©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of a Novel Collagen-binding Protein from Streptococcus pyogenes Strain 6414 (*)

(Received for publication, March 29, 1994; and in revised form, September 16, 1994)

Livia Visai (1) Silvia Bozzini (1) Giuseppe Raucci (2) Antonio Toniolo (3) Pietro Speziale (1)(§)

From the  (1)Department of Biochemistry, University of Pavia, Via Bassi 21, I-27100 Pavia, Italy, (2)Menarini Ricerche Sud, Via Tito Speri 10, I-00040 Pomezia, Roma, Italy, and (3)Institute of Medicine and Public Health, University of Pavia, Viale Borri 57, I-21100, Varese, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In this report we have analyzed the binding of collagen to Streptococcus pyogenes strain 6414. This binding was rapid, specific, and involved a limited number of receptor molecules (11,600 copies per cell). When the proteins in a streptococcal lysate were blotted onto a nitrocellulose filter and probed with I-labeled collagen, a prominent collagen-binding protein of 57 kDa was identified as well as minor 130-150-kDa components. The major 57-kDa protein was isolated by affinity chromatography on collagen-Sepharose followed by gel filtration chromatography. The 57-kDa protein purified from S. pyogenes was used to raise a monospecific antibody which also reacted with a collagen-binding protein of similar molecular size isolated from Streptococcus zooepidemicus. The two collagen-binding proteins from streptococci have a similar amino acid composition and isoelectric points. Isolated collagen-binding protein was specifically recognized by I-collagen in a solid-phase binding assay and displayed an affinity for the ligand quite similar to that exhibited by intact bacteria (K= 3.1 versus 3.5 times 10M, respectively). Surface-labeled bacteria attached to microtiter wells coated with different collagen types and the 57-kDa protein blocked the adhesion to collagen substrate. We propose that the 57-kDa protein is an adhesin involved in the attachment of streptococci to host tissues.


INTRODUCTION

Interactions of cells with the surrounding extracellular matrix play important roles in numerous physiological and pathological events. In higher animals, cell-matrix interactions involve binding of cells to collagens, proteoglycans, and various glycoproteins such as fibronectin and laminin.

Collagens are widely distributed proteins in vertebrate tissues and at least 14 genetically different types of collagen have been discovered. In the organisms the collagens play a structural role and influence biological processes such as cell attachment(1, 2) , proliferation(3) , as well as cell differentiation during organogenesis (4) and hematopoiesis(5) .

Membrane proteins with collagen binding properties have been suggested to act as collagen receptors and have been purified from osteoblastoma cells(6) , fibroblasts(7) , endothelial cells(8) , platelets(9) , and chondrocytes(10) .

Several studies have shown that a variety of pathogenic bacteria interact with extracellular matrix components including fibronectin (11) , fibrinogen(12) , laminin(13) , and collagen. For instance strains of coagulase-negative staphylococci (^1)and strains of Staphylococcus aureus, particularly those isolated from patients with septic arthritis or osteomyelitis, have been reported to express collagen receptors(14) . The binding of collagen to staphylococci has been characterized in some detail(15) , and the isolation(16) , cloning, and sequencing of a collagen receptor from S. aureus has been performed(17) . Furthermore, evidence that the staphylococcal collagen receptor functions as a colonization factor of cartilage and as a potential virulence determinant in septic arthritis has been reported(18) .

Within the genus Streptococcus, there are species, mainly among the groups A, B, C, and G, which bind collagens(19) . This binding has been postulated as a factor contributing to the development of a number of infections. For example binding of Streptococcus mutans strains to collagen has been proposed to play a role in the pathogenesis of root caries(20) , and the ability of Streptococcus pyogenes to bind collagen type IV may be an important virulence factor in determining post streptococcal glomerulonephritis(21) .

A collagen receptor from streptococci has previously not yet been isolated or characterized. In this study we report on the isolation and characterization of a 57-kDa collagen-binding protein from S. pyogenes 6414 which can act as an adhesin and mediate the adherence of streptococcal cells to collagen rich tissues.


EXPERIMENTAL PROCEDURES

Chemicals

Type II collagen was purified from bovine nasal septum as described by Strawich and Nimmi(22) . Collagen was denatured by heating an aliquot of the collagen stock solution at 60 °C for 30 min immediately before using. Isolation of native collagen types I-IV was as described previously(15) . Fibronectin was purified from human plasma(23) .

