©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Critical Residues in the Ligand-binding Site of the Staphylococcus aureus Collagen-binding Adhesin (MSCRAMM) (*)

Joseph M. Patti (§) , Karen House-Pompeo , Jeffrey O. Boles , Norma Garza , S. Gurusiddappa , Magnus Höök

From the (1) Albert B. Alkek Institute of Biosciences and Technology, Center for Extracellular Matrix Biology, Texas A& University, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have identified a discrete collagen-binding site within the Staphylococcus aureus collagen adhesin that is located in a region between amino acids Asp and Tyr. Polyclonal antibodies raised against a recombinant form of the collagen adhesin inhibited the binding of collagen type II to S. aureus. When overlapping synthetic peptides mimicking segments of the adhesin fragment were tested for their ability to neutralize the inhibitory activity of the antibody only one peptide, CBD4 was found to be active. CBD4 bound directly to collagen and at high concentrations inhibited the binding of collagen to S. aureus. A synthetic peptide derivative of CBD4 lacking 2 carboxyl-terminal residues (Asn, Tyr) had no inhibitory activity. The importance of these residues for collagen binding was confirmed by biospecific interaction analysis. Mutant adhesin proteins NA and YA exhibited dramatic changes in collagen binding activity. The dominant dissociation rate for the binding of mutant adhesin protein NA to immobilized collagen II decreased almost 10-fold, while the YA and the double mutant exhibited even more significant decreases in affinity and apparent binding ratio when compared to the wild type protein.


INTRODUCTION

Staphylococcus aureus cells can colonize many different host tissues and cause various types of infections such as endocarditis, pneumonia, wound infections, osteomyelitis, and septic arthritis. Adherence of staphylococci to host tissues involves a family of adhesins that recognize extracellular matrix components and which we have named MSCRAMMS (Microbial Surface Components Recognizing Adhesive Matrix Molecules) (1) . The expression of specific MSCRAMMs appears to be needed for the colonization of different types of tissues. For example, staphylococcal strains recovered from the joints of patients diagnosed with septic arthritis or osteomyelitis almost invariably express a collagen-binding MSCRAMM, whereas significantly fewer isolates obtained from wound infections express this adhesin (2) . Similarly, S. aureus strains isolated from the bones of patients with osteomyelitis often have an MSCRAMM recognizing the bone-specific protein, bone sialoprotein (BSP) (3) .

We have previously reported the cloning, sequencing, and expression of a gene cna, encoding a S. aureus collagen-binding MSCRAMM (4) . The cna gene encodes an 133-kDa adhesin that contains structural features characteristic of surface proteins isolated from Gram-positive bacteria. We have demonstrated that the collagen-binding MSCRAMM is required and sufficient for the adherence of S. aureus to collagen-coated artificial substrates as well as to cartilage, a tissue rich in type II collagen (2) . All strains expressing the collagen-binding MSCRAMM were able to adhere to cartilage, whereas those strains lacking the MSCRAMMs did not adhere. Preincubation of S. aureus with polyclonal antibodies raised against the purified adhesin or saturation of the cartilage substrata with soluble recombinant collagen-binding MSCRAMM resulted in a complete inhibition of bacterial attachment (2) .

S. aureus colonization of the articular cartilage within the joint space appears to be an important factor contributing to the development of septic arthritis. The importance of the collagen-binding MSCRAMM in the pathogenesis of septic arthritis was examined by comparing the virulence of two sets of S. aureus isogenic mutants in an animal model (5) . Greater than 70% of mice injected with CNA strains (i.e. a clinical isolate expressing the collagen-binding MSCRAMM or a negative strain into which the cna gene had been introduced) developed clinical signs of arthritis, whereas less than 27% of the animals showed symptoms of disease when injected with CNA strains (i.e. a strain lacking the cna gene or a strain in which the cna gene had been inactivated through homologous recombination). Taken together these results demonstrate that the collagen-binding MSCRAMM plays an important role in the pathogenesis of septic arthritis induced by S. aureus.

Recently, we have localized the ligand-binding site within the N-terminal half of the collagen-binding MSCRAMM (6) . By analyzing the collagen binding activity of recombinant proteins corresponding to different segments of the MSCRAMM, we identified a 168-amino-acid long protein fragment (corresponding to amino acid residues 151-318) that had appreciable collagen binding activity. Short truncations of this protein in the N- or C terminus resulted in a loss of ligand binding activity but also resulted in conformational changes in the protein as indicated by circular dichroism spectroscopy. These results raised the possibility that the ligand-binding site of the MSCRAMM is contained within a short segment of amino acids and that flanking sequences are required for the proper folding of these residues in the ligand-binding site.

