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Originally published In Press as doi:10.1074/jbc.M502896200 on July 21, 2005

J. Biol. Chem., Vol. 280, Issue 39, 33228-33239, September 30, 2005
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Streptococcus pyogenes Collagen Type I-binding Cpa Surface Protein

EXPRESSION PROFILE, BINDING CHARACTERISTICS, BIOLOGICAL FUNCTIONS, AND POTENTIAL CLINICAL IMPACT*{boxs}

Bernd Kreikemeyer1, Masanobu Nakata, Sonja Oehmcke, Caroline Gschwendtner, Jana Normann, and Andreas Podbielski12

From the Department of Medical Microbiology and Hospital Hygiene, Hospital of Rostock University, Schillingallee 70, D-18057 Rostock, Germany

Received for publication, March 16, 2005 , and in revised form, July 12, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Streptococcus pyogenes collagen type I-binding protein Cpa (collagen-binding protein of group A streptococci) expressed by 28 serotypes of group A streptococci has been extensively characterized at the gene and protein levels. Evidence for three distinct families of cpa genes was found, all of which shared a common sequence encoding a 60-amino acid domain that accounted for selective binding to type I collagen. Surface plasmon resonance-based affinity measurements and functional studies indicated that the expression of Cpa was consistent with an attachment role for bacteria to tissue containing collagen type I. A cpa mutant displayed a significantly decreased internalization rate when incubated with HEp-2 cells but had no effect on the host cell viability. By utilizing serum from patients with a positive titer for streptolysin/DNase antibody, an increased anti-Cpa antibody titer was noted for patients with a clinical history of arthritis or osteomyelitis. Taken together, these results suggest Cpa may be a relevant matrix adhesin contributing to the pathogenesis of S. pyogenes infection of bones and joints.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptococcus pyogenes (group A streptococci (GAS)3) belongs to the group of bacterial pathogens that causes the most frequent purulent infections in humans. The majority of GAS-associated diseases involves the tonsils, pharynx, and the skin predominantly of the lower legs. However, GAS can infect every organ and type of tissue in the human host. The attachment and colonization of GAS are thought to be mediated by defined surface molecules, predominantly proteins, that interact either with structures on the eukaryotic cell surface or with components of the intercellular matrix (1). These latter molecules are commonly described as MSCRAMMs, an acronym for "microbial surface components recognizing adhesive matrix molecules" (2). Matrix protein binding is generally accepted as a crucial mechanism for bacteria to colonize and eventually infect their eukaryotic hosts.

Among the different MSCRAMMs of pathogenic Gram-positive cocci, the fibronectin binding category has been characterized the most extensively (3–5). However, by using distinct MSCRAMMs, these bacteria are also able to specifically attach to collagen, laminin, elastin, and other matrix components (1). Because more than 20 types of human collagen exist (6), type-specific collagen binding could explain why a given Gram-positive coccus could infect only certain tissue types and organs.

The current concept of S. pyogenes attachment and colonization has identified a central role for fibronectin-binding structures (3–5). This concept is supported by the findings that surface expression of fibronectin-binding proteins is a common characteristic of S. pyogenes isolates. A number of distinct gene products can account for this activity, and the profile of expression can be influenced by environmental conditions. Two major fibronectin adhesins, proteins F1/SfbI and protein F2, have been extensively characterized, and there is experimental evidence to suggest an association between their expression and tissue tropism. In addition to binding fibronectin, these molecules have also been reported to interact with certain forms of collagen (7–9).

To date, the best studied collagen-binding protein of Gram-positive cocci is the Cna surface protein of Staphylococcus aureus. The mature Cna protein (~135 kDa) contains two domains, the collagen type I-binding 500 amino acid (aa) A-region and a repeated B-region, for which no defined function has been ascribed (10–14). A 19-kDa subdomain within the A domain was found to be responsible for the majority of collagen binding activity. The amino acid residues involved in collagen binding and the crystal structure of the subdomain have been experimentally determined (15–17). The Cna collagen-binding site forms an extended groove that exactly accommodates the collagen triple helix. This interaction is similar to collagen-binding proteins of the integrin family (18).

The cna gene has been detected in the genomes of approximately half of the S. aureus strains studied (19–21). Expression of Cna reaches its maximum during the exponential growth phase and enables an S. aureus isolate to attach to collagen-expressing tissues, e.g. cartilage (10). The results from several animal infection models suggest that Cna-expressing S. aureus strains can induce and promote septic arthritis, endocarditis, keratitis, and osteomyelitis (22–26). Recently, a Cna homologue was detected in the animal pathogen Streptococcus equi (27).

Among the family of Streptococcaceae, two orthologous collagen-binding MSCRAMMs have been characterized at a molecular level. These are the collagen types I- and IV-binding Ace protein in enterococci (28–33) and the collagen type I-binding antigen I/II family surface components in strains of the Streptococcus mitis and Streptococcus mutans groups (34–45).

The ability of S. pyogenes strains to bind to collagen type IV has been well established (46–50). However, the importance of this interaction alone or in concert with fibronectin binding to the bacterial physiology and pathogenesis of streptococcal infection has not been systematically studied.

Recently, Podbielski et al. (51) identified a gene from S. pyogenes strain 591 (serotype M49) that encoded a protein that directly bound to collagen type I. This gene, designated cpa (collagen-binding protein of group A streptococci) was present in about 30% of 68 different S. pyogenes serotype strains analyzed (52). Analysis of the genomic neighborhood of cpa in serotypes M1, M6, M12, and M49 strains led to the identification of a highly recombinatorial region, containing genes encoding fibronectin and collagen-binding proteins as well as the T antigen. Bessen and Kalia (53) designated this locus as the FCT region.

In different S. pyogenes strains, the content of the FCT region can vary among different serotype strains. FCT regions typically include a gene for a RofA/Nra, a stand-alone regulator, as well as genes for several surface proteins involved in intercellular matrix attachment. Six of the seven serotype strains for which the complete sequence of the FCT region is available contain a five-gene cpa operon. This operon is composed of the cpa gene followed by a potential signal peptidase gene, a srtC sortase gene, and two genes encoding unknown factors (53–57). To date, the importance of this gene locus in responding to environmental conditions and in establishing attachment and colonization of S. pyogenes at different body sites remains speculative.