Bovine serum albumin, ovalbumin, fetuin, bovine IgG, protein A, and protein G were from Sigma. Iodogen was from Pierce Chemical Co. Todd Hewitt Broth was supplied by Difco. Carrier-free I (specific activity, 15 mCi/µg) was purchased from Radiochemical Centre, Amersham, UK.

Bacterial Cultures

The strain used for this study, S. pyogenes 6414, was obtained from Dr. M. Hook (Institute of Extracellular Matrix Biology, Texas A& University, Houston, TX). Streptococcus zooepidemicus strain S(z) III and S. pyogenes strain Sp 1-4065 were provided by Dr. M. Lindberg (Swedish University of Agricultural Science, Uppsala, Sweden). The strains were grown in Todd-Hewitt Broth at 37 °C for 16 h.

After harvesting, bacteria were suspended in 0.13 M sodium chloride, 0.02% sodium azide, and 10 mM sodium phosphate, pH 7.4 (PBS), (^2)washed, and adjusted to a cell density of 10 cells/ml using a standard curve relating the A to the cell number determined by counting cells in a Petroff-Hausser chamber. The cells were then heat-killed at 88 °C for 10 min and then stored at -20 °C until used.

Radiolabeling of Collagen and Bacteria

Collagen was labeled by the IODO-GEN method following the procedure recommended by the manufacturer (Pierce). The specific activity of the radioactively labeled ligand was estimated to be 3 times 10^6 cpm/µg. Radiolabeling of bacteria was performed as reported(24) . The specific activity of the bacterial suspension was 10^5 cpm/3.7 times 10^5 cells of S. pyogenes 6414.

Binding of I-Collagen to Bacteria

The binding of I-labeled collagen to streptococci was quantitated as described previously(11) . Briefly, 5 times 10^8 bacteria were incubated with 5 times 10^4 cpm of I-labeled collagen in 0.5 ml of PBS containing 0.1% bovine serum albumin and 0.1% Tween 80 to block aspecific binding to cells and tubes. The mixture was rotated in an end-over-end mixer for 1 h at 22 °C. The reaction was stopped by the addition of 3 ml of ice-cold PBS containing 0.1% Tween 80, and the tubes were centrifuged at 1400 times g for 15 min. After aspiration of the supernatant, the remaining pellet was analyzed for radioactivity in a -counter. Radioactivity recovered in the tubes incubated in the absence of bacteria (background) was subtracted from that of the samples containing bacteria. The background radioactivity (600-800 cpm) was similar to that obtained in controls where I-labeled collagen was incubated with bacteria in the presence of 100 µg of unlabeled collagen. Samples and controls were run in duplicate.

Solubilization of Collagen-binding Proteins

Cells of S. pyogenes 6414 were suspended in 50 mM Tris-HCl, pH 7.6, containing 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mMN-ethylmaleimide, to a density of 5 times 10 bacteria/ml. The suspension was sonicated (4 times 1.2 min), and bacterial debris were removed by centrifugation (30 min times 10,000 times g). The supernatant was further clarified by centrifugation for 1 h at 90,600 times g and then stored at -20 °C until used.

Incubation of Bacteria with Trypsin

Streptococci (10 cells/ml) were suspended in phosphate buffer and digested with trypsin (1 µg/ml) at 37 °C for 30 min. The reaction was stopped by heating for 3 min at 100 °C. Cells were pelleted by centrifugation, washed, suspended in NaCl/P(i), and finally assayed for collagen binding. Trypsin-treated cells showed a 75% reduction in collagen binding ability relative to control cells. The supernatant from the trypsin-treated cells (denoted trypsinate) was employed for immunoblotting assays.

Electrophoresis and Blotting

Electrophoresis in 10% SDS-polyacrylamide gel electrophoresis (PAGE) was carried out according to Blobel and Dobberstein(25) . Radioactive components were visualized by autoradiography of dried gels with X-Omat A-R film (Eastman Kodak Co.). Blotting was performed essentially using the procedure of Towbin et al.(26) . Proteins separated by electrophoresis were electrotransferred for 2 h at 200 mA onto nitrocellulose membranes (Bio-Rad).