In the current study we have sought to further define the ligand-binding site in the S. aureus collagen-binding MSCRAMM and have identified a 25-amino-acid peptide that directly inhibits the binding of S. aureus to I-labeled collagen type II. Furthermore, site-directed mutagenesis of the collagen-binding MSCRAMM has revealed 2 specific residues critical for ligand binding activity.


EXPERIMENTAL PROCEDURES

Bacteria and Growth Conditions

Escherichia coli strain JM101 was used as the bacterial host for plasmid cloning and protein expression. Luria Broth (Life Technologies, Inc.) was used as the culture medium for E. coli JM101. S. aureus strain Phillips, originally isolated from a patient diagnosed with osteomyelitis, was used in the collagen binding studies. S. aureus was cultured in brain heart infusion broth (Difco, Detroit, MI) at 37 °C with constant rotation, harvested by centrifugation, and resuspended in phosphate-buffered saline (PBS,() 10 mM phosphate, 150 mM NaCl, 0.02% azide, pH 7.4). Bacteria were resuspended to a final density of 1 10 cells/ml as determined by comparing the OD of the sample with a standard curve relating OD to cell number.

Collagen

Bovine collagen type II was a gift from Dr. Richard Mayne, University of Alabama at Birmingham. NaI, specific activity 15 mCi/µg, was obtained from Amersham Corp. Collagen was labeled with I by the chloramine-T method (7) as described previously (8) . Estimated specific activity of type II collagen ranged from 2 10 to 5 10 counts/min/µg protein.

Peptides

Synthetic peptides () were synthesized by a solid-phase method on a p-benzyloxybenzyl alcohol resin using Fmoc chemistry and a model 350 Multiple Peptide Synthesizer from Advanced ChemTech Inc. (Lousiville, KY). Fmoc-amino acids were purchased from Advanced ChemTech Inc. All coupling reactions were mediated through diisopropylcarbodiimide in presence of 1-hydroxy-benzotriazole as the coupling reagent. Deprotection of Fmoc group was accomplished with a solution of 40% piperidine in dimethylformamide. Side chains were protected with the following groups: t-butyl (Asp/Glu and Ser/Thr/Tyr), t-butyloxycarbony (Lys), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Arg) and trityl (Asn/Gln/His).

After the completion of synthesis, peptide resins containing the coupled peptides were washed thoroughly with dimethylformamide, ethanol, and ether, then dried in a vacuum desiccator. Trifluoroacetic acid containing anisole, thioanisole, and water (2.5% each) was used to cleave the peptides from the resin as well as to assist in the removal of side chain protecting groups. The resins were filtered and the peptides precipitated with cold anhydrous ether. The precipitate was washed an additional three times with anhydrous ether and dried. Peptides were analyzed by reverse-phase HPLC on a Waters 625 Liquid Chromatography system using a C analytical column. The purity of the peptides as assessed by HPLC was greater than 90%. Peptides (5 mg/ml) were solubilized in dimethyl sulfoxide and diluted into the appropriate assay buffer prior to their use in binding and inhibition assays.

Antibodies

Polyclonal antibodies against the purified recombinant collagen-binding MSCRAMM segment CBD(151-297) were produced in rabbits (HTI Bio-Products, Ramona, CA). The IgG fraction of serum (CBD(151-297) IgG) was purified by affinity chromatography on a column of immobilized Protein A/G (Pierce) according to the manufacturer's recommendations.

Construction of Expression Plasmids

A series of expression plasmids were constructed using the vector pQE-30 (Qiagen Inc., Chatsworth, CA). Recombinant proteins expressed from this vector contain a N-terminal tail of 6 histidine residues. The derivation of expression constructs pQE-504 (CBD(151-318)) and pQE-441 (CBD(151-297)) have been previously described (6) . To construct pQE-1500 (CBD(30-529)), a cna fragment was amplified from S. aureus FDA 574 genomic DNA (4, 6) by polymerase chain reaction (PCR) together with flanking oligonucleotides, primer A and primer D (). PCR and the subsequent cloning of the amplified gene product into the expression plasmid were performed as described earlier (4, 6) .

