In summary, this study identifies Shr as being a new microbial surface component recognizing adhesive matrix molecules in GAS that mediates attachment to epithelial cells and contributes to the infection process.


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INTRODUCTION
 
Group A streptococcus (GAS), also known as Streptococcus pyogenes, is a versatile human pathogen that colonizes the mucosal surfaces of the upper respiratory pathways and the epidermis (9). GAS usually produces self-limited infections such as pharyngitis, impetigo, and erysipelas. Infrequently, GAS also causes severe invasive infections in a variety of body sites, including bacteremia, necrotizing fasciitis, and myositis. In addition to the immediate damage caused by primary GAS infections, some episodes lead to the development of streptococcal toxic shock syndrome or to autoimmune complications such as glomerulonephritis and acute rheumatic fever, resulting in renal injury and inflammation of connective tissue, respectively. Acute rheumatic fever can trigger additional manifestations in the form of rheumatic heart disease and the neurological syndrome Sydenham's chorea (48). The wide spectrum of GAS-related illnesses makes it an intriguing pathogen and underscores the complexity of GAS interactions with human tissues and the immune response.

The initial, nonspecific interaction of GAS with the host tissues is provided by lipoteichoic acid. This amphipathic surface molecule has been proposed to counteract the electrostatic repulsion between the bacteria and host surfaces. The subsequent step in adherence involves high-affinity binding to host components and is mediated by a large number of adhesins (6, 22, 50). These include M proteins from several serotypes, F and F-like proteins, the hyaluronic acid capsule, and the recently described pili (1, 28). Some of the adhesins in GAS bind directly to molecules on the host cell surface. For example, M6 and the hyaluronic acid capsule bind to the keratinocyte receptors CD46 and CD44 (17, 37, 42), and the GAS collagen-like protein Scl1 interacts with the {alpha}2β1 integrin expressed by human epithelial cells (7). More often, GAS adhesins attach the bacterium to the extracellular matrix (ECM) or to plasma components that in turn function as a bridge between the bacterium and receptors on the host cells. This mechanism of bacterial attachment is very common among pathogenic gram-positive cocci, and the bacterial proteins involved are called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (39). GAS expresses several surface molecules that bind collagen or laminin (10, 14, 24, 52). However, many more GAS adhesins target the ECM component fibronectin (22, 50). At least 11 fibronectin-binding proteins have been identified in GAS thus far, including F1 (SfbI), F2 (PFBP), Fbp54, SOF, and several M proteins. While a few of these GAS proteins (such as Fbp54) are ubiquitous and encoded by most serotypes, others are found only in some or even in a single serotype. Moreover, most of the fibronectin-binding proteins in GAS are individually regulated and are expressed under unique growth conditions or in response to certain environmental stimuli (22, 23).

The production of infection by GAS and its survival in the host environment are dependent on its ability to acquire essential nutrients such as iron. GAS growth in vitro in iron-restricted medium is supported by whole blood, serum, myoglobin, hemoglobin, and the hemoglobin-haptoglobin complex but not by the ferric carrier proteins transferrin and lactoferrin (13, 16, 40). Hence, heme, the most abundant iron form in mammals, serves as a major source of iron for this hemolytic pathogen. The sia operon is an iron-regulated operon in GAS involved in heme acquisition and transport. In addition to five genes with unknown function, the sia operon carries the shr, shp, and siaABC (htsABC) genes (4, 26). The SiaABC proteins are the components of an ABC-type heme transporter. Shp is a surface protein that was shown to acquire heme from hemoglobin and transfer it to apoSiaA (HtsA) while forming a stable complex with it (27). Shr, encoded by the first gene in the sia operon, is a 145-kDa protein that binds myoglobin, hemoglobin, and hemoglobin-haptoglobin complexes (4). Two near transporter (NEAT) domains are found in Shr (Fig. 1A) (2). While their functional role is not well understood, NEAT domains appear to share a common immunoglobulin-like fold, and some of them were found to bind heme (19, 29, 32, 35) and/or heme-containing proteins (8, 11, 32, 53).


Figure 1
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  		FIG. 1. Shr protein domains and cellular location. (A) Schematic representation of the Shr protein. The SMART algorithm (http://smart.embl-heidelberg.de) was used for the structural analysis of Shr. The location of protein domains (expressed as amino acid numbers) is shown. LP, leader peptide; NEAT 1, NEAT domain 1; TM, transmembrane domain. (B and C) Proteins prepared from NZ131 cells grown in THYB were analyzed by Western blotting using anti-Shr antibodies (B) or anti-M49 antibodies (C). T, total protein; CW, cell wall fraction; CM, cell membrane fraction. Full-length Shr and M49 are indicated by the arrows.

In this study, we explored Shr's function and investigated its contribution to GAS pathogenesis. We demonstrate here that in addition to its likely role in heme acquisition, Shr is an MSCRAMM that specifically binds to fibronectin and laminin and mediates bacterial attachment. We report that Shr is expressed in vivo and is important for GAS virulence in a zebrafish infection model.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. Escherichia coli strains DH5{alpha} and Top10 (Invitrogen) were used for cloning and gene expression. The clinical GAS (S. pyogenes) isolates used in this study were obtained from the Georgia Emerging Infections Program and are listed in Table 1. The other GAS strains used in this study were MGAS5005, an M type 1 strain (49); JRS6, an M type 6 strain (46); NZ131, an M type 49 strain (47); and ZE4912, a shr mutant constructed in NZ131 (provided by Bernard Beall, Centers for Disease Control and Prevention Respiratory Diseases Branch, Atlanta, GA) (Fig. 2). The shr mutation in ZE4912 is a deletion-insertion mutation made by replacing an internal 0.3-kb BglII fragment with the spectinomycin resistance gene aad9 (B. Beall, personal communication). We confirmed the structure of the shr mutation in the ZE4912 genome by sequence analysis of a DNA fragment carrying the mutant shr allele amplified from the ZE4912 chromosome with primers ZE245 (5'-GTGCCCACAAAACCAAGGCACAC-3') and ZE246 (5'-CAGTCGATGAGTATCGGCGAG-3'). Lactococcus lactis strain MG1363 was used as a heterologous host for the expression of the native Shr protein from plasmid pXL14. E. coli cells were grown in Luria-Bertani broth with agitation. GAS was grown statically in Todd-Hewitt broth with 0.2% (wt/vol) yeast extract (THY broth; Difco Laboratories). L. lactis was grown statically at 30°C in M17 medium (Difco Laboratories) supplemented with 0.5% (wt/vol) glucose. When necessary, spectinomycin at 100 µg/ml or kanamycin at 70 µg/ml was added to the medium.