For Western ligand blotting assay the membranes were incubated with 3% bovine serum albumin in PBS for 1 h at 22 °C and then probed overnight with 5 times 10^5 cpm of I-labeled collagen type II containing 0.1% albumin and 0.1% Tween 80. The membranes were washed extensively with 0.1% Tween 80 in PBS, dried, and exposed at -70 °C using a X-Omat A-R film.

For immunoblotting assays, the nitrocellulose sheets were blocked with 3% albumin in TBS (20 mM Tris, 0.5 M NaCl, pH 7.5) then probed with IgG overnight at 4 °C, followed by washing with TTBS (0.5 M NaCl, 20 mM Tris/HCl, 0.05% Tween 20, pH 7.5). Subsequently the sheets were incubated with anti-mouse IgG antibodies conjugated with horseradish peroxidase (Bio-Rad) diluted 1:1000 in TTBS containing 1.0% albumin.

Amino Acid Analysis

The material isolated from collagen-Sepharose was subjected to SDS-PAGE and electrophoretically transferred to Immobilon-P membrane (Millipore, Bedford, MA) according to the procedure described by Matsudaira (27) and stained with Coomassie Brilliant Blue. The band corresponding to the 57-kDa protein was excised and subjected to gas-phase hydrolysis in 6 N HCl containing 1% (v/v) phenol at 105 °C for 24 h. Amino acid analysis was carried out after precolumn derivatization procedure as reported by Cohen and Michaud(28) . using a Jasco (Japan Spectrometry) amino acid analyzer equipped with 980-PU pump and a 820-FP detector.

Amino-terminal Sequence

The 57-kDa protein electrophoretically adsorbed on Immobilon-P was excised and covalently attached to the membrane by using the SequeNet kit from Millipore and sequenced by a Millipore model 6625 protein sequencer.

Solid-phase Binding Assay

Adhesion of streptococci to surface coated collagen and binding of collagen to surface-immobilized 57-kDa protein were performed in microtiter plates (P.E.G.T. assay strips, Costar, Europe Ldt., Badhoevedorp, The Netherlands). In binding experiments wells were coated with 20 µl of protein solution (100 µg/ml), incubated overnight at 4 °C, and then subjected to blocking with 200 µl of 1% bovine serum albumin for 1 h at 37 °C. After washing with PBS containing 0.1% Tween 20 the wells were incubated with the indicated amounts of radioactively labeled collagen for 2 h at 37 °C. After extensive washing the amount of bound I-labeled collagen was quantitated in a -counter.

For adhesion assays, microtiter wells were coated with 50 µl of collagen type II (100 µg/ml) and blocked with bovine serum albumin as reported above. The wells were then overlaid with 10-20 µl of a suspension of I-labeled bacteria (3.3 times 10^7 cells/ml), incubated at 37 °C for 2 h, washed extensively with PBS containing 0.1% Tween 20, and counted. All of the samples were corrected for background values corresponding to the radioactivity recovered in wells coated with albumin alone.

Preparation of Antibodies to 57-kDa Protein

BALB/c mice (6-week-old females) were immunized by intraperitoneal injection with 15 µg of isolated 57-kDa protein emulsified in Freund's complete adjuvant. Three booster injections of the same material in incomplete adjuvant were administrated at 2-week intervals. Ten days following completion of the fourth cycle of injections, the mice were sacrificed, and the blood was collected. The blood was allowed to clot and spun at 2000 times g for 10 min. The IgG fraction of the serum was purified on a column of protein A-Sepharose (Pharmacia, Uppsala-Sweden), and adsorbed IgG antibodies were eluted with 3 M MgCl(2), dialyzed against PBS, and stored frozen in small aliquots.


RESULTS

Characterization of Collagen Binding to S. pyogenes Strain 6414

To study the streptococcal collagen receptor we selected a strain, S. pyogenes 6414, which in preliminary studies was shown to bind a high amount of collagen. This strain grown in brain-heart infusion or Todd-Hewitt broth more effectively bound I-collagen than when grown in Luria broth.

The binding of I-labeled collagen was very rapid and essentially completed before 10 min of incubation. Prolonged incubation did not result in additional binding of I-labeled collagen.

Heat-killing of streptococcal cells did not alter the binding kinetics and similar amounts of collagen bound to live and killed bacteria. Therefore, heat-treated bacteria were used throughout this study, and the cells were routinely incubated with the ligand for 60 min.

