The Fibrinogen-binding MSCRAMM (Clumping Factor) of Staphylococcus aureus Has a Ca2+-dependent Inhibitory Site*

David P. O'ConnellDagger , Tamanna Nanavaty§, Damien McDevitt§, Sivashankarappa Gurusiddappa§, Magnus Höök§, and Timothy J. FosterDagger par

From the Dagger  Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Republic of Ireland and the § Center for Extracellular Matrix Biology and the Department of Biochemistry and Biophysics, Institute of Biosciences and Technology, Texas A&M University, Houston, Texas 77030-3303

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The clumping factor (ClfA) is a cell surface-associated protein of Staphylococcus aureus that promotes binding of fibrinogen or fibrin to the bacterial cell. Previous studies have shown that ClfA and the platelet integrin alpha IIbbeta 3 recognize the same domain at the extreme C terminus of the fibrinogen gamma -chain. alpha IIbbeta 3 interaction with this domain is known to occur in close proximity to a Ca2+-binding EF-hand structure in the alpha -subunit. Analysis of the primary structure of ClfA indicated the presence of a potential Ca2+-binding EF-hand-like motif at residues 310-321 within the fibrinogen-binding domain. Deletion mutagenesis and site-directed mutagenesis of this EF-hand in recombinant truncated ClfA proteins (Clf40, residues 40-559; and Clf41, residues 221-559) resulted in a significant reduction of affinity for native fibrinogen and a fibrinogen gamma -chain peptide. Furthermore, Ca2+ (or Mn2+) could inhibit the binding of the fibrinogen gamma -chain peptide to Clf40-(40-559) and the adhesion of S. aureus cells to immobilized fibrinogen with an IC50 of 2-3 mM. In contrast, Mg2+ (or Na+) at similar concentrations had no effect on the ClfA-fibrinogen interaction. Far-UV CD analysis of Clf40-(40-559) and Clf41-(221-559) in the presence of metal ions indicated Ca2+- and Mn2+-induced differences in secondary structure. These data suggest that Ca2+ binds to an inhibitory site(s) within ClfA and induces a conformational change that is incompatible with binding to the C terminus of the gamma -chain of fibrinogen. Mutagenesis studies indicate that the Ca2+-dependent inhibitory site is located within the EF-hand motif at residues 310-321.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Staphylococcus aureus causes a wide range of opportunistic infections that range from superficial skin infections to life-threatening diseases including endocarditis, pneumonia, and septicemia. Adherence of bacteria to host matrix components that is mediated by bacterial surface adhesins is the initial critical event in the pathogenesis of most infections. The extracellular matrix (ECM)1 contains numerous glycoproteins and proteoglycans assembled into insoluble matrices that serve as substrata for the adhesion and migration of tissue cells. These processes involve integrins, a family of heterodimeric (alpha beta ) cell-surface receptors that recognize specific ECM proteins. It has become increasingly evident that bacteria, including S. aureus, also utilize the ECM as substrata for their adhesion by way of a family of adhesins called MSCRAMM (microbial surface components recognizing adhesive matrix molecules) (1) that specifically recognize host matrix components.

One important component of the ECM, also occurring in soluble form in blood plasma, is fibrinogen, a 340-kDa hexamer composed of 2alpha -, 2beta -, and 2gamma -chains linked by disulfide bonds. This protein is recognized by several integrins including the platelet integrin alpha IIbbeta 3. Activation of platelets and integrin alpha IIbbeta 3 results in fibrinogen-dependent aggregation in vitro and the formation of platelet-fibrin thrombi in vivo.

S. aureus contains several fibrinogen-binding proteins, one of which (clumping factor, ClfA) is primarily responsible for the clumping of bacteria in fibrinogen solutions and bacterial adherence to fibrinogen substrata (2). The gene encoding the fibrinogen-binding protein of S. aureus has been cloned, sequenced, and characterized in our laboratory (2). The clfA gene encodes a 933-amino acid protein that contains structural features characteristic of many cell surface-associated proteins from Gram-positive bacteria including a typical cell wall attachment region comprising an LPDTG motif, a hydrophobic transmembrane sequence, and a positively charged C terminus (Fig. 1). In addition, the protein contains a repeat sequence (region R) of 308 alternating aspartate and serine residues located just outside the cell wall attachment region. Region R is required for the surface display of the 520-amino acid-long region A, which contains the fibrinogen-binding domain (3, 4). Recombinant region A bound fibrinogen and strongly inhibited bacteria-fibrinogen interactions, as did anti-region A antibodies. Analysis of PCR-generated truncated proteins localized the binding domain to between residues 221 and 559 (3).


