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Infection and Immunity, July 2006, p. 4200-4213, Vol. 74, No. 7
0019-9567/06/$08.00+0     doi:10.1128/IAI.00493-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of an Antigen I/II Family Protein Produced by Group A Streptococcus

Shizhen Zhang,1 Nicole M. Green,1,2 Izabela Sitkiewicz,1 Rance B. LeFebvre,2 and James M. Musser1*

Center for Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute, and Department of Pathology, The Methodist Hospital, Houston, Texas 77030,1 Department of Pathology, Microbiology, and Immunology, University of California at Davis, Davis, California 956162

Received 27 March 2006/ Returned for modification 14 April 2006/ Accepted 24 April 2006


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ABSTRACT
 
Group A Streptococcus (GAS) is a gram-positive human bacterial pathogen that causes infections ranging in severity from pharyngitis to life-threatening invasive disease, such as necrotizing fasciitis. Serotype M28 strains are consistently isolated from invasive infections, particularly puerperal sepsis, a severe infection that occurs during or after childbirth. We recently sequenced the genome of a serotype M28 GAS strain and discovered a novel 37.4-kb foreign genetic element designated region of difference 2 (RD2). RD2 is similar in gene content and organization to genomic islands found in group B streptococci (GBS), the major cause of neonatal infections. RD2 encodes seven proteins with conventional gram-positive secretion signal sequences, six of which have not been characterized. Herein, we report that one of these six proteins (M28_Spy1325; Spy1325) is a member of the antigen I/II family of cell surface-anchored molecules produced by oral streptococci. PCR and DNA sequence analysis found that Spy1325 is very well conserved in GAS strains of distinct M protein serotypes. As assessed by real-time TaqMan quantitative PCR, the Spy1325 gene was expressed in vitro, and Spy1325 protein was present in culture supernatants and on the GAS cell surface. Western immunoblotting and enzyme-linked immunosorbent assays indicated that Spy1325 was produced by GAS in infected mice and humans. Importantly, the immunization of mice with recombinant Spy1325 fragments conferred protection against GAS-mediated mortality. Similar to other antigen I/II proteins, recombinant Spy1325 bound purified human salivary agglutinin glycoprotein. Spy1325 may represent a shared virulence factor among GAS, GBS, and oral streptococci.


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INTRODUCTION
 
Group A Streptococcus (GAS) is a human pathogen responsible for a myriad of infections ranging from pharyngitis and cellulitis to severe life-threatening invasive diseases, such as streptococcal toxic shock-like syndrome and necrotizing fasciitis. The molecular mechanisms underlying the ability of GAS to cause such a wide array of diseases are poorly understood. To enhance understanding of GAS pathogenesis, the genomes of 11 GAS strains representing eight different M protein serotypes (M1, M2, M3, M4, M6, M12, M18, M28) prevalent in GAS pharyngitis and invasive disease have been sequenced (3, 5, 17, 20, 43, 55, 58). The genome sequence of each strain has revealed previously undescribed cell surface and secreted proteins, provided new leads for pathogenesis research, and documented the crucial role of horizontal gene transfer in GAS evolution. For example, genome sequencing, comparative genomics, and molecular population genetics analyses have shown that prophages and other horizontally transferred elements are the primary source of variation in gene content among GAS strains (2).

We recently sequenced the genome of a serotype M28 GAS strain (20). Our interest in the analysis of a serotype M28 strain genome was stimulated by two factors. First, serotype M28 strains are among the top four M protein serotypes causing pharyngitis, invasive episodes, and other infections (1, 15, 34, 44, 52). Second, serotype M28 strains are significantly overrepresented among cases of puerperal sepsis (also known as childbed fever), a rare but serious postpartum infection (9, 11, 15, 18, 64, 68). The molecular mechanisms responsible for the enrichment of serotype M28 strains in puerperal sepsis are not known, symptomatic of the general lack of understanding of the pathogenic processes underlying intraspecies disease specificity.

We postulated that the chromosome of a serotype M28 strain would contain genes contributing to the overabundance of these GAS strains in puerperal sepsis cases. Consistent with this hypothesis, the genome of serotype M28 GAS strain MGAS6180 contained a novel 37.4-kb foreign genetic element designated region of difference 2 (RD2) (20). Comparative genetic analysis of RD2 found that the region is similar in gene content and organization to regions described as "genomic islands" in serotype III and V group B streptococcus (GBS) strains NEM316 and 2603 V/R (19, 62). RD2 also has multiple genes with orthologues in prophages and plasmids. The overall G+C content of RD2 (35.1%) is considerably lower than the average GAS value (38.3%) and closely approximates that of GBS (35.7%), which suggests that this element was acquired by horizontal gene transfer. More recently, the genomes of six other GBS strains (serotypes Ia, Ib, II, III, and V) were partially sequenced, revealing the presence of regions closely similar to RD2 (61). Importantly, GBS is the primary cause of neonatal invasive infections and also commonly colonizes the female urogenital tract (6, 14). Thus, the discovery of a genetic element in GAS common to GBS strains was very noteworthy and suggested that RD2 may be one factor in the enrichment of serotype M28 strains in puerperal sepsis. Consistent with this idea, RD2 is present in other GAS serotypes associated with puerperal infections, including serotypes M2, M4, M28, M48, M77, and M124 (20).

The RD2 element encodes seven proteins with conventional gram-positive secretion signal sequences; six of the seven proteins have not been previously identified in GAS. One of these proteins, M28_Spy1325 (Spy1325), has sequence similarity to members of the antigen I/II family of proteins initially identified in oral streptococcal species. Antigen I/II proteins are structurally complex, multifunctional adhesins that bind human salivary glycoproteins and assist colonization of the oropharynx (27, 28). Antigen I/II proteins also have been the subject of vaccine research and have been reported to confer protection against oral streptococci (50, 53).

The goal of the present study was to begin analysis of the role of Spy1325 in GAS-host interactions. We describe allelic variation in Spy1325 and demonstrate that Spy1325 transcript and protein are expressed during in vitro growth and in human and mouse GAS infections. We show that, analogous to other antigen I/II family proteins, Spy1325 binds salivary agglutinin glycoprotein (SAG). Furthermore, immunization with Spy1325 confers protection against GAS challenge in a mouse model of invasive infection. We hypothesize that acquisition of RD2 by certain GAS strains has expanded the pathogenic potential of these organisms and is a contributing factor in the overrepresentation of these strains in neonatal invasive infections and puerperal sepsis.