In the present study, Cpa expression, structure, and function are thoroughly characterized, and the results indicated that Cpa-associated collagen type I binding could be a critical factor for S. pyogenes infections at certain special sites such as the skeletal system. Because the ability of the bacteria to reach such sites requires other virulence traits, the Cpa protein is expected to be part of a more complex mechanism of streptococcal pathogenesis associated with deep tissue infection.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Culture Conditions—S. pyogenes (GAS) serotype M49, strain 591, is a skin isolate originally obtained from R. Lütticken, Aachen, Germany. The different M serotype GAS strains used in this study have been described by Podbielski et al. (58, 59) and by Kaufhold et al. (60). The Escherichia coli strain DH5{alpha} was purchased from Invitrogen and served as a host for plasmids pFW5-luc (Podbielski et al. (51)), pUCerm (Baev et al. (71)), and pMAL-c2 (New England Biolabs, Frankfurt/Main, Germany). The E. coli strain BL21 (DE3) was used as the host for plasmid pET28. Both strain and pET28 plasmid were obtained from Invitrogen.

The GAS wild type (wt) strain and cpa mutant derivatives were cultured in Todd-Hewitt (TH) broth or on TH agar (Oxoid-Unipath, Wesel, Germany). Both were supplemented with 0.5% yeast extract (THY) or in chemically defined medium (CDM).

GAS mutants harboring recombinant pFW–or pUCerm–plasmids were maintained in medium containing 60 mg of spectinomycin or 5 mg of erythromycin x liter–1, respectively, except when being used for functional analyses. GAS strains were grown as standing cultures at a temperature of 37 °C under a 5% CO2 to 20% O2 atmosphere unless otherwise indicated.

E. coli DH5{alpha} or BL21(DE3) isolates transformed with pFW5-luc, pUCerm, pMAL-c2, or pET28 derivatives were grown on disk susceptibility agar (Oxoid) supplemented with 100 mg of spectinomycin, 150 mg of erythromycin, 50 mg of ampicillin, or 30 mg of kanamycin x liter–1, respectively. All E. coli cultures were grown at 37 °C in ambient air except when used for expression of recombinant proteins.

Quantitation of Collagen Binding by GAS Wild Type Strains—Highly purified human collagen types I and IV (Sigma) were radiolabeled with 125I by utilizing the chloramine T method (61). Binding assays of labeled proteins to GAS were performed essentially as described in Kreikemeyer et al. (62, 63).

Assays were performed in triplicate within an experiment, and each experiment was repeated on three independent occasions. Results are shown as the mean of the entire nine measurements. Binding rates of >5% or ≥10% of the total collagen per assay were scored as relevant-specific or strong-specific binding, respectively.

Nucleic Acid Techniques and Sequence Analysis—Chromosomal and plasmid DNA preparations, genetic manipulations, and other conventional DNA techniques, including electroporation of GAS and E. coli strains, were done as described in Podbielski et al. (51). Nucleic acid sequences of the S. pyogenes serotypes M1, M3, M5, M6, M12, M18, and M49 FCT regions were obtained from Refs. 51, 53, and 64–67 and NC_002958 (GAS Sequencing Group at the Sanger Institute, UK, www.sanger.ac.uk/Projects/S_pyogenes/). Sequence alignments and secondary structure predictions were performed using the ClustalW program and Psipred prediction-, PHDsec-, SSpro8-, and PROF-programs (68–70).

Construction of Recombinant Vectors and GAS Strains—For the integration of a luciferase reporter box downstream of the cpa operon, a 1347-bp fragment comprising the last two open reading frames of the cpa operon but not the transcription terminator was PCR-amplified by using the forward/reverse primer pair 5'-GAT TAG TCA AAG AAT GAT GAT G-3'/5'-GGT TTT ATA GCC TAC TCT TCA-3' and by utilizing PstI and BlnI 5'-primer extensions. The resulting PCR product was cloned into the multiple cloning site of pFW5-luc (51). The resulting recombinant pFW-luc plasmid was integrated by a site-specific single crossover event into the strain 591 genome as described by Podbielski et al. (51), thereby duplicating the 3'end of the cpa operon. The correct insertion site was confirmed by using Southern blot hybridizations and appropriate PCR assays on genomic DNA preparations from wt and mutant strains (data not shown). Similarly generated mga- and nra-luc reporter fusions in the 591 strain have been described previously (51).

Because the cpa gene is the first gene of a five-gene operon, a simple cpa mutation by a single crossover approach would have polar effects on the four downstream genes. Therefore, a special recombinant plasmid (Fig. 1A) had to be constructed before using the established strategy for genomic integration of the plasmid. In a first round, an internal 834-bp cpa fragment was PCR-amplified by using the forward/reverse primer pair 5'-AAC ATT TTC CAT CCA AGT CAG A-3'/5'-TCC ACT GAG TAT GGC TCT GC-3' and via SacI/BamHI 5'-primer extensions, cloned into plasmid pUC-erm (71). In a second round, the cpa promoter region was PCR-amplified as a 447-bp fragment by utilizing the forward/reverse primer pair 5'-GGC ATG TAA TAG CTC-3'/5'-TCC TTC TAA ACT AAA GTA GCT TAG C-3' and via BamHI/SphI 5'-primer extensions, cloned immediately upstream of the cpa fragment into the recombinant pUC-erm plasmid. Integration of this new recombinant plasmid into the strain 591 genome via a single crossover event resulted in truncating the cpa gene with the loss of its coding sequence for the cell wall anchor region. In addition, this strategy resulted in another truncated cpa gene, devoid of its leader peptide region, and placed the four downstream genes under control of the homologous recombinant cpa promoter (Fig. 1A). Again the correct insertion site of the plasmid was confirmed using Southern blot hybridizations and appropriate PCR assays on genomic DNA preparations from wt and mutant strains (Fig. 1B).



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  		FIGURE 1.
A, generation of a serotype M49 S. pyogenes cpa mutant with a sustained orthologous transcription of the downstream cpa operon genes. In order to keep up a regular transcription of the 4 cpa-operon genes downstream of cpa, an insertion vector was constructed that contained an internal fragment of the cpa gene in direct combination with the cpa/nra promoter region ("Pcpa"). By introducing this vector into the genome of the wt strain, the cpa gene was truncated, and the cpa promoter was duplicated with one copy in front of the four other cpa operon genes. B, confirmation of the cpa mutant by Southern blot hybridizations. Genomic DNA from wt and cpa mutant bacteria was digested with EcoRI and subjected to agarose gel electrophoresis and subsequent stringent hybridization with a cpa-specific probe. The ethidium bromide-stained agarose gel (left) and the hybridized blot (right) is shown. C, SDS-PAGE and Western immunoblots with surface protein preparations from wt and cpa mutant bacteria. Surface proteins produced by an isoosmotic C1 phage lysin treatment were subjected to 12% SDS-PAGE and after Western blotting, Cpa and one degradation fragment were specifically detected by affinity-purified anti-Cpa49 antiserum. The Coomassie-stained gel (left) and the immunoblot (right) are shown. D, indirect immunofluorescence staining of whole wt and cpa mutant bacteria. Early stationary phase bacteria were exposed to affinity-purified anti-Cpa49 antiserum and, subsequently, to fluorescein-labeled anti-rabbit IgG antiserum. Representative pictures from fluorescence microscopy of the wt (left) and cpa mutant (right) bacteria are shown in the upper half. For comparison, the results from microscopy with visible light are presented in the lower half. {Delta}cpa, cpa mutant; m, size (kbp) or molecular mass (kDa) markers.