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Fig. 1.   Schematic model showing the domain organization of S. aureus clumping factor (ClfA), as described previously (3). S, signal peptide; A, fibrinogen-binding region; R, repeat region; W, cell wall-spanning region; M, membrane-spanning domain; +, positively charged tail. The putative EF-hand-like motif (residues 310-321) is indicated in black. The minimum fibrinogen-binding domain is located between residues 221 and 559.

In earlier studies, Hawiger and co-workers (5, 6) showed that a synthetic peptide mimicking the extreme C terminus of the fibrinogen gamma -chain inhibited fibrinogen-induced clumping of S. aureus cells. Recently, it was shown that purified recombinant ClfA protein specifically recognized these amino acid residues and that a synthetic peptide corresponding to this domain effectively inhibited binding of fibrinogen to recombinant ClfA (7). Interestingly, this same synthetic peptide also interacts with the alpha -subunit of the platelet integrin alpha IIbbeta 3 (8, 9). The gamma -chain-binding site has been mapped to a region of the alpha IIb polypeptide that contains a sequence motif resembling the Ca2+-binding EF-hand motif found in many eukaryotic Ca2+-binding proteins (10). The EF-hand motif consists of 13 residues, with coordination typically supplied by oxygenated residues at positions 1, 3, 5, 7, and 12 and by a solvent molecule hydrogen-bonded to residue 9 (Fig. 2A) (11). These residues form a coordination sphere for the cation and are flanked by alpha -helices. Cooperative binding of multiple Ca2+ ions is not unusual, and more than one Ca2+-binding motif often can be found within the same protein. Analysis of the alpha -subunit of alpha IIbbeta 3 revealed the presence of four functional motifs similar to EF-hands, although they lack an oxygenated residue at position 12 (Fig. 2B) (31, 39). Chemical cross-linking experiments provided direct evidence for the role of an alpha IIb EF-hand-like sequence in fibrinogen binding (10) (Fig. 2B). In addition, a peptide corresponding to this EF-hand sequence bound to fibrinogen in a divalent cation-dependent manner (12). Taken together, these data provide evidence for the involvement of EF-hand-like sequences in the divalent cation-dependent binding of the fibrinogen C-terminal gamma -chain peptide. Further biochemical characterization of integrin alpha IIbbeta 3 has demonstrated that Ca2+ binds to two distinct classes of sites: high affinity binding sites that promote ligand binding and low affinity binding sites that inhibit ligand binding (13).


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Fig. 2.   Metal ion-binding motifs in integrins and S. aureus clumping factor. A, the EF-hand consensus showing preferred residues at each position (11, 38). Acceptable residues are in parentheses; unacceptable residues are in braces. X indicates any residue. The boldface letters indicate cation-coordinating residues. B, metal ion-binding sites in the alpha IIb subunit of platelet integrin (adapted from Gulino et al. (39)). The asterisk indicates sequence identified as the fibrinogen C-terminal gamma -chain-binding site in alpha IIb (10). C, divalent cation-binding motif in region A of ClfA. The boldface letters indicate putative cation-coordinating residues.

In this report, we propose a mode of interaction between ClfA and the fibrinogen gamma -chain peptide that exhibits some similarities to alpha IIbbeta 3-ligand interactions. A potential divalent cation-binding EF-hand motif was identified in ClfA (Figs. 1 and 2C). It differs from the EF-hand consensus (Fig. 2, A and B) at only one residue, a non-cation-coordinating residue. We demonstrate that region A of ClfA can bind Ca2+ and that the interaction between region A and fibrinogen is inhibited by millimolar concentrations of Ca2+. In addition, we show using far-UV spectroscopy that Ca2+ ions affect the secondary structure of the fibrinogen-binding region of ClfA. Site-specific mutants of ClfA with a modified EF-hand were generated and shown to have a lower affinity for fibrinogen compared with the wild type. In addition, the effects of Ca2+ were reduced in these mutant proteins. Together, these studies indicate that Ca2+ plays a regulatory role in the interaction of fibrinogen and region A of ClfA.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Bacteria and Growth Conditions

Escherichia coli XL1-Blue (14) was used as the bacterial host for plasmid cloning and protein expression. E. coli cells harboring plasmids were routinely grown in L-broth, Terrific broth, and L-agar (15). Ampicillin (100 µg/ml) was incorporated as appropriate. S. aureus strain Newman was grown in Trypticase soy broth or agar.