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MATERIALS AND METHODS
 
Bacterial strains and culture conditions. GAS strain MGAS6180 (serotype M28) was isolated in 1998 from a patient in Texas with invasive disease (20). Strain MGAS10270 (serotype M2) was isolated in Texas in 2002 from a patient with pharyngitis (4). All other GAS strains used in this study were isolated from patients from diverse geographic localities with either pharyngitis or invasive disease (Table 1). GAS strains were grown on Trypticase soy agar containing 5% sheep blood (Becton Dickinson and Company, Franklin Lakes, NJ). Bacteria were incubated at 37°C in an atmosphere of 5% CO2. GAS grown as liquid cultures used Todd-Hewitt yeast (THY) broth under the conditions described above.


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  		TABLE 1. Characteristics of group A Streptococcus strains analyzed

Identification of antigen I/II family streptococcal orthologs. The amino acid sequence of Spy1325 was used as a BLASTP query against the NCBI database to identify orthologous proteins. Multiple amino acid sequence alignments were performed with CLUSTALW (63), and a phylogenetic tree was constructed using MacVector (version 8.0).

Analysis of allelic variation in the Spy1325 gene. Chromosomal DNA was isolated from overnight cultures of GAS (Table 1) using a modified phenol-chloroform extraction procedure. The full-length Spy1325 gene (~4 kb) was amplified with the primer set M28_1325Fwd/M28_1325Rev (Table 2). PCR was performed in a 25-µl reaction volume under the following conditions: 95°C for 5 min, 95°C for 30 s, 60°C for 30 s, and 72°C for 5 min (25 cycles) and a final extension step at 72°C for 7 min. PCR analysis of the region of Spy1325 encoding the alanine- and proline-rich repeats was performed with primer pairs Spy1325_Seq3/Spy1325_Seq4 and Spy1325_Seq6/Spy1325_Seq8 (Table 2). Sequencing primers (Table 2) were designed based on the Spy1325 gene in strain MGAS6180. DNA sequence analysis was conducted by standard procedures with an ABI Prism 3730 DNA sequencer (Applied BioSystems) using full-length Spy1325 PCR product as template. Sequence data were assembled and compared to the MGAS6180 Spy1325 reference sequence using Sequencer (GeneCodes, Ann Arbor, MI).


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  		TABLE 2. Primers and probes used for analysis of Spy1325

Overexpression and purification of recombinant proteins. The coding sequences for full-length Spy1325 and various fragments thereof (rAR, rD, rNAD, rPC, rC; see Fig. 4A) were amplified from serotype M28 strain MGAS6180 or MGAS9538, both of which have identical Spy1325 alleles. Primer pairs used for amplification are listed in Table 2. PCR products were cloned into vector pQE-30 (QIAGEN) or pET-21b (Novagen). The pQE-30 vector incorporates a six-His tag at the amino terminus of the recombinant protein, while pET-21b does not. All constructs were sequenced to rule out the presence of spurious mutations. Escherichia coli strains harboring the recombinant plasmids were grown at 37°C in 2 to 4 liters of Luria-Bertani broth supplemented with the appropriate antibiotics. The expression of recombinant proteins was induced by the addition of 0.3 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) at an optical density at 600 nm (OD600) of 0.5 to 0.8, and growth was continued until early stationary phase. An AKTA Explorer instrument (Amersham Biosciences) was used for all chromatography procedures. Cells were harvested by centrifugation, suspended in 10 mM Tris-HCl buffer (pH 7.9), and sonicated for 15 min. Cell debris was removed by centrifugation, and the supernatant was filtered through a 0.45-µm membrane before being loaded onto the column.


Figure 4
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  		FIG. 4. Spy1325 has trypsin-resistant and trypsin-sensitive fragments. (A) Schematic of recombinant Spy1325 fragments used in analysis. r1325 (aa 40 to 1319), mature Spy1325 without cell wall anchor motif; rAR (aa 201 to 434), alanine-rich repeats; rD (aa 435 to 646), divergent region; rNAD (aa 40 to 646), N-terminal, alanine-rich, and divergent regions; rPC (aa 647 to 1319), proline-rich repeats and conserved region; rC (aa 976 to 1319), conserved region. (B and C) Purified rSpy1325 (r1325) and fragments thereof were incubated in digestion buffer in the absence (B, –) or presence (C, +) of trypsin (10 ng/ml) for 17 h. Proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue. The arrowhead in panel C indicates trypsin.

His-tagged proteins were purified by affinity chromatography followed by ion-exchange chromatography and hydrophobic interaction chromatography. For recombinant proteins without the His tag, ion-exchange chromatography was first employed, followed by hydrophobic interaction chromatography. The purity of full-length, mature Spy1325 was increased by a final size-exclusion chromatography step. After the final purification step for each of the recombinant proteins, peak fractions were pooled, buffer exchanged to phosphate-buffered saline (PBS), quantified, aliquoted, and stored at –80°C. Details of chromatography procedures are given below. All columns were purchased from Amersham Biosciences.

Affinity chromatography. A HisTrap HP affinity column (5 ml) was equilibrated with 25 mM Tris-HCl (pH 7.9) containing 100 mM NaCl. Recombinant protein (full-length rSpy1325 and rPC) was eluted with a linear gradient of 25 mM Tris-HCl (pH 7.9) containing 200 mM imidazole and 100 mM NaCl. Fractions were analyzed for the presence of recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and peak fractions were pooled and dialyzed overnight at 4°C against 10 mM Tris-HCl (pH 7.9) before being loaded onto the ion-exchange column.

Ion-exchange chromatography. Recombinant protein samples (rSpy1325, rAR, rD, rNAD, rPC, and rC) were dialyzed against 10 mM Tris-HCl (pH 7.9), filtered through a 0.45-µm membrane, and loaded onto a Q Sepharose FF column (10 cm by 1.6 cm) equilibrated with 10 mM Tris-HCl buffer (pH 7.9). Proteins were eluted using a step gradient of 10 mM Tris-HCl (pH 7.9) containing 250 mM NaCl. Fractions were analyzed for the presence of recombinant proteins by SDS-PAGE, and peak fractions were pooled. The rC protein fragment was run through both Q Sepharose and DEAE Sepharose fast flow (FF) columns before the hydrophobic interaction chromatography step.