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  		TABLE 1. Analysis of Shr expression on the surface of GAS clinical isolates


Figure 2
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  		FIG. 2. Successful inactivation of shr in strain ZE4912 and mutant complementation in strain ZE4924. (A) Schematic representation of the sia operon and the shr mutation in ZE4912. The shr mutation in strain ZE4912 consists of a small deletion and an insertion of the spectinomycin resistance gene aad9. Strain ZE4924 is a merodiploid strain, which carries both the mutant and the wild-type alleles of shr in the chromosome (see Materials and Methods). (B to D) RNA and proteins from wild-type strain NZ131 (lane 1), shr mutant strain ZE4912 (lane 2), and shr-complemented strain ZE4924 (lane 3) were prepared and analyzed. (B) Total RNA was extracted, loaded (1 µg/well) on an agarose gel, and examined for integrity. Reverse transcription-PCR products obtained with recA-specific primers (used to control for equal amounts of RNA template) or with siaA-specific primers were analyzed by agarose gel electrophoresis. (C) Total proteins were prepared and analyzed by Western blotting with anti-SiaA serum. (D) Proteins from the membrane fraction were analyzed by Western blotting with anti-Shr serum. Full-length Shr is indicated by the arrow.

Nucleic acid methods. Chromosomal and plasmid DNA extraction and DNA manipulations including restriction digests, cloning, and DNA transformation into E. coli, GAS, or L. lactis were done according to the manufacturer's recommendations and with standard protocols as previously described (12, 41). For RNA extraction and analysis, GAS cells were harvested at the logarithmic growth phase, and total RNA was prepared using the RiboPure-Bacteria kit (Ambion). RNA was quantified spectrophotometrically, and its integrity was examined by agarose gel electrophoresis. The absence of DNA contamination was verified by PCR. cDNA was produced by Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's specifications. One microgram of RNA was used in the reverse transcription step, and 1/20 of the reaction mixture served as a template for 25 cycles of PCR. Primers SRAR (5'-CTGATGCTACTGCCATAGCAG-3') and SRAL (5'-GCGTTCAGGAGGTCTAGCTC-3') were used in the analysis of the recA gene, and primers 204A-Rev (5'-TCTGGAATGGCATGAGCTGTTC-3') and 204A-Rev (5'-TCTGGAATGGCATGAGCTGTTC-3') were used for the analysis of siaA transcription.

Construction of shr-complemented strain ZE4924. To complement the shr mutation in ZE4912, we cloned the native shr gene into temperature-sensitive plasmid pJRS700 (33). A 4.5-kb fragment amplified from the NZ131 chromosome with primers ZE170 (5'-TTTTTTATCGATTTAGCTCTTGCTGACTAG-3') and ZE174 (5'-TTTTTTATCGATTATGCAGTAGTGACATCTC-3') was cloned into the ClaI site of pJRS700, generating plasmid pXL14. Since ZE4912 cells harboring pXL14 did not grow well at 30°C (possibly due to the high gene dosage of shr carried by pXL14), we constructed a merodiploid strain (ZE4924), which contains both the mutant and the wild-type alleles of shr in the chromosome. Strain ZE4924 was made in ZE4912 by Campbell insertion. Plasmid pXL14 was introduced into ZE4912 cells. Clones in which pXL14 was integrated into the chromosome (by homologous recombination between the mutant shr allele in the chromosome and the wild-type copy on pXL14) were selected on kanamycin plates at 37°C (the nonpermissive temperature). The resulting chromosomal structure was verified by PCR analysis.

Antibodies. Rabbit polyclonal antibodies against Shr and rabbit anti-SiaA serum were previously described (4). Rabbit polyclonal antibodies against M protein type 49 (a killed whole-cell vaccine that was absorbed extensively to produce M49-specific antiserum) were provided by Bernard Beall (Centers for Disease Control and Prevention Respiratory Diseases Branch, Atlanta, GA). Rabbit immunoglobulin G (IgG) against GAS type-specific carbohydrates was purchased from Abcam, Inc. Rabbit antiserum for the metal-dependent repressor MtsR (5) was raised against purified MtsR using the same method employed for the production of Shr antiserum (4). Western blot analysis determined that the MtsR antiserum specifically recognizes purified MtsR protein and reacted with protein bands at the correct size in protein extract from wild-type strain NZ131 but not from mtsR mutant strain ZE491 (5; data not shown). Mouse antiserum against Lactococcus lactis surface components was raised by intraperitoneal injection with 108 CFU of MG1363 cells. A booster injection was given 10 days after the first injection. Antiserum was collected 10 days after the booster.

Preparation of GAS cell fractions. GAS cell fractionation was done as previously described, with minor modifications (38). Essentially, GAS was grown overnight in THY broth containing 20 mM glycine. Cells from 14-ml cultures at an optical density at 600 nm (OD600) of 1.0 were harvested, washed with phosphate-buffered saline (PBS), and resuspended in 0.5 ml of either TEA [10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, and 0.75 µg/µl of the protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF); Roche] for the preparation of total cellular proteins or TEAR (TEA with 30% raffinose) for cellular fractionation. The cell suspensions were treated with the muralytic enzyme mutanolysin (500 U) for 1 h at 37°C to dissolve the cell wall. For total protein extract, the mutanolysin-treated cells were boiled in sample buffer for 10 min. For cell fractionation, the protoplasts were separated from the cell surface fraction by centrifugation, resuspended in TEA, and lysed by successive cycles of freezing (–80°C) and thawing (37°C). Membrane proteins were separated from the soluble intracellular proteins by centrifugation and were dissolved by boiling in sample buffer. The protein extracts were standardized with respect to the cell number in the corresponding culture and were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Shr, M protein type 49 (M49), or SiaA was detected by Western blotting as described previously (4).