 
For generating recombinant plasmids to hyperexpress Cpa49 fragments as well as the mature Cpa1 and Cpa49 proteins, various sections of the cpa49 and cpa1 genes were PCR-amplified using the following forward/reverse primer pairs: mature Cpa1, aa residues 48–719, 5'-GGT TGG ATC CAA GAC TGT TTT TGG-3'/5'-GTA GGA ACT CGA GGC TCT TTA TT-3'; mature Cpa49, aa residues 47–703, 5'-GCG TTC GGA TCC GAA GAA CAA TCA G-3'/5'-GAT CTC TGC AGT TTT CAA AAG TG-3'; N-terminal Cpa49-1, aa residues 47–532, 5'-GCG TTC GGA TCC GAA GAA CAA TCA G-3'/5'-TTA AAT TCT CTC GAG ACA GCT AGG G-3'; N-terminal Cpa49-2, aa residues 47–359, 5'-GCG TTC GGA TCC GAA GAA CAA TCA G-3'/5'-GGA TTT TCC ACT GCA GAA CCA TC-3'; N-terminal Cpa49-3, aa residues 47–185, 5'-GCG TTC GGA TCC GAA GAA CAA TCA G-3'/5'-GGA TTA ACT GCA GCT GAA TCA GTA TAC C-3'; C-terminal Cpa49, aa residues 168–703, 5'-GAA AGG GAT AGG ATC CCT AAA CGC-3'/5'-GAT CTC TGC AGT TTT CAA AAG TG-3'; internal Cpa49-1, aa residues 183–535, 5'-GAT TCA GCT CAA ATT AAT CCG-3'/5'-TTA TTC TTT TGT GAC AGC TAG GGT-3'; internal Cpa49-2, aa residues 339–535, 5'-GCG GAG CCG ATT AAG TTT AGA G-3'/5'-TTA TTC TTT TGT GAC AGC TAG GGT-3'; internal Cpa49-3, aa residues 339–466, 5'-GCG GAG CCG ATT AAG TTT AGA-3'/5'-GTG CTT TAA GAA GTC TTC TGG-3'; internal Cpa49-4, aa residues 407–536, 5'-GTT GTC TAC TGC TTC AAT GCT-3'/5'-TAA TTC TTT TGT GAC AGC TAG-3'. By employing the appropriate 5'-primer extensions, the resulting PCR products were cloned into the BamHI/PstI or BamHI/XhoI sites of plasmids pMAL-c2 and pET28, respectively. The inserted fragments in each recombinant plasmid were confirmed by completely sequencing the cloned fragments on both strands.

Quantitative Assays for Luciferase Activity—For assessment of the luciferase activity of the cpa-luciferase reporter fusions, the GAS luc reporter strains were grown in freshly inoculated THY or CDM broths without antibiotic supplements as agitated cultures at 200 rpm in ambient air, or as standing cultures under a 5% CO2/20% O2 atmosphere or, after prereduction of the media, under a 2% CO2/2% H2/96% N2 atmosphere using the Anoxomat anaerobic culture system (Mart Microbiology, Lichtenvoorde, The Netherlands).

To determine the potential specific induction of cpa transcription by the presence of collagen in the culture medium, human collagen type I (Sigma) dissolved in PBS was mixed with an equal amount of pepsin and digested overnight at room temperature. Both undigested and digested collagen was added at final concentrations of 0.1 mg/liter when starting the bacterial cultures for the luciferase assays.

For measurement of luminescence, 1-ml aliquots of the bacterial cell suspensions were withdrawn at hourly intervals and processed as described by Podbielski et al. (51). All reported data are representative of at least three independent experiments.

Purification of WT and Recombinant Cpa Fragments—For detection of surface-attached Cpa protein, whole wt and cpa mutant GAS grown to early stationary phase were washed twice in 50 mM sodium acetate buffer, pH 5.5, and adjusted to an A600 nm of 2.0 in 1 ml of sodium acetate buffer. GAS were pelleted by centrifugation and suspended in 100 µlof the same buffer containing 30% raffinose and 5 mM EDTA. Streptococcal C1 bacteriophage lysin was added to the suspension at a concentration of 100 units/ml and incubated for 30 min at room temperature with constant rotation. Protoplasts were then sedimented by centrifugation at 13,000 x g for 20 min at 4 °C, and the supernatant containing the cell wall fragments plus the wall attached proteins was stored at 4 °C until further usage.

The recombinant Cpa fragments were expressed as fusion proteins carrying either a maltose-binding protein (pMAL-c2) or a His tag (pET28) depending on the expression plasmid. Hyperexpression of each polypeptide was achieved by growing the recombinant bacteria in LB broth (Invitrogen) as shaking cultures (200 rpm) at 37 °C to mid-exponential phase. Subsequently, 1 mM isopropyl 1-thio-{beta}-D-galactopyranoside was added for induction. After continuing the incubation for another 4 h, the E. coli cells were harvested and processed according to the manufacturers' protocols (pMAL-c2, New England Biolabs) or (pET28, Invitrogen). Purification of the recombinant proteins was achieved by absorption to either a composite amylase/agarose matrix (New England Biolabs) or to nickel-nitrilotriacetic acid resin (Qiagen, Hilden, Germany) as outlined in the manufacturers' instructions. The purity of the recovered affinity-purified recombinant proteins and polypeptides was determined by 12% SDS-PAGE and Coomassie Blue or silver staining (data not shown).

Characterization of WT and Recombinant Cpa Fragments—A total amount of 500 µg of purified Cpa49-maltose-binding protein fusion was used to generate a rabbit polyclonal anti-Cpa49 antiserum by Euro-Gentec (Seraing, Belgium) according to their standard protocol.

An enzyme-linked immunosorbent assay (ELISA) format was used to study the interaction of mature Cpa and Cpa fragments with soluble collagen types I, II, or IV. 96-Well ELISA plates (Greiner Bio-One, Solingen, Germany) were coated overnight at 4 °C with 2.5 µg/well human collagen types I, II, or IV (Sigma) in PBS. Plates were blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at 37°Cand washed three times with 0.05% Tween 20/PBS before use.