Hydrophobic interaction chromatography. The recombinant protein samples (rSpy1325, rAR, rD, rNAD, rPC, and rC) were precipitated by adding (NH4)2SO4 to 70% saturation at 25°C. The precipitate was collected by centrifugation (18,000 x g, 4°C, 20 min), suspended in 10 ml of 0.8 M (NH4)2SO4 in 10 mM Tris-HCl (pH 7.9), and loaded onto a HiPrep 16/10 Octyl FF (high sub) column (10 cm by 1.6 cm) equilibrated with 1 M (NH4)2SO4 in 10 mM Tris-HCl (pH 7.9). Protein was eluted with a step gradient of 1 to 0 M (NH4)2SO4 in 10 mM Tris-HCl (pH 7.9), and the fractions were analyzed by SDS-PAGE. rPC was purified using HiPrep 16/10 Phenyl Fast Flow (high sub) instead of HiPrep 16/10 Octyl Fast Flow. For rAR and rD, ion-exchange chromatography was repeated with a DEAE FF column (10 x 1.6 cm) after use of a Hi Prep 16/10 Octyl FF.

Size exclusion chromatography. Following hydrophobic interaction purification, the rSpy1325 fractions were pooled, and the protein was concentrated with a 10-kDa Centricon Plus-20 filter (Millipore, Bedford, MA) by centrifugation at 3,200 x g for 15 min at 4°C. The concentrated protein was loaded onto a Superdex 200 column (30 cm by 1 cm), and eluted fractions were analyzed by SDS-PAGE.

Generation of anti-Spy1325 antisera. CD-1 Swiss mice were immunized with 50 µg of purified, recombinant Spy1325 (rSpy1325) in TiterMax adjuvant (TiterMax USA, Inc., Norcross, GA). Mice were boosted on day 28 with 25 µg of rSpy1325 in PBS, and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). Antisera used for domain-interaction ELISAs were generated in rabbits by Bethyl Laboratories (Montgomery, TX).

Analysis of in vivo Spy1325 expression and domain interaction ELISAs. To determine if Spy1325 is expressed in vivo in the mouse, Spy1325 and recombinant fragments were analyzed by Western immunoblotting with mouse serum (pre- and 30 d postinfection) at a dilution of 1:1,000 and detected with horseradish peroxidase-conjugated anti-mouse secondary antibody (Bio-Rad). To determine if Spy1325 was expressed during human GAS disease, rSpy1325 and fragments thereof, including rAR, rD, rNAD, rPC, and rC, were used to coat MaxiSorp ELISA plates (Nunc) at 0.5 µg/well, at 4°C overnight. Paired acute- and convalescent-phase human sera were collected from 40 patients with pharyngitis under a protocol approved by the Baylor College of Medicine Institutional Review Board. Human patient serum was used at a dilution of 1:2,000, and secondary alkaline phosphatase (AP)-conjugated anti-human antibody (Antibodies Incorporated, Davis, CA) was used to detect bound primary antibody. Plates were read at 405 nm. An OD405 reading greater than twice the value of the PBS negative control was considered a positive reaction. For domain interaction assays, ELISA plates were coated with 0.5 µg of purified recombinant Spy1325 fragments, and 1 mM of the fragment of interest was added as probe. The appropriate antibody (N or C terminal) was used to detect bound probe protein, and secondary AP-conjugated anti-rabbit antibody (Bio-Rad) was used to detect bound primary antibody. Plates were read at 405 nm.

TaqMan real-time reverse transcriptase PCR analysis. Strains MGAS6180 (serotype M28) and MGAS10270 (serotype M2) were grown in liquid THY medium to an OD600 of 0.2 (early exponential phase), 0.5 (mid-exponential phase), 1.2 (late exponential phase), or 1.8 (stationary phase). Total RNA was isolated with an RNeasy Mini kit (QIAGEN), and cDNA was generated with Superscript III reverse transcriptase and random primers (Invitrogen) as described previously (67). The cDNAs were used as templates in TaqMan real-time PCR with gene-specific primers and probes (Table 2). Amplification reactions were performed as specified by the manufacturer of Platinum Quantitative PCR SuperMix-UDG (Invitrogen) and analyzed with a 7500 Real-Time PCR System instrument (Applied Biosystems) with standard settings recommended by the manufacturer. The transcript level of Spy1325 was standardized to the expression of proS and compared to the expression during early exponential growth phase ({Delta}{Delta}CT method).

Mutanolysin extraction of cell surface proteins. Strain MGAS10270 (20 ml) was grown to stationary phase (OD600 = 1.8). Cells were pelleted and washed twice with Tris-EDTA, pH 8.0, containing 1 mM phenylmethylsulfonyl fluoride. Cell pellets were suspended in Tris-EDTA containing 20% sucrose and lysozyme (3 mg/ml) with and without mutanolysin (300 U/ml). The cells were incubated with gentle agitation at 37°C for 2 h, and bacteria were removed by centrifugation. The extracted sample from 2.4 ml of culture was analyzed by Western immunoblotting with Spy1325-specific mouse antisera.

Detection of Spy1325 in the culture supernatant of GAS. GAS strains were grown to mid-exponential or stationary phase, and bacterial cells were removed by centrifugation. The supernatant was precipitated with 30% ice-cold trichloroacetic acid (Sigma-Aldrich) on ice for 30 to 60 min. Precipitated protein was collected by centrifugation at 18,000 x g for 15 min at 4°C. Protein pellets were washed twice in ice-cold acetone, dried in a speed vacuum for 10 min, and suspended in PBS. Proteins were analyzed by Western immunoblotting with Spy1325-specific antisera.

Trypsin digestion of Spy1325 fragments. rSpy1325 and fragments thereof were suspended in digestion buffer (100 mM Tris-HCl, pH 8.5, with 1 mM CaCl2) to a final concentration of 1 µg/µl. Trypsin was diluted to 0.1 µg/µl in 10 mM HCl and further diluted to 20 ng/µl in digestion buffer before use. Equal volumes of trypsin and recombinant Spy1325 fragments were mixed and incubated at 37°C for 17 h. Digested samples were separated on a 4 to 15% gradient gel and stained with Coomassie blue.