The specificity of collagen binding by streptococci was examined by incubating bacterial cells with I-labeled type II collagen in the presence of excess amounts of a variety of unlabeled proteins. Addition of unlabeled type II collagen to the incubation mixture effectively blocked the binding of labeled ligand. Other proteins tested, including alpha1-acid glycoprotein, fetuin, ovalbumin, fibronectin, protein G, protein A, and rabbit IgG antibody to the collagen receptor on S. aureus, did not affect ligand binding.

When increasing concentrations of native and denatured type II collagen and gelatin were tried as inhibitors of I-collagen binding to bacteria we found that the native collagen was a more potent inhibitor compared to the denatured forms (Fig. 1) indicating that native collagen displayed a higher affinity than denatured collagen for the streptococcal collagen binding component. The binding of I-labeled collagen to S. pyogenes was essentially irreversible, i.e. iodinated collagen which bound to bacteria during a preliminary incubation period of 1 h was only marginally displaced from the cells on addition of 100 µg of unlabeled collagen to the incubation mixture.


Figure 1: Effect of native and denatured collagen on I-collagen binding to S. pyogenes 6414. Bacteria were incubated for 1 h with I-labeled collagen in the presence of increasing concentrations of native (circle), denatured collagen type II (times), and gelatin (bullet), and the amount of bound radiolabeled collagen was determined. Inhibition is expressed as percentage of I-labeled collagen bound to bacteria in the absence of any potential inhibitor.



Incubation of streptococci with increasing concentrations of I-labeled collagen showed that the cells could be saturated with labeled ligand (Fig. 2). The amount of ligand bound to the bacteria increased to a maximum of 570 ng of collagen per 1 times 10^8 cells of S. pyogenes 6414. If we assume collagen has a molecular weight of 2.85 times 10^5 and that collagen is bound only to specific receptors, we can calculate an average number of 11,600 binding sites per cell. Since Scatchard plot analysis requires the binding reaction to be reversible, the necessary requirements to calculate the K(d) value are not fulfilled. However, an apparent dissociation constant of 3.5 times 10M can be estimated from the concentration required for half-maximal binding of the ligand.


Figure 2: Saturability of binding of I-labeled collagen to S. pyogenes 6414. Heat-killed bacteria (1 times 10^8) were incubated with increasing amounts of I-labeled collagen (specific activity, 14,000 cpm/µg) for 1 h. Background values were determined for each concentration of added collagen and subtracted from the incubation mixtures containing bacteria.



In a similar study using cells of S. zooepidemicus, strain S(Z) III, binding of radiolabeled collagen was highly specific, saturable, and with a calculated apparent K(d) value of 5 times 10M, which is of the same order of that found in S. pyogenes 6414.

Solubilization and Purification of Collagen-binding Proteins from S. pyogenes 6414

Mild digestion of streptococcal cells with trypsin markedly reduced binding of I-labeled collagen, suggesting that the bacterial collagen receptor contained a protein component. To assess whether the interaction of collagen with bacteria is mediated by specific protein components, a crude lysate obtained by sonication of bacteria was fractionated by SDS-PAGE and subjected to Western blot analysis with I-collagen. Western ligand blotting showed that the major collagen-binding proteins in the lysate have molecular masses of 57 and 130-150 kDa (see Fig. 4D, lane 1).


Figure 4: Analysis of proteins obtained at different steps of collagen receptor purification. A, fractions obtained from affinity and gel filtration chromatography were subjected to electrophoresis on a 10% SDS-PAGE gel in non-reducing conditions and stained with Coomassie Brilliant Blue. Lane 1, unfractionated lysate of S. pyogenes 6414; lane 2, unbound material (Ia); lane 3, material washed out by 0.5 M NaCl (IIa); lane 4, material eluting with 2 M guanidinium chloride (IIIa); lane 5, pool Ib, and lane 6, pool IIb, from Sephacryl S-200 HR. Arrows and numbers on the left indicate the migration distances and molecular masses of standard proteins. B and C, immunoblot detection of collagen-binding proteins with anti-57-kDa protein antibodies (B) and with a nonimmune IgG (C). Bound IgG was detected with peroxidase-conjugated goat anti-mouse-immunoglobulin. D, materials from the collagen receptor purification steps after separation in the polyacrylamide gel were electroblotted onto nitrocellulose membranes and probed with I-labeled collagen. Lanes in B, C, and D are numbered as reported in A. Lane 7 in B is a lysate from the collagen receptor-negative strain Sp 1-4065.