Site-directed Mutagenesis

Plasmid DNA purified from E. coli containing pQE-1500 served as the template in the mutagenesis studies. Residues Asn and Tyr in CBD(30-529) were changed to alanine residues individually or as a pair by overlap extension PCR (9) using the oligonucleotides listed in . In two separate reactions, flanking PCR primer A (BamHI restriction site incorporated into the oligonucleotide) and primer D (SalI restriction site incorporated into the oligonucleotide) were used in conjunction with appropriate internal primers B and C, respectively, to generate PCR products AB and CD. The PCR products were purified by agarose gel electrophoresis and the two overlapping PCR fragments AB and CD were combined with flanking primers A and D and used in a second PCR reaction. The amplified PCR product was agarose gel purified (EluQuik; Schleicher & Schuell), digested with BamHI and SalI, ligated into expression vector pQE-30, and transformed into E. coli JM101 cells. Transformants were screened for the proper plasmid construction by restriction digest analysis. The DNA sequence in the region of the mutation was determined by the dideoxy-chain termination method (10) using [-S]dATP (Amersham) and Sequenase 4.0 (United States Biochemical Corp., Cleveland, OH). Mutants possessing the correct nucleotide changes were selected for further investigation.

Expression and Purification of Recombinant Proteins

Recombinant collagen adhesin proteins, CBD(151-297), CBD(151-318), CBD(30-529), and the site-directed mutants N232Y, Y233A, and N232A:Y233A constructed from CBD(30-529) were expressed in E. coli and purified to homogeneity using immobilized metal chelate affinity chromatography (6) . Only freshly purified proteins were used for the analysis when it was observed that the proteins exhibited altered binding characteristics upon prolonged storage.

I-Collagen Binding Assay

The binding of I-labeled collagen type II to S. aureus strain Phillips in the presence of different potential inhibitors was quantified essentially as described previously (8) . Briefly, I-collagen was added to a tube containing 10 cells of S. aureus in a final volume of 0.5 ml of PBS containing 0.1% BSA and 0.1% Tween 80, pH 7.4, and incubated at 25 °C, end-over-end for 1 h. After centrifugation at 1350 g for 15 min, the supernatants were removed by centrifugation and radioactivity associated with the bacterial pellets was quantitated in a gamma counter. Duplicate samples were used for each data point.

Introduction of potential inhibitors involved different preincubation steps including: (A) incubation of bacterial cells for 30 min at 25 °C with IgG purified from immune and preimmune sera from rabbits immunized with CBD(151-297) to assess the inhibitory activity of the antibody. (B) A two-step preincubation procedure to determine the ability of synthetic peptides to neutralize the inhibitory activity of the antibody. First, 100 µg of each peptide was incubated with CBD(151-297) IgG (12 µg) for 30 min, and then 10S. aureus cells were added. The incubation was continued for an additional 30 min at which time the I-labeled collagen was added. (C) To test the ability of synthetic peptides to bind to collagen and inhibit bacterial binding, the peptides were preincubated with I-labeled collagen for 30 min at 25 °C before addition of 10S. aureus cells. Because these experiments were conducted over several months the amount of I-labeled collagen bound by S. aureus and the extent of inhibition by CBD(151-297) IgG varied somewhat from experiment to experiment. However, within a specific experiment extremely small variations were observed.

ELISA

The reactivity of CBD(151-297) IgG, purified from preimmune and immune rabbit sera, was tested against the synthetic CBD peptides () by ELISA. Immulon-4 96-well plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with 50 µl of each peptide (100 µg/ml in PBS) overnight at 4 °C. The plates were washed three times with PBS, containing 0.05% Tween 20 (PBST). A 0.1% BSA solution was added to the wells to block any remaining protein binding sites. After 1 h at room temperature, the wells were washed again three times with PBST, and 50 µl of CBD(151-297) IgG (80 µg/ml) was added to the wells and allowed to bind for 90 min at room temperature. The wells were again washed with PBST and alkaline phosphatase-conjugated goat anti-rabbit IgG (50 µl) (Bio-Rad) diluted 1:3000 in PBST was incubated with the peptide-coated wells for 90 min at room temperature. The samples were washed, and 100 µl of the alkaline phosphate substrate p-nitrophenylphosphate (Sigma) was added to the wells for 50 min at 37 °C. The A was monitored in a microplate reader (Molecular Devices Corp., Menlo Park, CA). A of the BSA-coated wells was used to assess nonspecific binding and was subtracted from A of the peptide samples. Each peptide was tested in triplicate.