Mouse vaccination studies. Groups of male CD-1 Swiss outbred mice (Charles River Laboratories, Wilmington, MA) were immunized subcutaneously with PBS, 50 µg of rNAD, 50 µg of rPC, or a mixture of 25 µg of rNAD and 25 µg of rPC in TiterMax adjuvant. A single boost of 25 µg of recombinant protein in PBS was given on day 28. Mice were challenged on day 42 with serotype M28 strain MGAS6180 (7.6 x 108 CFU/mouse) grown to mid-exponential phase. Mice were monitored every 3 h for the first 48 h and then daily for 10 days. Mortality or near mortality was recorded, and Kaplan-Meier survival curves were generated. Statistical analysis was performed using GraphPad Prism 4 (GraphPad Software, San Diego, CA).

Purification of SAG. SAG (also known as gp340; encoded by DMBT1) was purified from human saliva by adsorption to GAS by a method modified from Jenkinson et al. (29) as described below. Whole human saliva stimulated by Parafilm was collected from healthy adult volunteers into chilled tubes under a protocol approved by the Baylor College of Medicine Institutional Review Board. The saliva sample was clarified at 20,000 x g for 15 min and stored at –80°C until needed (13). Strain MGAS6180 was grown in 2 liters of THY medium to late exponential phase, harvested by centrifugation, and washed once with PBSC buffer (10 mM Na2HPO4-KH2PO4 [pH 7.2] containing 0.1 M NaCl, 0.05 M KCl, and 1 mM CaCl2). Saliva was clarified by centrifugation and diluted with an equal volume of PBSC. GAS was suspended in diluted saliva at a density of ~5 x 1010 CFU/ml (OD600 = 30), mixed by gentle agitation (37°C, 1 h), and centrifuged at 3,300 x g at 4°C for 15 min. Bacteria were washed with PBSC and suspended in PBS containing 1 mM EDTA (PBSE) to release bound proteins. The PBSE extraction procedure was repeated, and the supernatant was filtered through a 0.45-µm membrane and concentrated with a 10-kDa Centricon Plus-20 filter (Millipore) by centrifugation at 3,200 x g for 15 min at 4°C. The concentrated sample was loaded on a Superdex 200 column (30 cm by 1 cm; Amersham Biosciences). High-molecular-weight fractions were collected, separated by SDS-PAGE (7.5% gel), and transferred to nitrocellulose for Western immunoblotting. The membrane was incubated with anti-gp340 monoclonal antibodies (MBL International, Woburn, MA), washed three times in PBST, and detected with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (heavy plus light chains) (Bio-Rad) and a Super Signal West Pico detection kit (Pierce). The concentration of purified SAG was estimated by the Bradford method with protein assay reagent (Bio-Rad).

Far-Western immunoblotting of SAG and rSpy1325. Purified rSpy1325 and five fragments corresponding to rAR, rD, rNAD, rPC, and rC (1.5 µg/well) were separated by SDS-PAGE (4 to 15% gradient gel) and transferred to a nitrocellulose membrane. The membrane was incubated with purified SAG protein (1 µg/ml) in the presence of 1 mM Ca2+. After incubation with SAG, membranes were washed three times with PBST for 15 min and incubated with 1 µg/ml anti-gp340 monoclonal antibody (MBL International) for 1 h at room temperature. Membranes were washed with PBST, incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (heavy plus light chains) (Bio-Rad), and visualized with a Super Signal West Pico detection kit (Pierce).


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RESULTS
 
Spy1325 is an antigen I/II family protein. BLASTP and multisequence comparison of Spy1325 against the NCBI nonredundant protein database and subsequent phylogenetic analysis revealed the presence of several groups of related proteins (Fig. 1A). One group is composed of Spy1325 and five inferred and uncharacterized apparent cell surface proteins from GBS (Streptococcus agalactiae). These putative proteins are encoded by genes located in RD2-like regions of the GBS genome (19, 61, 62). Each of these putative orthologues has an amino-terminal secretion signal sequence and a carboxy-terminal LPXTG cell wall anchoring motif. The overall average amino acid identity of Spy1325 and these proteins is 38 to 40% based on pairwise global protein sequence alignments. The second closest-related group of proteins is the well-characterized antigen I/II family of cell surface proteins produced by oral streptococci (27, 28). These proteins all share a similar architecture (Fig. 1B), including a secretion signal, an alanine-rich repeat region, a divergent region, and a proline-rich repeat region followed by a C-terminal conserved region and a cell wall anchor motif. A third group of phylogenetically related proteins includes the agglutinin receptor from GBS (61, 62) and two proteins of unknown function produced by Streptococcus suis. The fourth group of more distantly related proteins is comprised of plasmid-encoded proteins made by Enterococcus faecalis, which are reportedly involved in bacterial aggregation and adhesion (69).


Figure 1
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  		FIG. 1. Spy1325 is a member of the antigen I/II protein family. (A) Phylogenetic relationships of Spy1325 and orthologous proteins. Spy1325 is most closely related to a group of five inferred and uncharacterized cell surface proteins in GBS (S. agalactiae) (group I). The second closest-related group of proteins is composed of antigen I/II protein family members (group II). The tree was generated with MacVector, version 8.0. Numbers indicate the genetic distance between Spy1325 and various orthologous groups. Species and strain designations are given in parentheses. (B) The molecular architecture of Spy1325 is conserved among antigen I/II family proteins. Comparison of Spy1325 with SspB of Streptococcus gordonii showing the signal peptide (S), N-terminal region (N), alanine-rich repeats (AR), divergent region, proline-rich repeats (PR), conserved region, and the cell wall anchor motif (CWA). Spy1325 and SspB contain an LDV (Leu-Asp-Val) integrin-binding motif in the conserved region. SspB is representative of antigen I/II family proteins. The percent amino acid identity between the domains is indicated.