Purification of rShr (His6-Xpress-Shr). Recombinant Shr (rShr) was purified from E. coli cells harboring plasmid pCB1 as previously described (4), with the following modifications: the cells were resuspended in a solution containing 20 mM Tris (pH 8.0), 100 mM NaCl, 0.1% Triton X-100, 0.5 mg/ml lysozyme, and a protease inhibitor cocktail (Complete Mini, EDTA free; Roche) and incubated on ice for 30 min. The bacteria were then lysed by sonication, and rShr was purified over a nickel column (HisTrap HP; GE Healthcare) and subsequently desalted by use of a HiTrap desalting column (GE Healthcare). The purified protein was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with anti-Shr antibodies and quantified by the Bradford assay (Bio-Rad).

ELISA. Three types of enzyme-linked immunosorbent assays (ELISAs) were used in the binding experiments.

(i) ELISA with immobilized bacteria. ELISA with immobilized bacteria was performed to study the availability of Shr on the bacterial cell surface. GAS or L. lactis cells from cultures grown overnight were harvested, washed with PBS, and used to coat 96-well EIA/RIA microplates (Costar; Corning Inc.) overnight at 4°C. The supernatant was then aspirated, and the plates were washed with PBS containing 0.05% Tween 20 (PBST). After blocking with PBST and 5% skim milk for 1 h at 37°C, the wells were washed with PBST and incubated with the primary antibody for 1 h at 37°C. The microplates were next washed with PBST and incubated with secondary antibodies conjugated to alkaline phosphatase (AP) for 1 h at 37°C. The reaction was developed using p-nitrophenyl phosphate system, and the absorbance was read at 405 nm.

(ii) ELISA with immobilized ligands and purified Shr protein. ELISA with immobilized ligands and purified Shr protein was used to investigate the binding of Shr to host proteins. The microplates were coated with the ligands (25 µg/ml), washed, and blocked as described above. Purified Shr (in PBST-1% bovine serum albumin [BSA]) was added to the coated wells in increasing concentrations. After 1 h of incubation at 37°C, Shr antiserum (diluted in PBST-1% BSA) was added to the wells, and the plates were incubated for 1 h at 37°C. The wells were then washed with PBST, and binding was detected with AP-conjugated secondary antibody as described above.

(iii) Analysis of L. lactis cell binding to immobilized ligands by ELISA. For analysis of L. lactis cell binding to immobilized ligands by ELISA, the microplates were coated with the ligands (25 µg/ml), washed, and blocked as described above. Samples of L. lactis cultures were harvested, washed with an equal volume of PBS, diluted into the desired concentrations in PBST-1% BSA, and applied to the wells. Following 1 h of incubation at 37°C, the plates were washed with PBST and reacted with the L. lactis antiserum. Binding was detected with secondary AP-conjugated antibody as described above.

Detection of anti-Shr antibodies in convalescent mice. Mice were injected subcutaneously with 2 x 107 CFU of wild-type strain MGAS5005 as previously described (43) and were monitored for 27 days. The infected mice developed subcutaneous lesions, which healed over time in the surviving animals (~80% of the injected mice). On the 28th day postinoculation, the surviving mice were bled by cardiac punctuation, and the serum was obtained by centrifugation. ELISA was used to determine the presence and the titer of anti-Shr antibodies in the mouse sera. Ninety-six-well EIA/RIA microplates (Costar; Corning Inc.) were coated with 1 µg/ml purified Shr. Following coating at 4°C overnight, the plates were washed with PBST and blocked with 1% (wt/vol) BSA in PBST for 1 h at 37°C. The plates were then washed with PBST and incubated for 1 h at 37°C with mouse sera at different dilutions. Following incubation and washing, AP-conjugated anti-mouse antibodies diluted 1:1,000 in blocking buffer were added to the plates and incubated for 1 h at 37°C. Bound antibodies were detected by using the p-nitrophenyl phosphate system. Plate contents were incubated at room temperature for 30 min and read at 405 nm.

HEp-2 cell culture and adherence assay. HEp-2 cells were cultured in 24-well tissue culture plates with nutrient mixture medium (RPMI) supplemented with 10% fetal bovine serum at 37°C in an atmosphere with 5% CO2. For adherence assays, cells grown to a semiconfluent state (~105 cells/well) with antibiotics (2% penicillin-streptomycin; Thermo Scientific Hyclon Antibiotics) were washed with PBS and inoculated with GAS cells (107 CFU/well) harvested at the mid-log phase (OD600 of 0.3 to 0.4), washed with PBS, and resuspended in RPMI medium. The bacteria were incubated with the tissue cultures for 2 h at 37°C; at that time, nonadherent bacteria were removed by successive washes with PBS. To determine the number of adherent bacteria, HEp-2 cells were detached with 100 µl of 0.05% trypsin for 5 min at 37°C and lysed with 400 µl per well of 0.025% Triton X-100. The bacterial number in each sample was determined by plating onto THY agar plates. Each adherence experiment was performed in triplicate or quadruplicate.