Circular Dichroism

Average secondary and tertiary structures of the recombinant collagen-binding MSCRAMM proteins were monitored by far-UV and near-UV circular dichroism (CD) on a Jasco J720 spectropolarimeter. The spectropolarimeter was calibrated with a 0.1% (w/v) 10-camphorsulfonic acid-d solution. Four spectra, representing CD data from 190 to 250 nm (far-UV CD) were recorded at 25 °C in a 0.2-mm path length quartz cell and averaged. Likewise, four spectra representing CD data from 250 to 320 nm (near-UV CD) were collected at 25 °C in a 1.0-cm path length quartz cell and averaged. Protein concentrations of 12 µM in 10 mM Tris, pH 7.5, were used for near- and far-UV CD data acquisitions. Molar ellipticity was expressed as degcm/mol.

Biospecific Analyses

Surface plasmon resonance measurements utilizing the BIAcore system (Biosensor AB, La Jolla, CA) were used to determine the equilibrium constants and kinetic rates for the interaction of CBD(30-529) and the mutant derivatives with immobilized bovine type II collagen. Sensor chip CM5 and the Amine Coupling Kit for the immobilization of bovine type II collagen were also obtained from Biosensor AB. Briefly, the detector monitors mass changes on a biospecific surface containing immobilized ligand molecules (L) in real time using the optical phenomenon surface plasmon resonance (11) . The binding of analyte (A) (present in solution) to immobilized ligand is monitored versus time for a series of analyte concentrations. HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.05% P-20 surfactant) was the buffer used for the binding studies. Concentration ranges for monitoring the binding of each analyte depended on the measured affinities and are reported in I. The association phase begins when the analyte is injected over the immobilized ligand; flow rate 10 or 20 µl/min. The dissociation phase begins when the injection of analyte ends and is characterized by a decrease in the response values with time. The dissociation of analyte from ligand was measured using the higher concentrations in the indicated ranges in order to minimize rebinding. Dissociation rates were measured using two different concentrations of analyte and five different flow rates from 15 to 450 µl/min; this was done in order to ensure that the complex, multiphasic rates reported were independent of analyte concentration and flow rate. Regeneration of the collagen-coated matrix was carried out by passing 100 mM HCl over the sensor chip surface for 5.0 min, followed by HBS buffer flow.

Immobilization Procedures

Pepsin-treated bovine type II collagen was covalently coupled to research grade sensor chip CM5 via primary amine groups using the following protocol. Carboxymethylated dextran was activated by derivatization with 50 mMN-hydroxysuccinimide mediated by treatment with 200 mMN-ethyl-N`-[(dimethylamino)propyl]-carbodiimide for 4.0-5.0 min. Following activation, a 25-30 µg/ml solution of collagen type II in 10 mM maleic acid, pH 6, was passed over the surface for 4.0-5.0 min at 5 µl/min and resulted in between 900-1500 response units (RU) of immobilized collagen. Non-covalently associated collagen was washed from the matrix by HBS buffer flow, and unreacted sites on the activated dextran surface were blocked by treatment with 1 M ethanolamine hydrochloride for 5 min.

Data Analysis

A plot of equilibrium response versus analyte concentration ([A]) can be analyzed via traditional Scatchard analysis (v/[A] versus v) (6, 12) where v represents the molar binding ratio (moles A/moles L). The slope of this line gives the association constant (K) with the x intercept being the maximum apparent binding ratio (BR). In our case, these plots were often biphasic (6) . Another method to determine equilibrium constants from these biphasic plots is to non-linearly curve fit the data to the expression

On-line formulae not verified for accuracy

where j represents the jthclass of binding sites and Kjrepresents the dissociation constant of the jthclass. Both Scatchard analysis and the non-linear curve fit gave comparable solutions when used to fit the equilibrium data. Dissociation rate constants were determined from non-linear curve fitting analysis (14, 15) of the acquired data using software specifically designed for BIAcore data and available in the BIAevaluation 2.1 package (Pharmacia Biosensor).