One noteworthy feature of Spy1325 is the presence of an LDV (Leu-Asp-Val) integrin-binding motif in the distal half of the protein. The LDV motif mediates binding of proteins to {alpha}4ß1 and {alpha}4ß7 integrins (39). Integrins are heterodimeric protein receptors located in the eukaryotic cell membrane which function in the attachment of cells to the extracellular matrix. Integrin-binding motifs present in bacterial and viral adhesins assist pathogen adherence to and entry into host cells (8, 12). An analysis of available sequence data revealed that SspB and SspA antigen I/II proteins, the lactococcal and enterococcal aggregation substance, the GBS agglutinin receptor, and the uncharacterized GBS protein SAG2021 also contain LDV motifs.

Analysis of Spy1325 allelic variation. A salient feature of the antigen I/II family proteins is the presence of alanine- and proline-rich regions, which are also present in Spy1325. The alanine-rich region of Spy1325 (amino acids [aa] 179 to 434) consists of 25% alanine residues and contains three contiguous copies of a 78-amino-acid motif (Fig. 1B). The proline-rich region has 40-amino-acid repeat blocks in addition to flanking, nonrepetitive, proline-rich sequences. Because the alanine- and proline-rich regions contain repeat segments, it is possible that variation exists in the number of repeats due to recombination and/or slipped-strand mispairing events. These processes are well described for pathogenic bacteria, including GAS (22, 36, 70). To determine if variation exists in the number of repeats present in GAS, PCR was used to screen 92 strains of diverse M protein serotypes (M2, M4, M28, M48, M50, M77, and M124) for size variation in the regions of Spy1325 encoding both the alanine- and the proline-rich repeat segments (Table 1). Strains were chosen based on previous confirmation of the presence of RD2 (20). No size difference was identified in the PCR amplicon corresponding to the segment of Spy1325 encoding the alanine-rich repeats. In contrast, we identified three size variants of the PCR amplicon corresponding to the segment of Spy1325 encoding the proline-rich repeats (Fig. 2A). DNA sequence analysis determined that the size variation was due to the presence of two, three, or four copies of a 120-bp repeat segment encoding 40 amino acids (Table 1 and Fig. 2B). The great majority of strains (85/92; 92%) had three copies of this repeat region.


Figure 2
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  		FIG. 2. Allelic variation in Spy1325 in natural populations of GAS. (A) Agarose gel electrophoresis showing three amplicon size variants corresponding to sequence variation in the proline-rich region. Size (in base pairs) is indicated on the left, and arrowheads denote an amplicon size corresponding to two, three, or four proline-rich repeats. All strains are serotype M28 except where indicated. L, 100-bp ladder, lane 1, MGAS8423; lane 2, MGAS8448; lane 3, MGAS11097; lane 4, MGAS11116; lane 5, MGAS9664; lane 6, MGAS9707; lane 7, MGAS10890; lane 8, MGAS10270 (serotype M2); lane 9, MGAS6180. (B) PCR and DNA sequence analysis identified seven distinct Spy1325 alleles among GAS strains of diverse M protein serotypes. Ninety-two percent of strains contained three proline-rich repeats (Spy1325.1), whereas four and three strains had two or four repeats (Spy1325.2 and Spy1325.3), respectively. Four nucleotide substitutions, each resulting in an amino acid replacement, also were identified (Spy1325.4 to Spy1325.7). nt, nucleotide.

To determine if additional allelic variation existed in Spy1325, we sequenced the entire gene in a subset of 51 of the 92 strains. Only four single nucleotide polymorphisms, all resulting in amino acid replacements, were identified (Fig. 2B). Thus, based on the combination of variation in proline repeat segments and amino acid substitutions, a total of seven distinct alleles of Spy1325 were identified in the strains studied. Spy1325 is therefore extremely well conserved and has a much lower level of allelic variation than that normally present in many GAS genes from different M protein serotypes (48). This result suggests that Spy1325 has been recently acquired by multiple GAS strains by horizontal gene transfer.

Analysis of Spy1325 expression. To determine if Spy1325 was expressed in vitro, we used real-time TaqMan quantitative PCR. RNA was isolated from strain MGAS6180 (serotype M28) and MGAS10270 (serotype M2) grown to early, mid-, and late exponential and stationary growth phases. These two strains have identical growth characteristics in THY liquid broth (Fig. 3A), virtually identical RD2 elements (4), and identical Spy1325 alleles. The level of Spy1325 transcript in each strain was low during early and mid-exponential growth phases (Fig. 3B). In serotype M28 strain MGAS6180, the Spy1325 transcript level remained low until stationary phase, whereas in serotype M2 strain MGAS10270, the Spy1325 transcript level increased in the late exponential growth phase and peaked in the stationary phase. The increase in the relative amount of transcript also differed dramatically between the two GAS strains, with increases of 4-fold and 15-fold for strains MGAS6180 and MGAS10270, respectively (Fig. 3B).


Figure 3
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  		FIG. 3. Characterization of Spy1325 in vitro expression. (A) Growth curve of strain MGAS6180 (serotype M28) and strain MGAS10270 (serotype M2) in THY broth. (B) The Spy1325 gene is transcribed in vitro. cDNA prepared from strains MGAS6180 and MGAS10270 harvested at early (EE), mid-exponential (ME), late exponential (LE), and stationary (S) phase was analyzed by real-time TaqMan quantitative PCR for Spy1325 transcript level. Values are expressed as the increase (n-fold) in transcript standardized to proS. The data represent values obtained with two independently isolated RNA samples analyzed in quadruplicate. (C) Spy1325 is present on the cell surface of GAS. MGAS5005, which does not contain the Spy1325 gene, and strain MGAS10270 (serotype M2) were harvested at stationary phase, and cell-wall proteins were extracted with (+) or without (–) mutanolysin. Proteins were separated by SDS-PAGE and analyzed by Western immunoblotting with Spy1325-specific antisera. (D) Spy1325 is present in the culture supernatant of various M protein serotype strains. Precipitated proteins prepared from culture supernatants from mid-exponential-phase (upper panel) or stationary-phase (lower panel) cultures were separated by SDS-PAGE and analyzed for the presence of immunoreactive Spy1325 with mouse antisera raised against purified rSpy1325. Strains tested: MGAS5005 (M1), MGAS6180 (M28), MGAS9455 (M28), MGAS9538 (M28), MGAS10208 (M28), MGAS10270 (M2), MGAS10318 (M2), MGAS10422 (M2), MGAS9482 (M77), MGAS11932 (M124), MGAS12431 (M124).