Recombinant purified Cpa fragments were biotinylated using the EZ-link-sulfo-NHS-biotin kit (Pierce) according to the manufacturer's instructions and subsequently dialyzed against PBS. To measure binding, each biotinylated Cpa fragment (17 pmol dissolved in 100 µl of PBS) was added to individual collagen-coated wells. After 1 h of incubation at 37 °C and three washes with PBS, 100 µl of an avidin-horseradish peroxidase (HRP) conjugate (Bio-Rad) diluted 1:1,000 in PBS was added to detect bound Cpa fragments. The incubation was continued for another hour at 37 °C and after four washes with PBS. Bound enzyme conjugate was quantified using an appropriate chromogenic substrate (Bio-Rad).

For Western immunoblotting studies, purified surface proteins, recombinant Cpa, or recombinant Cpa fragments were transferred to polyvinylidene difluoride Immobilon P membranes (Millipore, Eschborn, Germany) by semidry blotting techniques. Following transfer, membranes were blocked for 1 h in PBS containing 10% skimmed milk and then incubated for 1 h with the anti-Cpa affinity-purified polyclonal antiserum. Following a washing step, bound antibody was detected using an HRP-labeled anti-rabbit IgG and either an immobilized substrate or a chemiluminescence reporter system.

Immunofluorescence Detection of Surface Cpa—Bacteria, wt, or paired cpa mutant GAS from early stationary phase were incubated with either affinity-purified polyclonal anti-Cpa49 antiserum or the corresponding preimmune serum for 30 min at room temperature, Following a washing step, bound antibody was detected by reactivity with an Alexa Fluor 488®-labeled goat anti-rabbit IgG (MoBiTec, Göttingen, Germany) using a BX60 fluorescence microscope and 100 x 1.3 or 60 x 1.25 UplanFI objectives (Olympus, Hamburg, Germany).

GAS Adherence to Immobilized Collagen—Adherence assays to quantify binding of GAS wt and mutant strains to immobilized collagen was performed in 96-well microtiter plates (Greiner Bio-One). Plates were coated overnight at 4 °C with 5 µg/well human collagen types I and IV (Sigma) in PBS and blocked with 1% BSA in PBS for 1 h at 37 °C. After three washes in 0.05% Tween 20/PBS, early stationary phase GAS strains diluted to an A600 nm of 0.4 in PBS were incubated in the coated wells for 30 min at 37 °C. In preceding control experiments, the number of bacteria used was established to be within the linear range of the collagen-bacteria interaction.

Adherence was determined, after four washes with PBS, by incubating with a goat anti-group A streptococci-HRP conjugate (Dunn Labortechnik GmbH, Asbach, Germany) diluted 1:5,000 in PBS. Following incubation for 1 h at room temperature, the plate was washed four times with PBS, and residual bound antibody was detected by using a 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase EIA substrate kit (Bio-Rad). After 10 min, the reaction was stopped with 0.5% H2SO4, and the absorbance at 450 nm (A450 nm) was measured. As a specificity control, GAS strains were also incubated in noncoated and BSA-coated wells. Inhibition of adherence of GAS binding to immobilized type I collagen by Cpa fragments was measured by preincubation of a collagen-coated plate with 100 µlof0.1 µmol of Cpa fragment solution for 2 h at 37 °C prior to conducting the GAS binding assay.

Inhibition of GAS binding by the anti-Cpa antiserum was assessed by preincubating the bacteria with 1:2, 1:5, 1:10, or1:100 dilutions of the antiserum in PBS at room temperature for 1 h and washing the cells in PBS before continuing the experiments as described above. As a control, bacteria were preincubated with a 1:2 dilution of preimmune serum or with PBS alone.

The results of the adherence assays are reported as the mean of at least three independent experiments. The data were statistically evaluated by means of the Mann-Whitney U test.

Surface Plasmon Resonance Protocols—All surface plasmon resonance (SPR) measurements were performed at 25 °C using a Biacore 3000 (Biacore AB, Uppsala, Sweden) equipped with research-grade CM5 sensor chips. Ligands (collagens types I, II, and IV and fibronectin) were immobilized on the flow cell surfaces of the chips to densities of 500–1000 response units using standard amine-coupling chemistry. An unmodified flow cell served as a reference surface. To immobilize ligands to a pre-set target level and to prepare the reference surface, the software tool "Application Wizard-Surface Preparation" was used (BIA-core 3000 Instrument Handbook). To collect binding data, the analytes, i.e. the mature Cpa1 and Cpa49 proteins or the corresponding Cpa fragments, dissolved in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4, flowed over the ligand and reference surfaces at concentrations of 0, 10, 25, 50, 100, 200, and 500 nM and at a flow rate of 30 µl/min. Each analyte-ligand complex was allowed to associate and dissociate for 5 and 10 min, respectively. The ligand surface was regenerated with a 15-s injection of 0.2% SDS at the end of each binding cycle, and each analyte concentration was tested in duplicate. The data sets were double-referenced (72) and globally fit to a simple interaction model in CLAMP (73) to obtain kinetic rate constants. When appropriate, data sets were also fit to a heterogeneous surface model.

Eukaryotic Cell Adherence and Internalization and Determination of Eukaryotic Cell Viability—Bacterial adherence and internalization to eukaryotic cells was determined by an antibiotic protection assay following the protocol of Molinari et al. (74). Briefly, early stationary phase bacteria were suspended in modified Eagle's medium supplemented with 10% fetal calf serum and added to HEp-2 cells grown overnight to confluence at a multiplicity of infection between 10 and 50. After 2 h, the eukaryotic cells were washed with PBS. One-half of the cells were lysed with distilled water, and the number of bacteria in the lysate was assessed by viable counts. The other half of eukaryotic cells was exposed to culture medium supplemented with 10 mg/liter penicillin and 100 mg/liter gentamicin for another 2 h. These cells were then washed and lysed, and the bacterial numbers were counted as above. The results are presented as the mean of four independent experiments and were analyzed for significance by the Mann-Whitney U test.

Viability of eukaryotic cells in the presence of GAS was determined by using the live/dead stain (Molecular Probes, MoBiTec) according to the manufacturer's instructions by observing the cells at 600-fold magnification. At least nine microscopic fields containing a minimum of 50 cells were examined in each assay. The results were assembled from three independent experiments.

Assessment of Anti-Cpa Antibody Titers in Patient Sera—For these experiments, patient sera were collected over a period of 10 years from three different German University hospitals (Aachen, Ulm, Rostock) and were stored at –80 °C until used for the experiments. All sera were first subjected to measurements of anti-streptolysin O antibody (ASL) and anti-DNase B antibody (ADB) titers according to the manufacturer's protocols (Biomerieux, Nürtingen, Germany). Patient sera with ASL/ADB titers ≤180/≤200 IU/ml, respectively, were considered negative, whereas patient sera with ASL/ADB titers >200 and/or >250 IU/ml, respectively, were classified as positive.

Antibody to Cpa was measured in an ELISA in which 96-well microtiter plates were coated with 0.25 µg/well purified, mature Cpa1 and Cpa49 proteins, blocked with BSA, and washed with Tween 20/PBS. The coating conditions were identical to those used for coating 96-well microtiter plates with collagen for GAS adherence studies described above.