RESULTS

Antibodies to MSCRAMM Fragments Inhibit Collagen Binding to S. aureus

In previous studies, we attempted to identify the ligand-binding site of the S. aureus collagen-binding MSCRAMM by examining the binding activity of recombinant MSCRAMM fragments of progressively decreasing size. Truncations beyond a 168-amino-acid long segment, CBD(151-318), resulted in loss of collagen binding activity but also affected the folding of the resulting proteins as indicated by CD spectroscopy. Thus it is possible that the ligand-binding site is contained within a short segment of CBD(151-318), but due to the improper folding of the protein the collagen-binding site is not in an active form. To explore this possibility, we decided to try an antibody inhibition-neutralization approach. A similar strategy was used successfully to monitor the purification of the native collagen-binding MSCRAMM from S. aureus cells (8) . To generate an inhibiting antibody, we used as an antigen CBD(151-297), a recombinant version of the largest segment that did not bind collagen and exhibited an altered conformation. In this way, we would minimize generating inhibiting antibodies which recognize conformational dependent epitopes. An inhibiting monoclonal antibody generated against a biologically active collagen-binding MSCRAMM recognized a conformational dependent epitope and was of limited use in identifying the binding site.()

Rabbits were immunized with CBD(151-297) as described under ``Experimental Procedures.'' Sera were also collected prior to immunization and tested for reactivity to CBD(151-297). The reactivity of the antisera with different segments of CBD(151-297) was tested in an ELISA using a series of eight 25-amino-acid long synthetic peptides () with partially overlapping sequences as targets. As shown in Fig. 1, purified IgG reacted strongly with peptides 2, 3, 5, 6, and 7 and weakly with peptides 1, 4, and 8. When preimmune IgG was tested with the CBD peptides, little reactivity could be detected. The different peptides have not been compared with respect to their quantitative binding to the microtiter plate, and therefore the data presented in Fig. 1should not be used for a quantitative comparison of their immunological properties. However, the relative immunological reactivity of the different peptides correlated closely with their antigenic index using the algorithm of Jameson and Wolf (16) .


Figure 1: Reactivity of CBD(151-297) IgG with synthetic CBD peptides. The ability of CBD(151-297) IgG to recognize individual CBD peptides was quantitated by ELISA. Microtiter wells were coated with CBD peptides (100 µg/ml) in PBS. After incubation with the peptides, the wells were washed, and the remaining binding sites were blocked with BSA. Purified IgG (80 µg/ml) from immune CBD(151-297) sera (shaded boxes) or preimmune sera (open boxes) was then added to the peptide-coated wells. The amount of IgG bound was detected by the addition of alkaline phosphatase conjugated goat anti-rabbit IgG as described under ``Experimental Procedures.'' The amino acid sequences of the peptides are shown in Table I.



Purified CBD(151-297) IgG inhibited the binding of S. aureus to I-labeled collagen in a dose-dependent manner (Fig. 2). The amount of I-collagen bound by 10 bacterial cells was reduced over 50% by 5 µg and essentially completely inhibited by 10 µg of the purified immune IgG. Conversely, antibodies purified from preimmune sera did not possess significant inhibitory activity (Fig. 2). These results suggest that the CBD(151-297) antibodies recognize epitopes at or close to the active site of the MSCRAMM, thereby directly inhibiting or sterically interfering with collagen binding. A Synthetic Peptide Neutralizes the Inhibitory Activity of CBD(151-297) IgG-The different synthetic CBD peptides described above and shown to react with the CBD(151-297) IgG were assayed for their potential to neutralize the inhibitory activity of the antibody. The CBD(151-297) IgG (12 µg) was preincubated with a single dose (100 µg) of each peptide in step one. The S. aureus cells were then added and the preincubation continued. Finally, the I-labeled collagen type II was added. As shown in Fig. 3, peptide CBD4 neutralized 68% of CBD(151-297) IgG inhibitory activity, while the other peptides tested had little or no effect. These results suggested that only antibodies recognizing epitopes present in CBD4 were able to inhibit collagen binding to bacteria. Although other peptide sequences were more immunogenic than CBD4, the antibodies recognizing the corresponding epitopes were not inhibitory. These data suggest that the ligand-binding site of the MSCRAMM is located close to or within the sequence covered by peptide CBD4.


Figure 2: Purified CBD(151-297) IgG inhibits the binding of S. aureus to collagen type II. S. aureus was preincubated with increasing amounts of CBD(151-297) IgG (), or IgG purified from preimmune sera () prior to the addition of I-labeled collagen type II. Collagen binding to bacteria was quantitated as described under ``Experimental Procedures.'' Results are expressed as mean ± standard deviation. Each sample point was done in duplicate.