Zebrafish care and virulence assays. Care and feeding of zebrafish (Danio rerio) were done according to previously reported methods (34, 54). Streptococci were cultured overnight in THY broth with 2% (wt/vol) peptone (THYP) at 37°C, diluted 1:100 in THYP the next day, and incubated at 37°C. The cells were harvested at an OD600 of 0.3, washed once with THYP, and diluted to an appropriate concentration in fresh THYP. Streptococcal cells (10 µl) were aseptically injected into groups of six anesthetized male breeder zebrafish (Scientific Hatcheries). Following intramuscular (i.m.) infection, the fish were allowed to recover in 225 ml sterilized double-distilled water supplemented with aquarium salts (Instant Ocean; Aquarium Systems) at a concentration of 60 mg/liter in a 25°C incubator. Infected fish were monitored for 72 h, and death was recorded in intervals of 12 h. The 50% lethal dose (LD50) for infection was determined by the Reed-Muench method as previously described (34), where zebrafish were challenged over a range of 103 to 107 CFU of each of the streptococcal strains.

Competitive assay in the zebrafish model. The competitive index (CI) was defined as the change in the population ratio of two strains after growth in zebrafish muscle. NZ131 (wild type) and the mutant strain (ZE4912) were cultured separately as described above. Cells from each strain were mixed in a 1:1 ratio to a final concentration of 108 CFU/ml. Zebrafish were infected i.m. with 10 µl of this mixture, resulting in an infectious dose of 106 CFU. After 24 h, muscle lesions were collected from euthanized fish and homogenized in PBS. The homogenates were plated onto THY and THY-spectinomycin plates, and the ratio of the mutant to the wild-type strain in the lesion was determined.

Statistical analysis. A Student's t test was used to compare data sets derived from two groups to each other; a P value of ≤0.05 was considered to be significant. Analysis of variance (ANOVA) followed by the Tukey honest significant difference (HSD) post hoc test was used for multigroup comparisons; a P value of ≤0.05 was considered to be significant. Zebrafish survival data were analyzed by the method of Reed and Muench for the calculation of the LD50. Kaplan-Meier plots of zebrafish survival were used to compare infections by the wild-type, mutant, and complemented GAS strains. The statistical significance was evaluated by the log rank test (18).


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RESULTS
 
Shr is attached to the GAS cell membrane and is exposed to the extracellular environment. Shr is a large (1,275-amino-acid) protein found both in the culture supernatant and in association with whole cells (4). It has a functional leader peptide, and it has a putative transmembrane domain followed by a positively charged tail at its carboxy terminus (Fig. 1A). However, unlike proteins anchored to the surface of gram-positive bacteria, Shr lacks a cell wall-anchoring motif at its C terminus (45), suggesting that after it is exported to the cell surface, Shr may be bound to the cell membrane and not anchored to the cell wall. To determine the cellular location of Shr, GAS strain NZ131 (M type 49) was fractionated following digestion with the muralytic enzyme mutanolysin and examined by Western blot analysis. The Shr protein (at 145 kDa) was not released by this process but was found in the membrane fraction instead (Fig. 1B). As a control, the cell wall protein M49, represented by the monomer at 37 to 40 kDa (as predicted from the genome sequence) and the typical range of slower-migrating bands containing cell wall fragments (15), was found exclusively in the cell wall fraction, as expected (Fig. 1C). Therefore, Shr appears to remain associated with the cell membrane, probably by the transmembrane domain in its carboxy terminus.

The cellular location of Shr was further studied in an shr deletion-insertion mutant (ZE4912) (Fig. 2A) and the mutant strain complemented with shr expressed from its own promoter in the chromosome (ZE4924) (see Materials and Methods). To examine the effects of the shr mutation and of its complementation on the expression from the sia operon, we preformed reverse transcription-PCR and Western blot analysis on RNA and proteins extracted from isogenic strains NZ131, ZE4912, and ZE4924. As can be seen in Fig. 2, the transcription of the siaA gene (Fig. 2B) and the production of this protein (Fig. 2C) were not significantly altered in either the mutant or the complementation strain in comparison to the parent strain. Therefore, the mutation in shr does not seem to be polar on the downstream genes in the sia operon. On the other hand, cell fractionation and Western blot analyses showed that the shr mutation in ZE4912 resulted in the loss of Shr from the membrane fraction (Fig. 2D, lane 2) and in the formation of a truncated Shr fragment (25-kDa) that was secreted into the culture supernatant (data not shown). The complementation of shr in ZE4924 strain restored the presence of the Shr protein in the cell membrane fraction (Fig. 2D, lane 3). These observations are consistent with the suggestion that the carboxy terminus of Shr is required for the Shr association with the cell membrane and with our previous observation that Shr has a leader peptide that can target its secretion.

To test whether membrane-bound Shr is exposed on the cell exterior or is buried within the peptidoglycan layer, we conducted whole-cell ELISA using Shr antibodies. GAS cells grown to stationary phase were used to coat ELISA plates and reacted with anti-Shr serum. An antiserum against the repressor protein MtsR (5) was used as a control for intracellular proteins, and normal rabbit serum (NRS) was used as a control for any nonspecific reaction. As shown in Fig. 3, the Shr antiserum (black bars) reacted with the wild type (NZ131) and with the complementation strain (ZE4924). The specificity of the antiserum for Shr was demonstrated by its lack of reaction with an shr mutant strain (ZE4912). In all tested strains, no significant reactivity was obtained with the MtsR antiserum or the NRS (Fig. 3, stripes and white bars, respectively). As expected, the positive control antibodies, which specifically recognize GAS type-specific carbohydrates, reacted with all of the GAS strains examined (gray bars). Similarly to its interaction with NZ131 whole cells, Shr antiserum specifically recognized cells of strains JRS4 (M type 6) and MGAS5005 (M type 1) (data not shown). These observations suggest that Shr is exposed on the surface and accessible for interactions with the host extracellular environment.


Figure 3
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  		FIG. 3. Shr is exposed on the surface of GAS. Data are from ELISA with GAS cells immobilized onto microtiter plates. Wells coated with cells from the isogenic series of NZ131, ZE4912, and ZE4924 were reacted with antibodies recognizing GAS type-specific carbohydrates (gray bas), Shr antiserum (black bars), or control antibody MtsR antiserum (striped bars) or NRS (white bars) as described in Materials and Methods. Antibody binding to GAS surface antigens was detected by anti-rabbit (AP)-conjugated secondary antibodies. The reaction was developed and measured at 405 nm. Each bar represents the average of at least three repeats, and the standard deviation (SD) is represented by the error bars. The significance of the difference in the bindings of anti-Shr serum to the three strains was examined by ANOVA (P = 0.001), followed by the Tukey HSD test for each strain pair. The asterisk indicates that the difference between the strains is significant (P < 0.01 for each strain pair).