Patient sera were diluted 1:2,000 with PBS, added to two different wells per sample, and incubated at 37 °C for 30 min. As a control, each serum sample was also added to a BSA-coated well. Subsequent washing with PBS, incubation with a second goat anti-human IgG-HRP conjugate (Bio-Rad), and visualization with the peroxidase substrate kit were performed as described previously. The assay was repeated on two independent occasions.



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  		FIGURE 2.
Temporal transcription profiles of an S. pyogenes cpa promoter-luciferase reporter box fusion under different atmospheric conditions and in the presence of different growth media or factors. Panels A and B show the growth curves (measured by A600 nm values) and associated temporal luciferase activities (measured by relative light units) of a S. pyogenes-cpa-luc reporter fusion grown in THY and CDM medium under ambient air (aer), a 5% CO2/20% O2 atmosphere (CO2) or an anaerobic atmosphere (anaer). Panels C, D, and E display temporal luciferase activities of S. pyogenes cpa, mga, regulator, and nra regulator luc fusions, respectively, grown in CDM medium without (squares) or with supplementation of collagen type I (circles) or peptic digests of collagen type I (triangles).

 
For evaluation, the BSA background values were subtracted from the respective anti-Cpa values for individual patient sera. The mean of values from the two wells and from the two independent experiments was calculated. Standardized mean A450 nm values above 0.5 were considered positive.

Western immunoblot experiments with immobilized mature Cpa and Cpa fragments were performed as described above utilizing patient sera diluted 1:5,000 in blocking solution as the first antibody and goat anti-human IgG-HRP conjugate as the second antibody reporter system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Collagen Binding among GAS M Serotype Strains— To date, only limited studies of collagen types I and IV binding by S. pyogenes have been reported (46–50). Only one analysis for the presence of the cpa gene, which encodes a type I collagen-binding protein (51), has been performed on a different set of strains (52).

In the initial studies, binding of collagen types I and IV by S. pyogenes strains of 28 M serotypes that represented an epidemiologically relevant selection of bacteria contained in the preceding cpa epidemiology study (52) was determined. The results summarized in TABLE ONE demonstrate that 14 of 28 strains bound collagen type 11 to a high level, whereas 3 others bound significantly, and 3 isolates were negative. Collagen type IV interaction was less common with only 7 strains demonstrating significant binding and only 1 of those binding to a high level (TABLE ONE). All collagen type IV-binding strains also displayed significant to strong collagen type I binding. After complementing the results of Kreikemeyer et al. (52) on the molecular cpa epidemiology with the most recent accessible information about complete S. pyogenes genome sequences (65–67) and NC_002958 (GAS Sequencing Group at the Sanger Institute, UK, www.sanger.ac.uk/Projects/S_pyogenes/)), the cpa gene was found to be present in 13 and 5 of the collagen type I and IV binding isolates, respectively. There was no association between either the collagen-binding phenotype and/or the presence of the cpa gene with the typical initial infection site or nonpurulent sequelae caused by these strains (TABLE ONE).


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  		TABLE ONE
Binding of soluble, 125I-labeled collagen types I and IV molecules to S. pyogenes strains belonging to the group of epidemiologically relevant serotypes

For the origin of the S. pyogenes serotype strains see under `Experimental Procedures.' Epidemiologic relevance is listed according to Colman et al. (86), Johnson et al. (87), and Kaplan et al. (88). For details of the 125I-labeling of collagen and the quantifying of collagen binding by whole early stationary phase bacteria, see under `Experimental Procedures.' The genomic presence of the cpa gene is shown according to Kreikemeyer et al. (52) and to the publically available cpa sequences of serotype strains M1 (NC_002737 [GenBank] ), M3 (NC_004606 [GenBank] ), M5 (NC_002958), M6 (NC_006086 [GenBank] ), M12 (AF447492 [GenBank] ), and M18 (NC_003485 [GenBank] ). The epidemiologic association of single S. pyogenes serotype strains to clinical diseases such as pharyngitis, pyodermia, acute rheumatic fever (ARF), and acute glomerulonephritis (AGN) is presented according to Bessen et al. (89), Maxted (90), Wannamaker (91), and Widdowson (92).

 
Transcription Profiles of the Cpa Operon as Determined by Luciferase Reporter Gene Activity—To study the expression of cpa under different environmental conditions, a cpa operon-luc fusion was created. Previous studies have shown that coupling FCT region genes with a luciferase reporter gene is a useful approach to follow the temporal expression profiles of these comparatively low level transcribed genes (51, 55). In the initial experiments, bacteria grown in a complex media, THY, demonstrated maximally expression of the cpa operon under anaerobic conditions (Fig. 2B). Under CO2-enriched air or an aerobic atmosphere, the cpa operon transcription decreased by a factor 1.5–2 (Fig. 2). When using CDM broth in a CO2-enriched atmosphere, cpa expression was found to be lower by a factor 1.5 when compared with incubation in THY broth (Fig. 2B). Supplementation of CDM broth with full sized collagen type I or peptic breakdown products did not significantly alter the cpa operon expression (Fig. 2C).



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  		FIGURE 3.
Determination of the minimal collagen-binding site within the Cpa molecule. Recombinant full sized mature Cpa1 and Cpa49 as well as N-terminal, C-terminal, and internal Cpa49 fragments were used to assess the binding to immobilized collagen type I by an ELISA approach or SPR measurements. Collagen binding of the individual fragments is indicated by plus or minus signs. The N- and C-terminal amino acid (aa) residues contained in the individual fragments are specified by the respective position numbers of the Cpa1 or Cpa49 aa sequences.

 
Previous studies have also suggested that the regulators Mga and Nra could influence cpa expression (51). Consequently, recombinant S. pyogenes strains harboring mga- and nra-luc fusions were also tested for expression in response to exposure to collagen type I and peptic digests. These collagen type I preparations decreased the mga or nra transcription rates by factors of 3 and 4, respectively (Fig. 2, D and E).

Mapping of Collagen-binding Domains in Recombinant Cpa—The results presented in TABLE ONE indicate that the majority of cpa-carrying strains bind both type I and type IV collagen. In the next series of studies, the importance of the cpa gene product in binding to different types of collagen as well as the potential biological activity of these interactions was studied. Initially, recombinant mature Cpa1 and Cpa49 proteins and a number of internal Cpa49 fragments were tested for their specific reactivity with types I, II, and IV collagen. In these experiments fibronectin binding was included as a negative control.