Figure 3: Peptide neutralization of CBD(151-297) IgG inhibitory activity. Overlapping 25-amino-acid long CBD peptides (100 µg) derived from CBD(151-297) were assayed for their potential to neutralize the ability of CBD(151-297) IgG (24 µg/ml) to inhibit the binding of S. aureus to I-labeled collagen type II. For details see ``Experimental Procedures.'' The amino acid sequences of the peptides are shown in Table I.



To further investigate the interaction between peptide CBD4 and CBD(151-297) IgG and its affect on collagen binding, a fixed concentration of the antibody was incubated with an increasing amount of peptide CBD4 (Fig. 4). To make the assay more sensitive, we chose an CBD(151-297) IgG concentration (12 µg/ml) which resulted in a 50% reduction in collagen binding to 10S. aureus cells. Thus, a relatively small reduction in inhibition could be easily detected. At low concentrations, the peptide appears to neutralize the inhibitory activity, and in this experiment 100 µg of peptide CBD4 restored the level of collagen binding to S. aureus observed in absence of CBD(151-297) IgG. Somewhat surprisingly, addition of more peptide CBD4 resulted in a dose-dependent decrease in collagen binding to S. aureus (Fig. 4).


Figure 4: Effect of peptide CBD4 on the ability of CBD(151-297) IgG to inhibit the binding of S. aureus to collagen type II. An increasing amount of peptide CBD4 was added to CBD(151-297) IgG (12 µg/ml), and the amount of I-labeled collagen bound by S. aureus was quantitated as described under ``Experimental Procedures.'' The solid vertical bar indicates the amount of I-labeled collagen bound by S. aureus in the absence of any inhibitor. Results are expressed as mean ± standard deviation. Each sample point was done in duplicate.



Peptide CBD4 Directly Inhibits Collagen Binding to S. aureus

To assess the role of amino acids 209-233 in collagen binding, peptide CBD4 was tested for the ability to directly inhibit the binding of I-collagen to S. aureus. Peptide CBD7 which reacted strongly with CBD(151-297) IgG in the ELISA assay (Fig. 3) was also tested. As shown in Fig. 5 , when increasing amounts of peptide CBD4 were incubated with I-collagen prior to the addition of S. aureus, binding to collagen was inhibited in a dose-dependent manner. Five µM CBD4 inhibited binding by over 50%. Peptide CBD7 had no inhibitory effect when it was preincubated with I-collagen. These data suggest that peptide CBD4 can bind soluble I-collagen and that CBD4 contains the residues that represent a collagen-binding site within the MSCRAMM protein.


Figure 5: Dose-dependent inhibition of I-labeled collagen binding to S. aureus by peptide CBD4. Increasing amounts of peptide CBD4 () or peptide CBD7 () were added to I-labeled collagen, and the binding to S. aureus was evaluated as described under ``Experimental Procedures.'' Results are expressed as mean ± standard deviation. Each sample point was done in duplicate.



Identification of Critical Residues within CBD4 Necessary for Collagen Binding Activity

To identify residues within CBD4 necessary for collagen binding we synthesized several smaller overlapping peptides (). A series of peptides that contained truncations of 5, 10, or 15 amino-terminal residues and one peptide that contained a 2 amino acid deletion at the carboxyl terminus were made. The peptides (10 µM) were assayed for their ability to inhibit the binding of I-labeled collagen to S. aureus (Fig. 6). Peptide CBD4 inhibited collagen binding by 76%, whereas all peptides containing amino-terminal truncations had little activity at the concentration tested. These data indicate that when as few as 5 residues are removed from the active site peptide, the ability to bind collagen is lost. Moreover, deletion of only the 2 carboxyl-terminal amino acids causes a complete loss of biological activity. This suggests that amino acids at the C terminus of CBD4, Asn or Tyr or both are integral member(s) of the active site of the collagen MSCRAMM.


Figure 6: Effect of CBD4 synthetic peptide derivatives on the binding of collagen binding to S. aureus. Binding of S. aureus to I-labeled collagen was evaluated in the presence of peptides (10 µM). The total amount of collagen bound was quantitated in the absence of any peptide. Results are expressed as mean ± standard deviation. Each sample point was done in duplicate. Amino acid sequences of the peptides are shown in Table I.