Shr is an MSCRAMM. The surface localization and exposure of Shr raised the possibility that it may function in bacterial adherence in addition to iron uptake. We therefore examined the interactions of Shr with ECM components using ELISA assays. Different ligands immobilized onto microtiter plates were reacted with increasing concentrations of purified Shr. Shr binding was detected using Shr antiserum (Fig. 4). As a control for nonspecific interactions, wells coated with transferrin or goat IgG were incubated with the Shr antiserum directly. Only low background binding (OD405 of 0.1 ± 0.01) (Fig. 4 and data not shown) of the Shr antiserum to the control wells was observed, demonstrating the specificity of the Shr antibodies. Although Shr did not bind to transferrin (Fig. 4, diamonds) and goat IgG (data not shown), Shr bound to immobilized fibronectin (Fig. 4, triangles) and laminin (Fig. 4, squares) in a dose-dependent and saturable manner.


Figure 4
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  		FIG. 4. Shr binds in vitro to ECM components. The Shr protein was added in increasing concentrations to microtiter plate wells coated with fibronectin (triangles), laminin (squares), or transferrin (diamonds) and incubated for 1 h at 37°C. Bound Shr was detected by anti-Shr antibodies and anti-rabbit AP-conjugated secondary antibodies. Each datum point stands for the mean ± SD (represented by the error bars) from data from four independent experiments done in duplicates.

Inhibition experiments demonstrated the specificity of binding of Shr to the ECM ligands fibronectin and laminin. When Shr was allowed to interact with immobilized fibronectin in the presence of increasing concentrations of soluble fibronectin, the binding of Shr to the plates was inhibited in a dose-dependent manner (Fig. 5A, triangles). On the other hand, transferrin, which was not recognized by Shr in vitro, could not compete with immobilized fibronectin for binding to Shr (Fig. 5A, diamonds). Similarly, soluble laminin inhibited the binding of Shr to wells coated with laminin (Fig. 5B). Laminin also competed with fibronectin for Shr binding (up to 40% inhibition) (Fig. 5A, squares). However, at similar molar concentrations, laminin was less effective at competition than was soluble fibronectin (80% inhibition). This demonstrates that the binding of Shr is specific to a subset of ECM components.


Figure 5
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  		FIG. 5. Inhibition of Shr binding to immobilized ECM ligands. (A) Binding of purified Shr to immobilized fibronectin as a function of the competitor concentration. Shr (3.5 nM) was preincubated for 1.5 h at room temperature with increasing concentrations of fibronectin (triangles), laminin (squares), or transferrin (diamonds) prior to its addition to fibronectin-coated wells. Bound Shr was detected with anti-Shr and anti-rabbit AP-conjugated secondary antibodies. (B) Binding of Shr to immobilized laminin as a function of the soluble laminin concentration. Data are the same as in A except that 3.5 nM of Shr was preincubated with increasing concentrations of laminin before incubation in the laminin-coated wells. In both A and B, each datum point represents the mean of data from six independent experiments done in duplicate, and the standard deviation is represented by the error bars. The competitor amount is expressed as a multiple of the molar concentration of Shr.

GAS expresses multiple fibronectin binding proteins on its surface (22, 50). To test to the ability of Shr to mediate bacterial binding to fibronectin in the absence of the other GAS fibronectin-binding proteins, we expressed Shr in a heterologous host. Plasmid pXL14, which carries the native shr gene, was introduced into L. lactis strain MG1363. The production of the Shr protein in Lactococcus was confirmed by Western blot analysis (data not shown), and the presentation of Shr on the cell surface was tested by ELISA. Microtiter plates were coated with cells of native L. lactis strain MG1363 or the recombinant strain that harbors pXL14 (MG1363/pXL14). The coated wells were reacted with the Shr antiserum (Fig. 6A, black bars), L. lactis antibodies (Fig. 6A, gray bars), or NRS (Fig. 6A, white bars). The Shr antiserum reacted significantly (P = 0.0037) only with bacteria that harbor the shr plasmid (pXL14), demonstrating specificity for Shr. As expected, the L. lactis antibodies interacted strongly and similarly with both strains MG1363 and MG1363/pXL14, and only weak binding of NRS to both strains was observed. Therefore, like in its native host, the Shr protein produced in L. lactis is exposed on the bacterial cell surface.


Figure 6
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  		FIG. 6. Shr expressed on the L. lactis cell surface promotes bacterial binding to fibronectin. Cells of wild-type lactococcal strain MG1363 and of a recombinant MG1363 strain harboring plasmid pXL14 (MG1363/pXL14) were tested for Shr surface presentation and cell binding to different ligands. (A) ELISA with immobilized L. lactis cells and anti-Shr serum. Wells coated with L. lactis cells were reacted with anti-L. lactis serum (gray bars), anti-Shr serum (black bars), or NRS (white bars) as described in Materials and Methods. (B) Analysis of L. lactis cell binding to immobilized fibronectin. L. lactis cells were reacted with wells coated with fibronectin (black bars) or transferrin (white bars) as described in Materials and Methods. Bound bacteria were detected with anti-L. lactis serum. Antibody binding was detected with secondary (AP) conjugated antibodies. Each bar represents the average of data for at least four repeats, and the SD is represented by the error bars. The asterisks indicate statistical significance for the differences between MG1363 and MG1363/pXL14 in Shr-antiserum binding (P = 0.0037 [A]) and in binding to immobilized fibronectin (P = 0.001 [B]).