By employing an ELISA approach, immobilized collagen of the three types was incubated with different purified and biotinylated Cpa fragments. Binding was determined, following appropriate washing steps, using an avidin-horseradish peroxidase reporter system. In agreement with earlier studies (51), Cpa was confirmed to bind to collagen type I but demonstrated no significant reactivity with type II or IV or fibronectin. By using the various defined constructs of Cpa, it was possible to map the collagen type I-binding domain to an internal amino acid region between amino acids 359 and 466 of the Cpa49 molecule (Fig. 3).

A similar series of binding studies was also carried out by using SPR to determine the affinity of the interactions. Collagen types I, II, and IV and fibronectin as a negative control were immobilized on the surfaces of biosensor chips in four separate flow cells. Binding responses were recorded as Cpa fragments flowed across these surfaces (supplemental Fig. s1). No increase in response was observed when these recombinant Cpa fragments flowed over immobilized fibronectin (data not shown). Additionally, in agreement with the ELISA study, there was no significant binding to chips coated with collagen II and IV when any of the Cpa proteins or fragments were tested (data not shown). Binding of Cpa and fragments to collagen type I could be documented (supplemental Fig. s1), and the pattern of reactivity was consistent with the binding detected by ELISA (Fig. 3).

Fitting the SPR profiles to interaction models yielded kinetic and affinity information for the different Cpa construct-collagen I interactions. Initially, each profile was analyzed based on a simple 1:1 interaction model. The responses for the internal polypeptides Cpa49-3 and mature Cpa49 were well described by this model (supplemental Fig. s2, A and B); however, the responses for the internal Cpa49-1 and -2 fragments were not ((supplemental Fig. s2, C and D). Instead, the profiles of these two fragments could be better described by a heterogeneous surface model, which assumed two different classes of binding sites within collagen type I. Based on the best data fit, the Cpa constructs bound collagen type I with affinities in the nanomolar to low micromolar range (TABLE TWO). These values are of the same magnitude reported for the interaction of collagen with other MSCRAMMs (15, 29). A lower affinity (KD1) was measured for fragment internal Cpa49-4, suggesting that part of the critical collagen-recognition domain had been lost in this truncated fragment, and internal Cpa49-1 and -2 fragments also displayed weaker affinity binding sites for collagen type I. The biological relevance of this observation is currently unclear.


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  		TABLE TWO
Kinetic and affinity parameters for Cpa-collagen type I interactions

The parameters were determined by surface plasmon resonance measurements using immobilized collagen type I as the ligand, and Cpa fragments as the analyte. For the description of the recombinant Cpa49 fragments, see under the "Experimental Procedures." ka, association rate constant; kd, dissociation constant; KD, dissociation equilibrium constant; fraction indicates the distribution of the two Cpa-binding site classes on the collagen target molecule.

 
Prediction of the Collagen-binding Site in Other Cpa Molecules—Evidence for sequence variation among cpa genes from different isolates has been noted (53). To identify conserved consensus sequences, the predicted aa sequences for Cpa1, -3, -5, -12, and -18 were aligned to the smallest collagen-binding Cpa49 fragment determined experimentally (Fig. 3). Two conserved consensus sequences were recognized within the calculated ~60-aa subsequence necessary for collagen reactivity (supplemental Fig. s3). The consensus sequences of these two boxes are: 361(V/i)EN(P/q)NKE***PY(S/t)(V/i)(E/t)A(Y/f)(N/s)D(F/m)380 and 392Y(G/a)KFYY(A/g)KN*(N/d)(G/k)(S/t)(S/n)Q(V/i)VYCFN(A/v)(D/n)L(H/k)SPP(D/e)S421. The amino acid residue numbers refer to the Cpa49 sequence; lowercase letters indicate residues in which one or two sequences deviate from the remainder, and asterisks indicate residues that display 3- or 4-fold variability among the six sequences. A secondary structure analysis of the two consensus sequences, utilizing four different programs, predicted one extended ({beta}-strand/sheet) section, with a medium probability, in the first box and two extended ({beta}-strand/sheet) sections in the second box, with a high probability.

When comparing the six complete 523–757-aa Cpa sequences (supplemental Fig. s4), a relatively well conserved 46–53-aa leader peptide region was identified. This portion is followed by an extremely divergent N-terminal section spanning aa residues 47–52 in Cpa5 and Cpa18 up to aa residues 47–270 in Cpa49 and aa residues 54–291 in Cpa1. The adjacent central region comprises about 240-aa residues and is most conserved between the six Cpa sequences. C-terminal to the central section is a stretch of ~30-aa residues that displays high variability. Based on their sequence homologies, the remaining ~210 C-terminal aa residues allow the distinction of three types within the family of Cpa molecules: Cpa5, -12, and -49, Cpa3 and -18, and Cpa1.

The collagen-binding portion of the Cpa49 sequence is located in the middle of the central highly conserved region. Immediately adjacent to the collagen-binding portion, the sequence of Cpa49 aa residues 289–344 display a 80% homology to the S. aureus Cna B-region. In Cna, the B-region is not involved in the Cna-collagen binding (13, 14).

Interaction of GAS Strains with Immobilized Collagen—The molecular characterization of Cpa indicates that the ability to bind type I collagen is of sufficient affinity to promote bacterial adherence to this ligand if the protein is surface-expressed at an appropriate level. To test the importance of Cpa on bacterial adherence to collagen type I, we attempted to create an isogenic mutant with a targeted insertion in the cpa gene. The simple approach of disrupting the cpa gene failed most probably due to the crucial function of one of the downstream genes in the cpa operon. Consequently, an alternative strategy was used to create a recombinant insertion vector that not only disrupted cpa but also introduced a second cpa promoter downstream of the truncated cpa gene. This approach yielded viable mutants that expressed wild type levels of the downstream cpa operon genes (data not shown). In addition, the cpa mutant demonstrated similar growth kinetics and final population densities to the wild type bacteria when grown in either THY or CDM broth (data not shown).

For the following set of experiments, the wt and cpa mutant strains were grown to the late exponential phase, when the cpa gene expression was close to maximum. Extracts of surface proteins from the wild type but not the cpa mutant contained proteins that reacted with a polyclonal anti-Cpa49 rabbit antiserum bacteria by Western immunoblot analysis (Fig. 1C). The wt material contained two immunoreactive bands, the Mr ~110,000 band corresponding to full sized Cpa and a potential breakdown fragment Mr ~80,000 (Fig. 1C).

Immunofluorescent studies were also conducted using the polyclonal anti-Cpa49 rabbit antiserum to determine surface expression of Cpa (Fig. 1D). Surface Cpa expression was observed among some but not all wild type bacteria, whereas surface staining of any bacteria carrying the cpa mutation could not be detected (Fig. 1D). The observation that only a minority of wt bacteria could be stained with the anti-Cpa49 antiserum suggests that even if commonly present within a S. pyogenes population, because of an open-loop-type regulation (75), the cpa gene may be expressed by only a subset of bacteria. In addition, only the external and thus older poles of dividing wt bacteria appeared to carry Cpa on their surface (Fig. 1), which would correspond to the documented peak expression of cpa during the transition growth phase.