Spy1325 has a cell wall-anchoring LPSTG motif, and analyses of related proteins, including antigen I/II family members and aggregation substance from E. faecalis, indicate that these proteins are expressed on the bacterial cell surface (27, 45). Western immunoblotting of mutanolysin-released proteins was used to determine if Spy1325 also is present on the GAS cell surface. The results demonstrated that immunoreactive Spy1325 was present in mutanolysin extracts of strain MGAS10270 (serotype M2) but not in extracts from a strain lacking the Spy1325 gene (Fig. 3C). These results are consistent with the hypothesis that Spy1325 is expressed on the bacterial cell surface.

We next used Western immunoblot analysis to determine whether Spy1325 was made by GAS strains of diverse M protein serotypes. Although the presence of the LPSTG motif suggests that Spy1325 is anchored in the cell wall, identification of GAS surface proteins in culture supernatant during in vitro growth has been reported (57). In addition, the antigen I/II family protein PAc produced by Streptococcus mutans was originally purified from the culture supernatant (49). Ten strains previously confirmed to contain RD2 and representing four GAS M protein serotypes were tested for Spy1325 production. Concentrated culture supernatants from mid-exponential and stationary-phase cultures were separated by SDS-PAGE and analyzed by Western immunoblotting with Spy1325-specific antisera (Fig. 3D). Culture supernatants from all 10 GAS strains containing the Spy1325 gene produced immunoreactive Spy1325, although the amount and timing of protein production varied among strains.

Spy1325 has trypsin-resistant and trypsin-sensitive fragments. Antigen I/II proteins derived their name from experiments conducted by Russell et al., who determined that PAc from S. mutans contained a protease-sensitive (antigen I) and a protease-resistant (antigen II) fragment (49). To determine whether Spy1325 had a similar digestion pattern, full-length recombinant Spy1325 (r1325) and various recombinant fragments thereof (Fig. 4A), were incubated with 10 ng/ml trypsin for 17 h and analyzed by SDS-PAGE. The digested products of full-length Spy1325 comprised two polypeptide fragments of approximately 75 and 45 kDa (Fig. 4B and C). Cleavage products of the same size were observed when the carboxy-terminal portion of the protein was used as substrate (rPC and rC). In contrast, the amino-terminal fragments of Spy1325, encompassing the entire amino-terminal half of the protein (rNAD), the alanine-rich repeats (rAR), or the divergent region (rD), were completely sensitive to trypsin. This result suggests that Spy1325 may adopt a conformation similar to that of antigen I/II proteins in addition to having primary sequence similarity.

Interaction between Spy1325 alanine- and proline-rich domains. The mouse monoclonal antibody Guy's 13 recognizes the antigen I/II protein PAc made by S. mutans and Streptococcus sobrinus (54). This antibody inhibits S. mutans colonization and dental caries formation in nonhuman primates (31) and prevents bacterial colonization in human clinical trials (37, 38). The epitope recognized by Guy's 13 is conformational, and the antibody requires the alanine-rich repeat region (A site, aa 170 to 218) and a region encompassing the proline-rich repeats (P site, aa 956 to 969) for binding (65, 66). This result implies that these two noncontiguous regions are positioned in close proximity in the native protein. In addition, ELISA-based assays have demonstrated the direct interaction of the alanine-rich repeat region and the proline-rich repeat region of PAc (51, 65), further supporting this contention. To determine if the alanine- and proline-rich repeats of Spy1325 bind to one another, we performed ELISA-based interaction assays with recombinant fragments of the protein. Polyclonal antisera directed against the amino-terminal half of the protein (aa 40 to 646) or the carboxy-terminal half of the protein (aa 647 to 1319) were generated in rabbits. ELISA plates were coated with three different fragments of Spy1325 comprising the amino-terminal half of the protein: the N-terminal region, including the alanine-rich repeat region and the divergent region (rNAD), or the alanine-rich or divergent region alone (rAR or rD). The C-terminal half of Spy1325 with or without the proline-rich repeats (rPC or rC) was added to the wells, and binding was assessed by the addition of the carboxy-terminal-specific antiserum. The C-terminal fragment containing the proline-rich repeats (rPC) bound all N-terminal fragments except for the divergent region (Fig. 5A), suggesting that the alanine-rich repeats were required for this interaction. Interestingly, binding to all N-terminal fragments except rNAD was abrogated in the absence of the proline-rich repeats. This result suggests that an additional motif within the C terminus other than the proline-rich repeats permits binding to the N-terminal domain of the protein. It is possible that the extended proline-rich region (aa residues 767 to ~810) mediates this interaction, but further investigation is needed to determine if this is the case. The converse experiment was performed with carboxy-terminal fragments as capture antigen with similar results (Fig. 5B).


Figure 5
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  		FIG. 5. The alanine- and proline-rich regions of Spy1325 interact. (A) Plates (96 well) were coated with purified recombinant fragments of Spy1325, including NAD (aa 40 to 646), AR (aa 201 to 434, alanine-rich repeats), or D (aa 435 to 646, divergent region). The C terminus with (PC, aa 647 to 1319) or without (C, aa 767 to 1319) the proline-rich repeats was added as probe, and polyclonal antiserum raised against the C-terminal half of the protein was used to detect bound protein. (B) The converse experiment, in which C-terminal fragments were bound to the plate and probed with N-terminal fragments, was performed. Polyclonal antiserum directed against the N-terminal region of Spy1325 was used to detect bound protein.

Spy1325 is produced in vivo in infected mice and humans. To test the hypothesis that Spy1325 is expressed in vivo, we used a mouse model of invasive soft-tissue infection. Mice were infected with a sublethal dose of serotype M28 strain MGAS6180, and sera were harvested at 4 weeks postinoculation. Western immunoblot analysis revealed that convalescent-phase sera (but not preinoculation sera) contained antibodies that recognized full-length rSpy1325 and the rAR, rNAD, and rPC segments (Fig. 6). Interestingly, the convalescent-phase sera were not reactive with the purified recombinant conserved region (rC, aa 767 to 1319) (data not shown).