Collagen Binding Activity of Defined MSCRAMM Mutants

The importance of amino acid residues Asn and Tyr in the MSCRAMM for collagen binding was examined by creating specific mutants of CBD(30-529) and characterizing the interactions of these mutants with immobilized type II collagen using the BIAcore. In the mutations made, the identified amino acid residues were replaced individually, (NA, YA) or as a pair, (NA:YA) with alanine, a residue that is not expected to interfere with the existing secondary structure. The corresponding base changes were made using overlap extension PCR as described under ``Experimental Procedures'' and the sequence changes confirmed experimentally. The recombinant proteins containing the mutations were purified to homogeneity by metal ion chelating chromatography. Structural analyses of the isolated proteins by near- and far-UV CD spectroscopy suggested that no significant changes in secondary or tertiary structure had occurred as a result of the mutations (data not shown).

The BIAcore sensorgrams for the binding of the different mutants (20 µM) to immobilized collagen type II are shown in Fig. 7. Under the conditions described in this study CBD(30-529) exhibits complex multiphasic interactions when analyzed using the BIAcore. True equilibrium was not obtained during the injection time period due to the slow dissociation rate (k). The association phase exhibits multiphasic binding with an interaction characterized by a fast k apparent at the start of the injection followed by a second phase characterized by a much slower k. The dissociation phase gives information about the k, and this rate is independent of the number of binding sites, analyte concentration, and flow rate. Analysis of these data indicates at least a three-component dissociation with the two fastest rates greater than 10 s and the slowest k at 5 10 s-1 (I). The association phase contains the information to determine the association rate (k) of the interaction, but it is also influenced by the dissociation rate, the number of binding sites for each interaction, and the concentration of the analyte. Because of the complexity of this interaction and the absence of measurable equilibrium data, it was not possible to determine the binding constant (K), apparent binding ratio (BR), and k.


Figure 7: BIAcore analysis of the interaction between recombinant collagen adhesin constructs and collagen. Sensorgrams indicating relative response versus time for the binding of CBD(30-529) and mutant proteins NA, YA, and NA:YA to immobilized collagen type II. The concentration of each protein assayed was 20 µM.



In a comparison of the three mutant proteins with wild type CBD(30-529), it is readily apparent that each of the introduced mutations affected the collagen binding properties of the generated proteins (Fig. 7, I). While the shape of the binding sensorgram remains essentially the same for mutant NA, analysis of the dissociation phase indicates that the slowest dissociation rate has increased almost 10-fold. Although a dissociation constant (K) cannot be determined, it appears that the affinity of this mutant for collagen type II has also been influenced by this mutation. Both the YA and the double mutant bind immobilized type II collagen weakly, and analysis of the available equilibrium information indicates dissociation constants greater than 10M and fast dissociation rates (>10 s). The YA mutant exhibits biphasic binding to collagen type II, but the highest rate could not be accurately determined because of protein concentration restrictions. Analysis of the data obtained with the double mutant produces a monophasic binding constant when evaluated by either Scatchard analysis or Equation 1. Additionally, the BR has decreased to approximately two to three high affinity sites.

From the Biosensor results it is apparent that residues Asn and Tyr are important for both the affinity and the specificity of CBD(30-529)'s binding to type II collagen. There does not, however, seem to be an additive effect of the double mutation when compared to the single mutations. It is possible that the individual MSCRAMM binding sites on type II collagen are heterogeneous thus giving rise to the multiphasic character of the measured interaction.

DISCUSSION

The adhesion of microorganisms to host tissues is the critical first step in the development of most infections. It has become increasingly clear that eukaryotic adhesive extracellular matrix components, that are normally involved in cell-matrix interactions within the host, also serve as ligands for pathogenic microorganisms. Collagens are major constituents of most extracellular matrices and different specialized tissues are usually characterized by a specific repertoire of different types of collagens (17) . For example, dentin from root surfaces contains predominately collagen type I, cartilage mainly consists of type II collagen, and collagen type IV is exclusively found in basement membranes.