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  		FIGURE 4.
Inhibition of the adherence of whole M1 or M49 S. pyogenes wt bacteria with immobilized collagen type I by using recombinant purified Cpa fragments (A) or polyclonal anti-Cpa49 rabbit antiserum (B and C).

 
Adherence of the wild type and the isogenic cpa mutant pair to immobilized collagen types I and IV was tested. The cpa mutant displayed a statistically significant 7-fold decrease in the number of adherent bacteria to collagen type I when compared with the wild type and an unaltered (background) interaction with collagen type IV (TABLE THREE).


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  		TABLE THREE
Adherence of whole S. pyogenes wt and cpa mutant bacteria to immobilized collagen types I and IV as determined by ELISAs

For details of plate coating with collagen and the ELISA protocol, see under "Experimental Procedures." The ELISA values were obtained by subtracting the control values from the values of the corresponding A450 nm measurements. The tests were performed on three independent occasions, and the results are presented as the mean values of these measurements.

 
To test the specificity of the collagen type I binding by the wild type organisms, the wells were preincubated with increasing amounts of recombinant mature Cpa1 or Cpa49 or collagen-binding Cpa49 fragments. Even the lowest concentration of Cpa molecules used in this assay (17 nM/liter) could efficiently inhibit the adherence of serotypes M1 and M49 wt bacteria to collagen type I. Cpa1 molecules were equally effective as Cpa49 molecules in this inhibition experiment, and only Cpa49 fragments that contained the collagen type I-binding site showed an inhibitory effect (Fig. 4A).

Adherence could also be blocked by preincubating the bacteria with polyclonal anti-Cpa49 rabbit antiserum but not normal rabbit serum prior to incubation with immobilized collagen type I. Serum dilutions of 1:2 and 1:5 led to a markedly decreased binding of both serotype strains, whereas serum dilutions of 1:10 and higher as well as the preimmune serum or buffer had no inhibitory effect (Fig. 4, B and C).



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  		FIGURE 5.
Western immunoblots with recombinant purified Cpa1 and Cpa49 fragments using a defined set of patient sera with or without anti-Cpa type-specific antibodies. Full sized mature Cpa1 and Cpa49 proteins as well as the Internal Cpa49-1–4 fragments were transferred to each Western blot and reacted with a selection of S. pyogenes patient sera that, according to quantitative ELISA measurements, contained no anti-Cpa antibodies or high titers of anti-Cpa1 and/or anti-Cpa49 antibodies. m, molecular mass marker.

 
Role of Cpa for Interactions between GAS and Eukaryotic Cells—All of the studies described to date have focused on the interaction of bacterial proteins or bacteria with isolated collagen type I. No studies have addressed the importance of the S. pyogenes collagen type I binding for interactions between bacteria and eukaryotic cells. To address this issue, the serotype M49 wt and the paired isogenic cpa mutant were tested for adhesion and internalization by HEp-2 cells over a 5-h incubation period. By employing an antibiotic protection assay and subsequent measurement of viable organisms, the wt strain displayed adherence and internalization rates of 12.8 ± 2.4% and 2.2 ± 0.4%, respectively, whereas for the cpa mutant strain, values of 17.6 ± 6.1% and 1.0 ± 0.3%, respectively, were obtained. Based on a p < 0.01 value, the increased adherence of the cpa mutant was not statistically significant in the Mann-Whitney U test, whereas in contrast its decreased internalization rate was found to be significant. Over the 5-h assay period, the host cell viability in the presence of the cpa mutant did not differ from that observed for the wt bacteria according to results from Live/Dead staining and fluorescence microscopy (data not shown).

Epidemiology of Anti-Cpa Antibody Presence in Patient Sera and Association with Clinical History—The potential role of Cpa in the disease course of streptococcal infections is difficult to determine, since the cpa gene is present in only one-third of GAS strains, and it is currently unknown if the protein is expressed during a purulent S. pyogenes infection. To address this concern, a series of serological assays was performed by using serum from 87 patients with highly positive ASL and/or ADB titers collected over a period of 10 years at three German University Hospitals separated by about 600–800 km. The results of these studies were compared with a second set of 40 patient sera that all displayed ASL and ADB titers at or below the detection threshold ("negative control"). The control sera were collected and examined by the routine diagnostic laboratory for assessment of a variety of antibody titers.

The ASL titers of the GAS patient sera ranged from <48 to 3493 IU (mean 922.7, median 648) and the ADB titers from <72 to 4470 IU (mean 1049.0, median 613). These sera were also tested in an ELISA format with Cpa1 or Cpa49 as the target antigen. The results of the anti-Cpa antibody measurements in the S. pyogenes and control patient sera are shown in TABLE FOUR.


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  		TABLE FOUR
Clinical background and Cpa reactivity of patient sera

 
Of note, among the 34 anti-Cpa-positive GAS patients, 18 displayed antibody titers above the cut-off for only one of the two Cpa antigens tested, i.e. either Cpa1 or Cpa49, suggesting the presence of distinct immunodominant epitopes on the related Cpa antigens. For 10 of these patients, the positive anti-Cpa-titer was <0.3 OD values above the cutoff, suggesting a weak or waning type-specific anti-Cpa antibody titer.

The reactivity of a set of four GAS patient sera (anti-Cpa1-positive/anti-Cpa49 positive; anti-Cpa1-positive/anti-Cpa49-negative; anti-Cpa1-negative/anti-Cpa49-positive; and antiCpa1/Cpa49-negative) was also tested using Western immunoblotting. In these studies only the mature Cpa fragments were detected by the patient antibodies, and their pattern of reactivity matched that observed by ELISA measurements (Fig. 5).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Attachment and colonization of a host by bacteria is a complex and dynamic process that involves key interactions between surface bacterial structures and host extracellular matrix. Analysis of these interactions has identified bacterial structures that bind to fibronectin, collagen, and potentially other important cellular or basement membrane targets. In the majority of studies of S. pyogenes, attachment through interactions between a serotype-specific array of fibronectin-binding proteins and their ligands seems to be the predominant factor for effective colonization. Consequently, this phenomenon has been extensively studied (4, 76, 77). However, fibronectin binding alone does not explain why only some GAS isolates tend to cause infrequent but nonetheless severe infections of defined tissues in the meninges, lungs, or the bones and joints.