Figure 6
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  		FIG. 6. Spy1325 is expressed in vivo in mice infected with serotype M28 strain MGAS6180. Purified rSpy1325 (r1325) and various purified recombinant protein fragments thereof were separated by SDS-PAGE and analyzed by Western immunoblotting. Sera obtained from mice infected with strain MGAS6180 (day 30, II) recognized rSpy1325 and various domain fragments, whereas preimmune sera (I) did not. (A) Purified rSpy1325; (B) rAR, alanine-rich repeat region (aa 201 to 434); (C) rPC, proline-rich, and conserved regions (aa 647 to 1319); (D) rNAD, N-terminal, alanine-rich, and divergent regions (aa 40 to 646).

In a previous study (20), we performed a preliminary analysis of human sera for reactivity against rSpy1325. Western immunoblot analyses demonstrated that seroconversion occurred in patients with pharyngitis or invasive infection caused by serotype M28 GAS (20). To extend these results, we analyzed paired acute- and convalescent-phase sera from 40 pharyngitis patients infected with serotype M2, M28, or M77 GAS strains. RD2 has been identified in strains of these serotypes, and all infecting GAS strains were analyzed by PCR to confirm the presence of RD2 (data not shown). As assessed by ELISA, 38% (15/40) of the sera from human patients recognized Spy1325 (Table 3). As in the mouse serum analysis, antibodies did not react to the conserved domain (rC). Taken together, the mouse and human serologic data confirm that Spy1325 is expressed in vivo in infected hosts.


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  		TABLE 3. Summary of ELISA data for sera from human patients

Protection of mice against invasive GAS infection by immunization with rSpy1325 fragments. Several reports show that vaccination with antigen I/II proteins protects against dental caries and bacterial colonization of the oral cavity (reviewed in reference 53). Because Spy1325 is made in vivo in infected hosts, we tested the hypothesis that immunization of mice with rSpy1325 fragments conferred protection against invasive GAS infection. CD-1 Swiss outbred mice were immunized with PBS, 50 µg rNAD, 50 µg rPC, or a mixture of rNAD and rPC (rNAD plus rPC, 25 µg each) and challenged with serotype M28 strain MGAS6180. No significant differences in survival between mice immunized with rPC and mice mock immunized with PBS were observed (Fig. 7). In contrast, mice immunized with rNAD or with rNAD plus rPC were significantly protected from GAS-mediated mortality (P value for rNAD, 0.0013; P value for rNAD plus rPC, 0.0081).


Figure 7
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  		FIG. 7. Immunization of mice with recombinant fragments of Spy1325 confers protection against lethal GAS challenge. CD-1 Swiss outbred mice were immunized subcutaneously with 50 µg rPC (aa 647 to 1319), 50 µg rNAD (aa 40 to 646), or a mixture of 25 µg each of rNAD and rPC or were mock immunized with PBS. Mice were challenged intraperitoneally with a lethal dose of serotype M28 strain MGAS6180 (7.6 x 108 CFU/mouse) and monitored for mortality or near mortality.

Spy1325 binds SAG. GAS aggregate in saliva (13, 35, 46), but the molecules responsible for this phenotype are as yet unidentified. Because antigen I/II proteins have been reported to contribute to bacterial aggregation (26, 27, 35), we tested the hypothesis that Spy1325 is involved in this process. Aggregation of strain MGAS6180 expressing Spy1325, but not strain MGAS5005 (serotype M1), which lacks the Spy1325 gene, was observed upon incubation with human saliva (data not shown). We next tested the hypothesis that GAS expressing Spy1325 bound saliva proteins. Strain MGAS6180 was incubated with human saliva, and bound proteins were eluted from the cell surface as described in Materials and Methods. The eluted saliva proteins were purified by size exclusion chromatography, and the fractions were analyzed by SDS-PAGE. A dominant high-molecular-weight protein was present (Fig. 8A, left panel). Many antigen I/II proteins bind SAG, which is identical to lung scavenger receptor gp340 (46). A monoclonal antibody directed against gp340 (SAG) was reactive with this high-molecular-weight protein, as shown by Western immunoblot analysis (Fig. 8A, right panel).


Figure 8
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  		FIG. 8. Spy1325 binds salivary agglutinin glycoprotein (SAG) complex. (A) The high-molecular-weight species eluted from the surface of GAS is SAG. Human saliva was adsorbed with serotype M28 strain MGAS6180, and eluted components were purified by size exclusion chromatography. Fractions were separated by SDS-PAGE (left panel) and analyzed by Western immunoblotting (right panel). The high-molecular-weight species reacted with anti-gp340 monoclonal antibody. Molecular masses and fraction numbers are indicated along the left and bottom, respectively. (B) Spy1325 contains two distinct SAG binding sites. Mature rSpy1325 and purified protein fragments (represented schematically in left panel) were separated by SDS-PAGE and analyzed by Coomassie brilliant blue staining (middle panel). Proteins were transferred to nitrocellulose, and the membrane was incubated with purified SAG and analyzed by Western immunoblotting with anti-gp340 monoclonal antibody (Far Western, right panel). SAG bound to mature Spy1325 (r1325), rNAD, and rPC fragments.

We performed a far-Western analysis to determine the regions of Spy1325 involved in SAG binding. Mature rSpy1325 and five polypeptide fragments (rAR, rD, rNAD, rPC, and rC) (Fig. 8B) were separated by SDS-PAGE, transferred to nitrocellulose membrane, incubated with purified SAG, and analyzed by Western immunoblotting with gp340-specific monoclonal antibody. SAG bound to full-length mature rSpy1325 (r1325) and fragments representing the amino-terminal half (rNAD) and the proline-rich repeat and conserved (rPC) portions of Spy1325 (Fig. 8B). In contrast, no binding was detected between SAG and rAR and rD, two fragments representing shorter segments of the amino-terminal half of the molecule, or rC, the conserved domain alone. Together, the results suggest that at least two regions of Spy1325 participate in SAG binding, one in the amino half and one in the carboxy half of the protein. These results are consistent with results reported for other antigen I/II polypeptides such as PAc and SspA made by Streptococcus mutans and Streptococcus gordonii, respectively (21, 26).