To isolate these proteins the whole lysate obtained by sonication of bacterial cells was loaded onto a collagen-Sepharose affinity matrix equilibrated with PBS. The column was washed with 10 mM phosphate buffer containing 0.5 M NaCl and proteins adsorbed to the affinity matrix were eluted with 2 M guanidinium chloride in PBS, dialyzed against water, and lyophilized (Fig. 3A).


Figure 3: Purification of collagen- binding protein from S. pyogenes 6414. A, affinity chromatography on collagen-Sepharose. A lysate of S. pyogenes 6414 was passed over a type II collagen-Sepharose 4B column (2.8 times 8 cm) equilibrated in 20 mM phosphate buffer, pH 7.4. After washing the column with 0.5 M NaCl in phosphate buffer, bound proteins were eluted with 2 M guanidinium chloride. Fractions were pooled as indicated by the bars and dialyzed against water. B, gel filtration chromatography on Sephacryl S-200 HR. Freeze-dried material (pool IIIa) from the affinity chromatography step was dissolved in a small volume of phosphate buffer containing 2 M guanidinium chloride and 0.1% n-octyl-beta-D-glucopyranoside and eluted at a rate of 22 ml/h through a column of Sephacryl S-200 HR (0.8 times 117 cm) equilibrated in the same buffer. Fractions were pooled as indicated by the bars.



Analysis by polyacrylamide gel electrophoresis showed that the material bound to collagen-Sepharose and subsequently eluted with guanidinium chloride contained a predominant component of molecular mass 57-kDa, a minor protein of 130-kDa and a mixture of low molecular mass peptides (Fig. 4A, lane 4). Further purification of the major protein was achieved by gel filtration chromatography on a column of Sephacryl S-200 HR, equilibrated with 2 M guanidinium chloride supplemented with 0.1% n-octyl-beta-D-glucopyranoside (Fig. 3B). This chromatography step resulted in the separation of the major 57-kDa component from the low molecular mass proteins (Fig. 4A, lanes 5 and 6).

When a streptococcal lysate was passed over a gelatin-Sepharose column and the column subsequently eluted with guanidinium chloride none of the above polypeptides were seen in the eluate. Moreover, when the flow-through from the gelatin column was then applied to a collagen-Sepharose column, a 57-kDa protein bound to the column and was eluted by guanidinium chloride demonstrating a preference of the 57-kDa protein for native type II collagen-Sepharose compared to the gelatin matrix.

Preliminary Characterization of the 57-kDa Protein

An antiserum generated by immunizing mice with purified 57-kDa protein was found to react with a single band both in the whole lysate and in the fractions purified by affinity chromatography and gel filtration chromatography steps, with mobility identical to the antigen (Fig. 4B). An additional faint band corresponding to the 130-kDa polypeptide was detected in the material retained by the collagen-Sepharose matrix, suggesting that this component is immunologically related to the 57-kDa protein. The 57-kDa protein was not present in lysate generated from the collagen receptor-negative strain Sp 1-4065 of S. pyogenes when probed in Western blot analysis with immune IgG (Fig. 4B, lane 7). Furthermore, nonimmune IgG did not give a detectable signal with the material blotted as reported in Panel B (Fig. 4C). When the immune IgG were included in a cell/collagen binding assay they did not inhibit the binding of I-ligand to intact bacteria.

The presence of collagen-binding components of similar molecular weight was demonstrated in experiments in which samples from different steps of purification were separated by electrophoresis, electroblotted onto a nitrocellulose membrane and then probed with I-labeled collagen (Fig. 4D). In the lysate, as well as in the materials adsorbed on collagen-Sepharose and further purified by gel filtration chromatography most of the radiolabeled collagen that bound nitrocellulose was associated with a 57-kDa component. Other components in the molecular range of 130-150 kDa were labeled with the ligand. The M(r) 130,000 protein may correspond to the band detected after Coomassie Blue staining of the gel as shown in Fig. 4A, lane 4.