The binding of recombinant L. lactis cells expressing Shr to fibronectin and to transferrin was investigated. The two ligands were immobilized onto microtiter plates, and the coated wells were incubated with bacteria. The binding of Lactococcus to the immobilized ligands was detected using the L. lactis antiserum. Only weak binding of strain MG1363 to fibronectin (Fig. 6B, black bars), transferrin (Fig. 6B, white bars), or uncoated wells (data not shown) was observed, demonstrating that L. lactis does not interact strongly with fibronectin or transferrin. In contrast, MG1363 cells harboring plasmid pXL14 demonstrated significant (P = 0.001) binding to immobilized fibronectin (Fig. 6B, black bars); these cells, however, did not bind to transferrin (Fig. 6B, white bars). Therefore, L. lactis cells expressing Shr gained the ability to specifically bind to fibronectin. This observation demonstrates that Shr can mediate cell binding to the ECM and supports its possible role as a bacterial adhesin.

To test the function of Shr in the adherence of whole GAS cells directly, the shr mutant (ZE4912) was compared to its wild-type parent (NZ131) as well as to the mutant complemented with the shr gene (ZE4924). The binding of GAS to HEp-2 cells was assayed following incubation at 37°C for 2 h. In comparison to the wild-type strain, the shr mutant demonstrated about a 40% reduction in adherence (Fig. 7). This difference is statistically significant (P < 0.05). Although the complemented strain bound to the HEp-2 cells better than the mutant, it bound only about 75% as well as the wild type. The difference in binding between the complemented strain and either the wild type or the shr mutant was not statistically significant. Thus, although complementation was not complete, Shr seems to act as an adhesin in GAS attachment to HEp-2 cells.


Figure 7
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  		FIG. 7. Adherence of GAS to cultured epithelial cells. Mid-log-phase cultures of the wild-type strain (NZ131), the shr mutant (ZE4912), and the complemented strain (ZE4924) were incubated with confluent HEp-2 cell monolayers for 2 h at 37°C under 5% CO2. Unattached bacteria were removed by washing, cells were detached and lysed, and the number of bound bacteria per well was determined by plating. Each bar represents the mean number of GAS cells attached as obtained from at least two independent experiments performed in triplicate. The standard error of the mean is expressed by the error bar. The significance of the differences in adherence among the three strains was examined by ANOVA (P = 0.025), followed by the Tukey HSD test for each strain pair. The asterisk indicates that the difference between NZ131 and ZE4912 is statistically significant (P < 0.05).

Shr production and surface presentation are conserved among clinical isolates. A collection of 17 clinical isolates representing seven different M serotypes was examined for Shr expression using whole-cell ELISA. As summarized in Table 1, Shr antiserum reacted with most of the isolates (14 out of 17) and produced a signal that was at least twofold higher than the background signal produced with the control serum NRS. However, a significant variation in the strength of the produced signal was found among the different clinical isolates. This range of reactions may result from differences in the amount of Shr protein produced by each strain or may be due to antigenic differences in Shr and subsequently weaker interactions with the Shr antiserum. The Shr sequence is very conserved among GAS genomes available in the database; however, the sequence variability may increase among clinical isolates. In summary, the Shr protein was present on the surface in 17 out of the 20 strains tested in this study (including NZ131, MGS5005, and JRS4), suggesting that this protein is important for the GAS infection process.

Shr is expressed by GAS in vivo and elicits an immune response in mice. In cells growing in vitro in the laboratory, shr expression is regulated by the availability of iron in the medium (5). To determine if the conditions in vivo during GAS infection in a murine model allow the expression of Shr, we asked if a challenge with GAS leads to the production of Shr antibodies in mice. Mice were injected subcutaneously with strain MGAS5005 (M type 1), and sera collected 28 days after inoculation were assayed by ELISA for Shr antibody titers. Purified Shr was used to coat microtiter plates, which were then reacted with mouse sera (Fig. 8). Sera from mice injected with GAS reacted strongly with purified Shr in most of the animals tested (9 out of 14), whereas none of the sera obtained from naïve mice were reactive against Shr. The conversion of most mice to Shr positive demonstrates that Shr is immunogenic and that in most of the mice, it was produced during infection in sufficient amounts and durations to elicit an immune response.


Figure 8
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  		FIG. 8. Shr antibody levels in mice following recovery from GAS infections. Mice were inoculated subcutaneously with a sublethal dose of MGAS5005 (M1). Serum was collected from each mouse 28 days following challenge, diluted 1:1,000, and tested for reactivity with purified Shr by ELISA as described in Materials and Methods. Each datum point represents the titer obtained from an individual mouse.

Shr is important for GAS pathogenesis in the zebrafish infection model. The use of zebrafish as an infection model for pathogenic streptococci was previously established. i.m. injection of zebrafish with the native fish pathogen Streptococcus iniae causes a systemic disease, while injection with GAS causes mostly local muscle lesions and necrosis (34). The effect of shr inactivation on virulence was investigated via the i.m. infection route, as previously described (33, 34). Groups of six zebrafish were challenged with 106 CFU per fish of cells of wild-type strain NZ131, the shr mutant (ZE4912), or the mutant complemented with shr (ZE4924) (Fig. 9). Injection with the wild-type strain resulted in rapid fish death, with fish survival beginning to decline as early as 12 h postinfection, reaching a final survival rate of 22% at 48 h postinfection (Fig. 9, filled squares). On the other hand, the shr mutant strain was attenuated for virulence; fish injected with the mutant strain (Fig. 9, open squares) exhibited a slower decline in survival, and the final survival rate was significantly higher than that of fish infected with the wild-type strain (62% survival at 48 h postinfection; P < 0.0012). The complemented strain (Fig. 9, filled triangles) demonstrated intermediate virulence; fish infected with this strain died at a lower rate than that of fish infected with the wild-type strain (100% survival at 12 h and 24 h postinfection). However, the final survival rate eventually declined to a level similar to that of fish infected with the wild-type strain (25% survival at 48 h postinfection). Determination of LD50 values was done by injecting each fish with a range of 104 to 107 CFU of each strain. Consistent with the attenuated phenotype observed for the shr mutant, the wild-type LD50 value was significantly lower (5 x 104 CFU per fish) than that of the mutant (2.5 x 106 CFU per fish), and an intermediate LD50 value (5.6 x 105 CFU) was observed with the complemented strain, demonstrating a partial complementation of virulence.