Prior to this investigation, several studies documented the capability of a minority of S. pyogenes strains to bind collagen type IV (46–50). By using a variety of genetic approaches and binding studies, the interaction of 28 representative M serotypes of S. pyogenes with type I and IV collagen has been analyzed. These studies have led to a clear association between isolates containing a cpa gene and the ability to bind type I collagen. These studies also confirmed that the Cpa protein was selective for binding to type I collagen and that the expression of the molecule did not correlate with the ability of a bacterium to bind to type IV collagen and suggest the existence of an independent type IV-binding molecule, potentially associated with the bacterial capsule (8). However, all collagen type IV-binding isolates were also found to bind to collagen type I. There was no clear association between GAS expressing either type I collagen-binding proteins or type I and type IV collagen-binding proteins and a typical initial infection site for the S. pyogenes strains used in this study (TABLE ONE).

Evidence for variation in the cpa gene sequence among strains was apparent and suggested the existence of at least three distinct gene families. All variants of the cpa gene contained a highly conserved domain that encompassed the collagen type I binding domain. Using the PCR methods in this study, the finding that a minority of strains that bound collagen type I appeared to lack a cpa gene raises the possibility that additional families of cpa genes may exist and that these isolates may in fact contain a conserved collagen-binding domain that has thus far not been identified. With the comparison of Cpa sequences in the present and previously published studies it became obvious that the frequency and extent of Cpa sequence variation as well as the pattern of variable and more conserved regions within the Cpa molecules correlate to variation rates also seen in other FCT region-encoded surface proteins, e.g. F1/SfbI and protein F2 (52).

Cpa binds only to collagen type I and not to other tested matrix components. The affinity of this interaction, as determined by surface plasmon resonance and other experimental approaches, is within the range of other known adhesins from Gram-positive cocci (55, 78–80) and is actually higher than that of the minor S. pyogenes fibronectin adhesin, SOF, for its ligand (81). An interesting feature was the evidence for the independent high and low affinity of Cpa-1 and -2 fragments for two different sites within the collagen type I molecule. The exact location of the low affinity domain cannot be precisely determined with the existing subclones and will require a more refined structure-function analysis.

Analysis of the structural characteristics of the stronger collagen binding activity identified the putative high affinity collagen-binding domain, which would account for the common functional activity (Figs. 3 and 4) among the variable cpa genes (supplemental Fig. s2). The probable secondary structure of the two conserved boxes inside the binding fragment is shared by diverse Cpa molecules (supplemental Fig. s1). The predicted presence of {beta}-strands/sheets within these boxes resembles the predicted structure of the collagen-binding 19-kDa fragment of the S. aureus Cna molecule (15, 16). These structures form a trench that exactly accommodates a collagen triple helix (16, 18). Most interestingly, the Cpa collagen-binding site shares no sequence homology with the corresponding Cna site, suggesting a convergent evolutionary strategy. This type of conservation of a functionally important secondary structure, despite extreme sequence variation, has been demonstrated previously as an evolutionary principle for other MSCRAMMs in Gram-positive cocci (82).

Developing a complete understanding of the role of Cpa in S. pyogenes infection will require a detailed analysis of the regulation of cpa gene expression, measurement of the qualitative and quantitative expression of the protein under different environmental conditions in the infected host. The present study clearly demonstrated that the in vitro expression of the cpa gene was maximal during the exponential growth phase and when bacteria were grown under a CO2-enriched or anaerobic atmosphere, i.e. the partial gas pressures typically encountered within the inflamed tissues. Surprisingly, in culture cpa was transcribed and translated at a low level by only a few bacteria within a given population, as determined by immunofluorescence microscopy.

Most interestingly, there was no consistent evidence for enhanced cpa expression in the presence of collagen type I peptide fragments as seen in other streptococcal species (44, 83, 84). However, the presence of collagen peptides did influence expression of two S. pyogenes regulatory genes, mga and nra. These regulators have been reported to have direct effects on cpa gene expression (51).

Studies of the importance of the Cpa expression on adherence to HEp-2 cells demonstrate little effect when compared with the predominant activity associated with expression of fibronectin-binding proteins (55). There was a small increase in attachment rate of the cpa mutant that was accompanied by a decrease in bacterial internalization. The decreased internalization rate of the mutant is at present not understood at the molecular level, because the uptake mechanism of the bacteria has only been partially elucidated. Besides the fibronectin-integrin interaction-triggered phagocytosis mechanism, there exists at least one other pathway involving caveolae formation (85). At present it is not clear if Cpa expression could contribute to the efficiency of either or both of these pathways or could represent a third mechanism that could be critical at certain sites of infection.

One approach to accessing the potential role of Cpa in different infections is to analyze the serological response of S. pyogenes-infected patients. Evidence for Cpa antibody was found in a number of serum samples, indicating that the bacterial protein is immunogenic and can be expressed during infection. An association between Cpa-seropositive patients and their disease course suggested a statistically significant association with a diagnosis of arthritis and/or osteomyelitis. This observation raises the intriguing possibility that collagen type I, which is predominantly found in the synovia and bones, may be targeted. A similar association between Cna expression and collagen type I-binding S. aureus strains with arthritis or osteomyelitis has been proposed (22, 24, 26). The hypothesis that specific MSCRAMM surface molecules that target type I collagen, like Cpa in S. pyogenes or Cna in S. aureus, may provide only a subset of these pathogens with a niche-specific advantage for infection of the skeletal system merits consideration and further study.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Figs. s1–s3. Back

1 Supported by Deutsche Forschungsgemeinschaft Grants Po391/9-1, Po391/9-2, Po391/11-1, Po391/11-2, and Po391/12-1. Back

2 To whom correspondence should be addressed. Tel.: 49-381-494-5900; Fax: 49-381-494-5902; E-mail: andreas.podbielski{at}med.uni-rostock.de.

3 The abbreviations used are: GAS, group A streptococci; aa, amino acid(s); ADB, anti-DNase B antibodies; ASL, anti-streptolysin O antibody; BSA, bovine serum albumin; CDM, chemically defined medium; Cpa, collagen-binding protein of group A streptococci; ELISA, enzyme-linked immunosorbent assay; FCT, fibronectin-/collagen-binding protein, T-antigen Coen's; HRP, horseradish peroxidase; MSCRAMM, microbial surface components recognizing adhesive matrix molecules; PBS, phosphate-buffered saline; SPR, surface plasmon resonance; wt, wild type. Back


   ACKNOWLEDGMENTS
 
We thank M. D. P. Boyle (Juniata College, Huntingdon, PA) for critically reading the manuscript and correcting it for clarity, style, and grammar. We thank R. Lütticken (Aachen, Germany) and H. Kuramitsu (Buffalo, NY) for providing the GAS strain and the pUCerm plasmid, respectively. We also thank V. Fischetti (New York, NY) for giving us the streptococcal C1 phage lysin, which greatly improved the surface protein preparation process from GAS. In addition, we thank M. O. Glocker (Proteome Center Rostock, Germany) for granting access to the BIAcore system.



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 DISCUSSION
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