To test if the 57-kDa purified protein represents a soluble form of the collagen receptor, the protein was analyzed for its ability to competitively inhibit ligand binding to intact bacteria. Binding assays in which increasing amounts (2.5-35 µg) of isolated 57-kDa protein were added to incubation mixtures containing S. pyogenes 6414 cells and I-labeled collagen, were performed. Binding was inhibited by the purified 57-kDa protein in a dose-dependent manner suggesting that this component competed with the cell bound collagen receptor for available sites in the ligand molecule. These data indicate that the 57-kDa protein is a soluble form of a collagen receptor. Amino acid composition analysis (Table 1) of the purified protein revealed a high molar percentage of leucine (14.5%) and Glu/Gln (combined: 11.8%). The NH(2)-terminal amino acid sequence of the protein was determined (Table 2) and found to represent a unique sequence when compared to amino acid data sequences stored in the Swiss-Prot data bank. Electrophoretic analysis (Fig. 4A) and staining with antibodies (Fig. 4B) indicated that the isolated collagen-binding protein may be composed of two closely spaced bands with molecular masses of 57- and 53-kDa. To investigate the relationship of these two components, the bands were excised from the nitrocellulose membrane and separately sequenced.The identity of the NH(2)-terminal sequences suggests that the smaller component was derived from the 57-kDa protein due to endogenous proteolysis during extraction and/or purification.





Cellular Location of the 57-kDa Protein

To determine whether the isolated 57-kDa protein is located on the surface of S. pyogenes an experiment was performed in which bacteria were I-labeled externally, sonicated, and the corresponding lysate loaded on a column of collagen-Sepharose.The column was washed with phosphate buffer, followed by 0.5 M NaCl, and the material bound to the affinity matrix was eluted with 2% SDS. An autoradiogram of electrophoresed material bound and eluted from the column showed the presence of a 57-kDa protein as a major component along with several labeled smaller proteins (Fig. 5A). More conclusive evidence that the 57-kDa protein is present at the surface of bacteria was obtained by probing material released by trypsin from cells of S. pyogenes 6414 with an anti-receptor antibody. A mixture of peptides ranging from 43 to 35 kDa was detected, whereas the trypsinate from S. pyogenes Sp 1-4065 did not reveal any detectable amount of anti-receptor antibody reactive protein. Furthermore, a preimmune antibody did not react with trypsin released proteins from S. pyogenes 6414 (Fig. 5B).


Figure 5: Cellular location of 57-kDa protein. A, a lysate obtained from I surface-labeled cells of S. pyogenes 6414 was loaded on a column of collagen-Sepharose. The column was washed with NaCl/P(i) followed by 0.5 M NaCl. Material bound to the affinity matrix was eluted with 2% SDS, analyzed by SDS-PAGE and visualized by autoradiography. B, trypsinates of strain 6414 (lane 1) and strain Sp 1-4065 (lane 2) after electrophoretic separation in non-reducing conditions were transferred to nitrocellulose membranes and probed with an anti-57-kDa protein antiserum. Immunostaining with the second antibody was performed as reported under ``Materials and Methods.'' In lane 3 the reaction of trypsinate from strain 6414 with nonimmune mouse IgG is reported as a control.



Complex Formation of Collagen with Solid-phase Adsorbed 57-kDa Protein

Further evidence of the collagen binding activity of the 57-kDa protein was obtained in assays where the purified protein coated on microtiter wells was shown to bind I-labeled collagen. Different unlabeled proteins were then tried as potential inhibitors. As expected, binding of I-labeled collagen to the immobilized 57-kDa protein was strongly inhibited by unlabeled collagen type II, whereas other proteins tested, e.g. fibronectin, fetuin, fibrinogen, bovine IgG, ovalbumin, and albumin did not affect binding (Fig. 6) demonstrating the specificity of the collagen-receptor interaction. A major portion (up to 70%) of I-labeled collagen bound to immobilized receptor could be displaced by subsequent addition of a large excess of unlabeled ligand, demonstrating a reversibility of ligand-receptor interaction in this system. Under the equilibrium conditions of the microtiter plate assay Scatchard analysis of collagen binding to solid-phase absorbed 57-kDa protein fitted a straight line suggesting the presence of one class of binding sites for collagen ligand(29) . The calculated value of the dissociation constant of the complex was 3.1 times 10M (Fig. 7).


Figure 6: Competition of I-collagen binding to solid-phase adsorbed collagen receptor by non-collagen proteins. Microtiter wells coated with 2 µg of 57-kDa protein were incubated with 10^5 cpm of I-labeled collagen in the presence of 2 µg of the proteins indicated. After 2 h, wells were washed and the ligand binding was quantitated as described under ``Materials and Methods.'' Data are expressed as the percentage of collagen binding relative to controls incubated in the absence of potential inhibitors. Vertical bars show the S.D. of triplicate samples.