Figure 9
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  		FIG. 9. Zebrafish survival following i.m. injection. Kaplan-Meier plots showing survival of zebrafish following i.m. challenge with 106 CFU of GAS wild-type (NZ131) (filled squares), shr mutant (ZE4912) (open squares), and complemented (ZE4924) (filled triangles) strains are shown. The asterisks represent statistically significant differences between ZE4912 (n = 18) and NZ131 (n = 56) (P = 0.0012) and between ZE4912 (n = 18) and ZE4924 (n = 12) (P = 0.03) by the log rank test.

The impact of Shr on the infection process in GAS was also evaluated by testing the ability of the shr mutant to compete with the wild-type strain for growth in the muscle tissue during coinfection. Zebrafish were inoculated by i.m. injection with 106 CFU of a mixed culture comprised of a 1:1 ratio of wild-type to mutant cells. The lesion area was excised 24 h postinfection, and the number of viable bacteria of each strain in the tissue was determined by comparing the bacterial recovery on plates with spectinomycin (the mutant's marker) to that on antibiotic-free plates. The CI is defined as the output ratio of mutant to wild-type bacteria divided by the input ratio of mutant to wild-type bacteria. The CI of the shr mutant was calculated to be 0.011 ± 0.002. The growth rate of the shr mutant in vitro in THYB was identical to that of the wild-type strain. In addition, the chromosomal mutation in shr was stable during growth in THYB in the absence of spectinomycin, as was evident from the plating efficiency (100%) of this strain on selective plates after overnight growth in the absence of antibiotics. Therefore, the low CI suggests that the inactivation of shr dramatically reduced the in vivo fitness of GAS in comparison to the wild-type strain in zebrafish tissue. Since Shr is found both on the cell surface and in the culture supernatant, the fact that the presence of the wild-type strain during infection could not rescue the attenuated phenotype of the mutant suggests that the surface-associated form of Shr is important for GAS fitness during infection.


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DISCUSSION
 
The human pathogen GAS exports numerous proteins to its surface, where they carry out the many tasks that mediate colonization and disease. Examples of these tasks include the escape and subversion of the immune response, adherence, and nutrient scavenging (6). Shr is a large, complex, and highly conserved surface protein in GAS (Fig. 1A) that does not share extensive sequence homology with other proteins found in the database (4). Although the functional role of Shr has not yet been deciphered, this protein has been shown to bind hemoproteins and is also suggested to mediate heme acquisition and transport in conjunction with Shp and the SiaABC heme transporter (4, 36). The major objectives of this study were to explore the function of Shr and its role in GAS pathogenesis in depth. The results show that the contribution of Shr to the GAS infection process extends beyond iron uptake to include interactions with the ECM and adherence to epithelial cells. Therefore, Shr represents a new MSCRAMM in GAS that is necessary for virulence in the zebrafish model of necrotizing fasciitis.

When GAS is grown in culture under laboratory conditions, Shr is found in the supernatant as well as in association with whole GAS cells. Even though Shr contains a leader peptide that can mark proteins for secretion (4), the mechanism that facilitates the association of Shr with the GAS surface remains unclear. The majority of surface proteins in gram-positive bacteria are covalently attached to the peptidoglycan by a sortase enzyme, which recognizes a C-terminal cell wall-sorting motif followed by a hydrophobic segment and a charged tail (31, 45). The LPXTG sequence serves as the sorting signal for the housekeeping sortase in GAS. Shr does not have an LPXTG motif in its C terminus or the QVPTG signal that is recognized by srtC2, an accessory sortase that is also produced by GAS strain NZ131 (3). Using cell fractionation and immunoblotting, this study demonstrated that Shr is not bound to the cell wall; rather, Shr is found in the membrane fraction (Fig. 1B). The absence of recognizable cell wall sorting signals in the Shr sequence is consistent with these findings. It seems likely that Shr remains associated with the cytoplasmic membrane after its export to the surface due to the putative transmembrane segment and charged tail found in its C terminus (Fig. 1A), as was found to be the case for ActA of Listeria monocytogenes (21). Shr is not present in the membrane of mutant GAS strain ZE4912, since the mutation is located in the 5' coding region of the shr gene and therefore results in the formation of a truncated protein secreted into the culture supernatant (Fig. 2 and data not shown). The secretion of the abridged Shr fragment supports the hypothesis that the C terminus in Shr is required for its membrane anchoring and therefore for its association with GAS cells.

The ability of anti-Shr serum to recognize Shr on the surface of whole GAS cells (strains NZ131 and ZE4924) (Fig. 3, black bars) shows that the wild-type Shr protein spans the cell wall in GAS and is exposed to the extracellular environment. Therefore, Shr may be able to interact with large extracellular molecules, in addition to small ligands capable of diffusing the cell wall. Since Shr appears to be anchored to the cytoplasmic membrane through its C terminus, it is possible that it can deliver heme from the extracellular compartment directly to the transport components found in the cell membrane. This heme transport scheme is different from the one proposed previously by Maresso and Schneewind for heme uptake by the Isd proteins in Staphylococcus aureus, where heme is relayed in a cascade fashion from surface-exposed NEAT proteins, such as IsdA, IsdB, or IsdH, to an IsdC protein that is found deeper in the peptidoglycan; heme is then transferred by IsdC to the IsdDEF membrane transporter for uptake (30).