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DISCUSSION
 
The molecular mechanisms that enable GAS to cause diverse infections are poorly understood. The repeated isolation of certain M protein serotypes from specific types of infections suggests that these nonrandom associations have a molecular underpinning. One goal in sequencing the genome of a serotype M28 GAS strain was to identify candidate genes that may contribute to the overrepresentation of this serotype in invasive infections, particularly puerperal sepsis (20). The identification of RD2, which has significant sequence similarity to putative pathogenicity islands identified in GBS, has provided a basis to explore serotype and disease specificity. As GBS is the primary cause of neonatal invasive infections, we hypothesized that the acquisition of this element by serotype M28 GAS strains has expanded the pathogenic potential of these strains, possibly by allowing these organisms to inhabit a new anatomic niche. Characterization of the proteins encoded by RD2 could reveal new information regarding the mechanisms responsible for a specific serotype-disease association. As a first step in analyzing the contribution of the RD2 element to GAS pathogenesis, we chose to study the Spy1325 protein. This protein was chosen for analysis because it has a typical gram-positive secretion signal sequence and a carboxy-terminal LPSTG motif, suggesting that it is expressed on the GAS cell surface and potentially interacts with host cell factors.

Several lines of evidence indicate that Spy1325 is a member of the antigen I/II family of streptococcal adhesins. Spy1325 is similar to antigen I/II proteins in overall domain architecture. Analogous to antigen I/II proteins, Spy1325 consists of a trypsin-sensitive and trypsin-resistant fragment and has two distinct regions that participate in SAG binding, and the alanine- and proline-rich domains of the protein interact. With few exceptions, the majority of antigen I/II proteins analyzed from oral streptococci contain three proline-rich repeats (27). The proline-rich region appears necessary for the proper folding and structural stability of the molecule (7, 51), contains multiple B- and T-cell epitopes, and is involved in binding SAG (28). In our PCR analysis of 92 GAS strains, we identified seven strains that contained either two or four proline-rich repeats. Considering the functional importance of this domain in intra- and intermolecular interactions, experiments are currently under way to determine the effect, if any, of the number of repeats on the affinity of Spy1325 for SAG and the antigenicity of the protein.

It is well known that expression of virulence factors in GAS and other pathogenic bacteria can vary dramatically among strains of the same serotype (16, 23, 42, 59, 71). To determine if this was the case for Spy1325, we compared the transcript level of Spy1325 during in vitro growth in serotype M28 strain MGAS6180 and serotype M2 strain MGAS10270. Although the Spy1325 transcript peaked during stationary phase for both strains, the levels of maximal gene transcript for the two isolates were very different. Variations in the amount of Spy1325 protein in the culture supernatant, between serotypes and between strains of the same serotype, also were observed. Inasmuch as the Spy1325 alleles and upstream sequences in strains MGAS6180 and MGAS10270 are identical (4, 20), these differences in transcript levels cannot be due simply to differences in the Spy1325 gene or regulatory sequences. The results suggest that the expression of Spy1325 is influenced by regulatory factors that are strain specific. Variation in Spy1325 expression may enhance GAS pathogenesis or survival, depending on the infected anatomic niche.

We believe it is possible that antigen I/II family proteins made by GAS, GBS, and oral streptococci exploit one or more shared molecular mechanisms to enhance host-pathogen interaction. In this regard, we note that Spy1325 and all characterized antigen I/II proteins made by oral streptococci bind SAG. SAG, also known as gp340, is encoded by the human DMBT1 (deleted in malignant brain tumor 1) gene (24, 25, 40, 41). How might SAG/DMBT1 contribute to serotype M28 GAS and GBS infections involving female urogenital sites and neonates? DMBTI is a member of the scavenger receptor cysteine-rich superfamily of proteins. Although originally identified because it is deleted in certain tumors, the exact function of DMBT1 is not known. DMBT1 is expressed in many tissues, including the salivary gland (SAG), lung (gp340), small intestine, uterus, and stomach, and fetal intestine, lung, and epithelia (40, 41). Thus, we speculate that GAS strains expressing Spy1325, and GBS strains expressing orthologous antigen I/II family proteins, may preferentially colonize vaginal and uterine surfaces by binding DMBT1 expressed at these sites. Studies are under way to test this hypothesis.

Approximately 15 million cases of GAS pharyngitis and 10,000 cases of invasive disease occur annually in the United States. The lack of a licensed vaccine has prompted the search for candidate antigens, and genome sequence data have facilitated the rapid identification of extracellular and cell wall-anchored GAS proteins (20, 33, 47). In the area of vaccine research directed toward caries prevention, active immunization with antigen I/II protein or synthetic peptides corresponding to the alanine-rich region of antigen I/II proteins protects against dental caries and suppresses colonization of oral streptococci (30, 32, 60). Similarly, we found that immunization of mice with a purified Spy1325 NAD fragment encoding approximately the first half of the protein (aa 40 to 646) conferred significant protection against lethal GAS challenge. The Spy1325 gene is extremely well conserved and is present in all serotype M28 GAS strains analyzed and a large percentage of serotype M2 and M77 strains (20). In addition, orthologues of Spy1325 are present in four of the five major GBS serotypes responsible for human disease (serotypes Ia, II, III, and V). Thus, it is possible that immunization with Spy1325 from GAS would protect against GBS disease (or vice versa), as is the case with C5a peptidase or the R28 and Rib proteins from GAS or GBS, respectively (10, 56).


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ACKNOWLEDGMENTS
 
We thank I. Abdi, G. Li, X. Pan, and R. A. Reich for technical assistance and advice and C. G. Granville and M. J. Nagiec for help with the mouse studies. We also thank E. A. Graviss for statistical analysis, S. B. Beres for help generating the phylogenetic tree, and K. E. Stockbauer for critical reading of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Center for Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute, The Methodist Hospital, B154, 6565 Fannin St., Houston, TX 77030. Phone: (713) 441-5890. Fax: (713) 790-6460. E-mail: jmmusser{at}tmh.tmc.edu. Back

Editor: D. L. Burns


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Infection and Immunity, July 2006, p. 4200-4213, Vol. 74, No. 7
0019-9567/06/$08.00+0     doi:10.1128/IAI.00493-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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