Figure 7: Scatchard plot analysis of collagen binding to solid-phase adsorbed collagen receptor. Microtiter wells, coated with 2 µg of 57-kDa protein, were incubated with increasing concentrations of I-labeled collagen as indicated. The specific binding of ligand was plotted according to the method of Scatchard (correlation coefficient = 0.97). The binding data for kinetic analysis were calculated by a nonweighted linear regression computer program. The inset shows the saturation binding isotherm of collagen to the receptor protein. B/F, bound over free collagen. Each data point is the average of three samples.



Comparison of Collagen-binding Proteins from S. pyogenes and S. zooepidemicus

We also isolated a collagen-binding protein from S. zooepidemicus S(Z) III, a member of group C streptococci, associated with infections in animals. The protein migrated on SDS-PAGE with an apparent M(r) of 57,000, similar in size to the collagen-binding protein purified from S. pyogenes (data not shown). The two proteins were found to be homologous by at least two criteria. First, amino acid analysis indicates that both the proteins have a similar amino acid composition (Table 1) and are acidic as indicated by isoelectric points (pI 5.0). Second, the proteins were shown to be immunologically related since antisera raised against the collagen-binding protein from S. pyogenes cross-reacted with the collagen-binding protein from S. zooepidemicus (Fig. 8), and antisera raised against the collagen-binding protein from S. zooepidemicus recognized the collagen receptor from S. pyogenes (data not shown).


Figure 8: Immunoblot of collagen-binding proteins from S. pyogenes and S. zooepidemicus. Collagen-binding proteins isolated by affinity chromatography of lysates of S. pyogenes 6414 (lane 1) and S. zooepidemicus S(z) III (lane 2) were subjected to SDS-PAGE under non-reducing conditions, transferred to nitrocellulose, and incubated with anti-57-kDa protein antibody. Bound antibody was detected as reported.



Collagens as Substrates of Bacterial Adhesion

The ability of S. pyogenes to bind collagen type II with high affinity prompted us to analyze the role of this interaction in bacteria attachment to collagen-containing substrates. Radiolabeled bacterial cells were incubated in microtiter wells coated with type II collagen or bovine serum albumin. Adhesion of streptococci to type II collagen containing substrata was time-dependent and reached a maximum after 2 h of incubation, whereas negligible amount of bacteria had attached to bovine serum albumin after 3 h of incubation (Fig. 9). Moreover, I-labeled streptococci adhered efficiently to surfaces coated with all collagens tested (types I, II, III, and IV) but not to ovalbumin (Fig. 10). This broad specificity indicates that the streptococcal receptor recognizes a common structure in the different collagen molecules. On the other hand, streptococci showed a marginal attachment to either denatured collagen or isolated alpha-chains, suggesting the importance of the triple-helical structure in the collagen substrates for adherence of streptococcal cells.


Figure 9: Kinetics of attachment of S. pyogenes strain 6414 to collagen-coated microtiter wells. Collagen-coated surfaces (5 µg/well) were overlaid with 3.3 times 10^5 cells of a suspension of I-labeled bacteria and incubated for the indicated periods of time. After extensive washing with phosphate-buffered saline containing 0.1% Tween 80, adherence of bacteria was determined by counting the wells in a -counter. Symbol circle shows bacterial adherence to 5 µg/well of bovine albumin.




Figure 10: Attachment of S. pyogenes 6414 to wells coated with different collagen types. Bacteria (6.6 times 10^5 cells)were incubated for 2 h at 37 °C in wells coated with 5 µg of native collagen, denatured collagen type II, gelatin, and isolated alpha chains of collagen type I. After extensive washing the number of attached cells was quantitated. The values are averages of incubations performed in triplicate.



The proteins tested as competitors of radiolabeled collagen binding to streptococci were also used as potential inhibitors of streptococcal adherence to collagen coated substrate. Among the proteins analyzed only collagen type II and the solubilized 57-kDa protein showed inhibitory activity (Fig. 11). Therefore, these data suggest that collagen may serve as a substrate of streptococcal adherence and the 57-kDa protein can act as a collagen adhesin.