Using solid-phase binding assays, we were able to demonstrate that Shr also binds the ECM components fibronectin and laminin (Fig. 4). Competition studies performed with soluble and immobilized fibronectin and laminin demonstrated that Shr interactions with these adhesive molecules are specific (Fig. 5). Therefore, Shr appears to have a broader spectrum of ligands than previously suggested. Shr availability on the GAS surface combined with its affinity for ECM components qualifies this receptor as a new MSCRAMM in GAS. The report also states that recombinant L. lactis cells that express Shr on the surface bind specifically to immobilized fibronectin (MG1363/pXL14) (Fig. 6), which is an activity not found in native L. lactis cells (MG1363) (Fig. 6). This observation shows that Shr is able to mediate bacterial attachment to the ECM and further supports its role as an MSCRAMM. Interestingly, the Shr amino acid sequence does not contain the typical fibronectin-binding repeats found in other fibronectin MSCRAMMs (22, 44). Thus, it is not clear which part of this large protein mediates the binding to fibronectin or to laminin. It was previously reported that the NEAT protein IsdA in S. aureus binds to several nonheme host proteins, including the ferric carrier transferrin (51), several matrix and plasma proteins, and hemoglobin (8). The results presented here demonstrate that Shr does not bind to transferrin, as it has a binding pattern similar but not identical to that of IsdA. The ligand binding in IsdA is attributed to its single NEAT domain, but the residues involved in binding to the serum and ECM proteins were not determined. The sequence homology between IsdA and Shr is limited to the NEAT regions and is quite low even within these domains. Moreover, Shr is a significantly larger and more complex protein than IsdA (145 and 38 kDa, respectively) and has a central leucine-rich repeat (LRR) segment, a unique amino-terminal region, and two NEAT domains. Additional analysis is required to determine the domains and the residues involved in the recognition of various Shr ligands.

The data presented show that Shr helps facilitate GAS attachment to HEp-2 epithelial cells, where a reduction in binding of about 40% was observed in the shr mutant. This decrease in adherence is statistically significant and can be partially restored in the complementation strain (Fig. 7). Since GAS expresses several adhesins that mediate binding to HEp-2 cells, it is not surprising that only a small reduction in adherence was observed in the shr mutant. As far as we can determine, Shr is the only adhesin reported to be induced in response to the iron restriction likely encountered during infection. Therefore, Shr's contribution to adherence may be more significant under such disease-specific conditions. Shr-dependent adherence may take place through fibronectin binding or additional serum-bridging molecules, as found for other GAS adhesins (22, 50). Alternatively, Shr may interact directly with a host cell receptor via a mechanism that may be assisted by its LRR domain (Fig. 1A). The LRR was previously suggested to provide a scaffold for the formation of protein-protein interactions (20), and the LRR domain in internalin was shown to be necessary and sufficient for binding to E-cadherin in L. monocytogenes (25).

It seems likely that shr complementation was not able to restore adherence to the level observed in the wild type due to the nature of the mutation and the method used for complementation. The shr mutation in ZE4912 results in the production of a truncated Shr fragment that is secreted into the extracellular medium. This Shr fragment, which is also produced by the complementation strain, may serve as competitor interfering with Shr-mediated attachment to epithelial cells and impairing adherence in the complementation strain. In addition, complementation strain ZE4924 is a merodiploid strain created by Campbell insertion. Since this type of mutation is unstable, it may excise and reduce the efficiency of complementation in the binding assays.

This study presents several experiments that suggest that Shr is important for GAS virulence. A single infection event was found to be sufficient to trigger a significant antibody response to Shr in convalescent mice, indicating that Shr is expressed in vivo in an adequate amount and an adequate duration to elicit a host response (Fig. 8). While the majority of the injected mice (9 out of 14) developed high Shr titers, the anti-Shr antibody levels in the remaining five mice were low. The observed variations in the antibody response following GAS challenge may result from differences in the time that it took individual mice to clear the bacteria, from variations in the efficiency of their immune response, or from both. Analysis of Shr production in different GAS strains, including a collection of 17 clinical isolates (Table 1), revealed that Shr could be detected on the surface of most of the examined strains. The inactivation of shr resulted in an attenuation of virulence in a zebrafish model (ZE4912) (Fig. 9), with the LD50 of the mutant being about 50 times higher than that of the wild-type strain. As in the adherence assay, only a partial restoration of virulence was observed with the complemented strain. Both the kinetics of survival in zebrafish postinfection and the LD50 value of complementation strain ZE4924 demonstrated an intermediate level of virulence in comparison to those of wild-type strain NZ131 and shr mutant strain ZE4712. This suggests that either an instability of the complemented strain or the production of both full-length and a truncated fragment of Shr by ZE4924 prevented a complete recovery of virulence. The recovery of the mutant strain from the lesion tissue was much less than that of the wild-type strain after coinfection (CI of 0.01). This observation demonstrates that the inactivation of shr results in a mutant strain that is significantly less fit than the wild-type parent strain. Shr is found both in the culture supernatant and on the cell surface. The inability of the wild-type strain to complement the in vivo growth defect of the mutant strain during coinfection of zebrafish muscle strongly suggests that it is the surface-anchored form of Shr that is important for the ability of GAS to persist in the host during infection.

In summary, given that Shr is a broad-spectrum surface receptor contributing to iron acquisition (4; data not shown), ECM binding, and adherence, its contribution to the disease process in GAS appears complex. Ongoing research is under way to further investigate this important multifunctional surface protein and to better understand its role in the disease process.


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ACKNOWLEDGMENTS
 
We are grateful to Bernard Beall for his generous gift of strain ZE4912 and the M49 antiserum. We thank Monica M. Farley, Sarah W. Satola, and the Georgia Emerging Infections Program for their help and for providing clinical isolates.

This work was supported by NIH NIAID award 5R01AI57877.


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FOOTNOTES
 
* Corresponding author. Mailing address: Biology Department, Georgia State University, P.O. Box 4010, Atlanta, GA 30302-4010. Phone: (404) 413-5401. Fax: (404) 413-5301. E-mail: zeichen{at}gsu.edu Back

{triangledown} Published ahead of print on 18 August 2008. Back

Editor: J. N. Weiser

{dagger} M.F. and Y.-S.H. contributed equally to this work. Back


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Infection and Immunity, November 2008, p. 5006-5015, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00300-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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