The ability of microorganisms to express an MSCRAMM that can specifically recognize and bind to a collagenous substrata may represent a significant virulence factor for the initiation of many different types of infections. Eighty to 90% of nongonococcal bacterial arthritis is monoarticular, and in most of these cases S. aureus is the predominant pathogen (18) . Localization of S. aureus to the joint and the subsequent colonization of the collagenous tissues appear to be a critical initial step in the development of septic arthritis. Scanning electron micrographs of tissues removed from both animals and humans with septic arthritis demonstrated the presence of S. aureus closely associated with cartilage matrix surfaces, particularly collagen fibers (19). Previous studies have shown that expression of the collagen-binding MSCRAMM is both necessary and sufficient for S. aureus to adhere to cartilage (2) . Moreover, S. aureus strains lacking the collagen-binding MSCRAMM were less virulent than their isogenic parental strains in a mouse model of septic arthritis (5). Bacteria other than S. aureus have also been shown to express collagen-binding MSCRAMMs. Strains of enteropathogenic Yersinia were shown to bind several collagen types with a high affinity (20, 21) . Collagen binding is mediated by YadA, a plasmid-encoded cell surface-associated multifunctional protein that was identified as a virulence factor (21) . Yersinia spp. have also been implicated as initiators of reactive arthritis (22) . Interestingly, immunofluorescence techniques have been used to demonstrate the presence of a number of Yersinia enterocolitica 0:3 antigens in synovial fluid cells (23) and tissues (24) from patients with Yersinia-triggered reactive arthritis. Although the presence of the YadA protein in these samples has not been specifically demonstrated, it is possible that its collagen binding ability may allow localization to the joint thereby contributing to the pathology of reactive arthritis. Many uropathogenic E. coli express O75X, a frimbriae that is highly specific for the 7S fragment of collagen type IV (25) . Purified 075X fimbriae injected into rats preferentially localized from the circulation to the glomerular basement membrane. 075X fimbriae binding to collagen type IV within the glomerular basement membrane is postulated to contribute to the pathogenesis of chronic glomerulonephritis (26) . Other bacteria that are reported to express collagen-binding MSCRAMMs include Klebsiella pneumonia(27) , Streptococcus mutans (28), group A, B, C, and G Streptococci (29) , Lactobacilli (30), and Trypansoma cruzi(31) .

In addition to adhesins from prokaryotic organisms, a variety of proteins expressed by eukaryotes have collagen binding activity. These proteins include cell surface receptors such as the (32) and (33, 34) integrins, anchorin II (35) , and extracellular matrix proteins such as the propolypeptide of von Willebrand factor (36) , fibronectin (37) , decorin (38) , and thrombospondin (39) . Although the number of proteins that have been described to bind collagen is quite large, the localization of specific collagen-binding sites within those polypeptides has been analyzed in only a few cases. The YadA protein from Y. enterocolitica contains a stretch of 22 amino acids located in the hydrophobic amino terminus that is necessary for binding collagen types I, III, IV, and V. A dodecapeptide (36) , derived from the propolypeptide of bovine von Willebrand factor (40) , has been shown to contain collagen binding activity.

We have previously localized the collagen-binding domain from the 133-kDa S. aureus collagen-binding MSCRAMM to a 168-amino-acid long segment (CBD151-318) (6) . Data presented in this article demonstrate that the collagen-binding domain is contained within amino acid residues Asn and Tyr. In addition, site-directed mutagenesis of this region revealed that residues Asn and Tyr are critical for high affinity collagen binding. Moreover, mutated proteins containing alanine substitutions at Asn and Tyr positions exhibited altered collagen-binding kinetics compared to the wild-type protein. Residues Asp-Gly are apparently also important for collagen binding since a truncated version of peptide CBD4 lacking these residues lost its collagen binding activity. Furthermore, an MSCRAMM mutant in which we had deleted residues Gly, Gly did not bind collagen. However, this mutated protein had undergone considerable conformational changes compared to the wild-type protein as indicated by CD analyses (data not shown). Therefore, the importance of these residues in direct collagen binding is unclear. To our knowledge this is the first report where specific amino acids within a collagen-binding protein have been identified that are directly involved for collagen binding activity. Studies are underway to determine the location of the critical residues in the folded collagen-binding MSCRAMM.

  
Table: Synthetic peptides derived from the sequence of the S. aureus collagen-binding MSCRAMM


  
Table: Oligonucleotides used for site-directed mutagenesis

The underlined nucleotides indicate the location of the mutation. Oligonucleotides were synthesized by the Gene Technologies Laboratory, Institute of Developmental and Molecular Biology, Texas A& University, College Station, Texas.


  
Table: 0p4in Concentration of MSCRAMM protein used in the binding studies (µM).(119)


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL 47313, and AI 20624 and by a post-doctoral fellowship from the Arthritis Foundation. 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: Albert B. Alkek Institute of Biosciences and Technology, Texas A& University, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-677-7556; Fax: 713-677-7576; E-mail: JPATTI@ibt.tamu.edu.

The abbreviations used are: PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay.

J. M. Patti and M. Hook, unpublished observation.


ACKNOWLEDGEMENTS

We thank A. Höök for technical assistance.


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