Figure 11: Influence of various proteins on the adherence of S. pyogenes strain 6414 to collagen coated wells. Bacteria (6.6 times 10^5 cells) were surface labeled with I and incubated for 2 h at 37 °C with collagen-coated microtiter wells in the presence of 2 µg of the proteins indicated at the left. Data are averages of incubations in triplicate. The amount of attached cells is given as a percent of the number of cells that attached to wells in the absence of proteins.




DISCUSSION

We have demonstrated that the binding of collagen type II to cells of S. pyogenes 6414 exhibits the properties of a typical receptor-ligand interaction. Binding of collagen was specific and saturable, and cells bound exogenous collagen with high affinity. Incubation of bacteria with proteolytic enzymes resulted in a rapid loss of collagen binding suggesting the protein nature and surface location of the receptor.

On the basis of these preliminary results we then isolated a 57-kDa collagen-binding protein from a streptococcal lysate by affinity chromatography on collagen-Sepharose followed by gel filtration chromatography. The collagen-binding protein behaved as one would expect of a collagen receptor. Binding of collagen type II to S. pyogenes was inhibited in a concentration-dependent manner by the purified 57-kDa protein. Furthermore, the isolated receptor protein bound collagen with the same high affinity as intact cells, and this binding was inhibited by unlabeled collagen but not by unrelated proteins. Conflicting results were found concerning reversibility of collagen binding to intact cells and isolated receptor molecule adsorbed onto microtiter wells. This could be explained if we assume that the conformation of the solubilized receptor differs from that of receptor on the surface of bacteria.

The 57-kDa protein could be labeled by external I-iodination of bacteria, and a streptococcal trypsinate was positively detected by an anti-57-kDa protein antiserum suggesting a surface location of the isolated collagen receptor. Taken together these results strongly suggest that the isolated 57-kDa protein is a cell surface receptor and responsible for the binding of collagen to streptococcal cells.

At the present time we do not know what relationship, if any, the 57-kDa protein may have with the other collagen-binding components (molecular mass of 130-150 kDa) identified in the lysate of S. pyogenes. Further studies are needed to establish the relationship between the two proteins.

Recent studies have shown the presence of collagen-binding receptors on other species of bacteria. The Dr fimbriae found on uropathogenic Escherichia coli strains with the serotype 075 have been found to bind type IV collagen(30) , while the enteropathogenic Yersinia enterocolitica and Yersinia pseudotubercolosis adhere to various collagen molecules through the YadA protein(31) .

A collagen-binding component with a relative molecular mass of 135,000 Da has been isolated from S. aureus(16) . The streptococcal collagen-binding protein we have isolated has structural and functional properties distinct from the staphylococcal receptor. The difference in size of the two receptors suggests structural uniqueness. Furthermore, an antibody raised against the staphylococcal collagen receptor and shown to effectively inhibit the binding of I-labeled collagen to S. aureus cells did not interfere with the binding of collagen to streptococcal cells. The staphylococcal collagen receptor has been suggested as a cartilage colonization factor, because it is needed not only for the adhesion of the bacteria to collagen-containing substrates, but also to cartilage, a tissue rich in collagen(18) . The preliminary results reported in this study indicate that the 57-kDa collagen-binding protein may behave as an adhesin. In fact, incubation of bacteria with the collagen-coated microtiter wells in the presence of the soluble receptor protein resulted in a specific, significant inhibition of bacterial attachment.

It still remains to be seen whether this in vitro interaction promoted by the 57-kDa protein facilitates the establishment of streptococcal infections and thus contributes to the pathogenicity of this microbe.


FOOTNOTES

*
This investigation was supported by the Ministry of University and Scientific Research and Technology, by Consiglio Nazionale delle Ricerche (to P. S.), and by Progetto Finalizzato FATMA (to A. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 0039-382-507241; Fax: 0039-382-507240.

(^1)
L. Visai, S. Bozzini, G. Raucci, A. Toniolo, and P. Speziale, unpublished results.

(^2)
The abbreviations used are: PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We are indebted to Dr. Magnus Hook who provided encouragement and critical reading of the manuscript and Dr. Fabrizio Ceciliani for amino acid analysis. We also thank Anna Vai for typing the manuscript, Dr. Giampaolo Minetti for helping with elaboration of graph and numerical data, and Dr. Katharine Dyne for revising the text.


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