Manipulation of DNA

Restriction and DNA modification enzymes were purchased from New England Biolabs Inc. or Promega and were used according to the manufacturers' instructions. DNA manipulations were performed using standard procedures (15).

Amplification of clfA Gene Fragments

PCR, with the oligonucleotides listed in Table I, was used to amplify specific clfA fragments from chromosomal DNA of S. aureus strain Newman. Genomic DNA was isolated as described previously (16). The oligonucleotides contained restriction enzyme cleavage sites at their 5'-ends to facilitate directional cloning. PCR was performed with a Perkin-Elmer DNA thermocycler. Reaction mixtures contained 50 ng of target DNA, 100 pmol of forward and reverse primers, 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.0), 2 mM MgSO4, 250 µM each dNTP, 0.1% Triton X-100, and 2 units of Vent DNA polymerase (New England Biolabs Inc.). The reaction mixtures were overlaid with 100 µl of mineral oil and amplified for 25 cycles consisting of 1 min of denaturation at 94 °C, a 1-min annealing period at a temperature depending on the primers used, and an extension of 1 min, 30 s at 72 °C. On completion of the 25 cycles, the reaction mixture was incubated at 72 °C for 10 min. After amplification, the PCR products were treated with the Wizard PCR-preps DNA purification system (Promega) and analyzed by agarose gel electrophoresis.

                              
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Table I
Synthetic oligonucleotides used for amplifying clfA gene fragments from S. aureus Newman genomic DNA and site-directed mutagenesis and deletion mutagenesis
Restriction endonuclease sites are underlined. The nucleotides in boldface indicate the location of the desired mutation.

Construction of Expression Plasmids

Amplified fragments of the clfA gene were cloned into the expression plasmid pQE30 (QIAGEN Inc.) to generate the constructs pCF40-(40-559) and pCF41-(221-559). Recombinant protein expressed from this vector contains an N-terminal extension of six histidine residues (His tag). These fragments of the clfA gene were also cloned into the expression plasmid pGEX-KG such that an in-frame fusion was formed with glutathione S-transferase (17).

Construction of a Deletion Mutant Plasmid

Residues 310-321 of ClfA corresponding to a putative Ca2+-binding EF-hand were deleted in Clf40-(40-559) and Clf41-(221-559). The deletion mutant plasmids were designated pCF51 and pCF64, respectively. Construction of pCF51 involved two separate PCRs. The first reaction amplified DNA encoding residues 40-309 using a reverse primer (DOCR3) with an XbaI site incorporated at the 5'-end and a forward primer (DOCF1) incorporating a BamHI site. The second reaction amplified DNA encoding residues 332-559 using a forward primer (DOCF2) incorporating an XbaI site and a reverse primer (DOCR1) incorporating a HindIII site. Following cleavage by XbaI and either BamHI or HindIII (depending on the reaction), the two products were ligated together and cloned into the expression vector pQE30 as described above. Creation of the XbaI site introduced a serine and an arginine residue at the site of deletion. pCF64 was constructed in the same way using the appropriate forward primer (F5) in the first reaction.

Site-directed Mutagenesis

Plasmid DNA purified from E. coli XL1- Blue cells containing pCF40-(40-559) and pCF41-(221-559) served as the template in the mutagenesis studies. The residues were mutated to alanine as single, double, or quadruple mutations (Table I). A novel method of mutagenesis was developed that involved two separate PCRs.2 The first reaction employed a flanking reverse primer (incorporating a HindIII site) and a forward primer that introduced the nucleotide mismatch required for the desired mutation and, in addition, incorporated a novel restriction site at the 5'-end of the oligonucleotide that would not affect the amino acid sequence of the final gene product. In the second reaction, a reverse primer incorporating the same silent restriction site mutation was used in conjunction with a flanking forward primer (with a BamHI site). Both gene fragments were digested at the common restriction site, ligated, digested with BamHI and HindIII, cloned into the expression vector pQE30, and transformed into E. coli XL1-Blue cells. Transformants were screened for the proper plasmid construction. The DNA sequence was verified by the dideoxy termination method (15) using alpha -35S-dATP (Amersham Corp.) and Sequenase 4.0 (U. S. Biochemical Corp.). The oligonucleotides used to construct the site-directed mutants are listed in Table I.

Expression and Purification of Recombinant Proteins

Recombinant plasmids were transformed into E. coli XL1-Blue cells and expressed as described previously (3). Fusion proteins containing the His tag were purified by immobilized metal chelate affinity chromatography. A iminodiacetic acid-Sepharose 6B Fast Flow column (10 × 1 cm; Sigma) was charged with 150 mM Ni2+ and equilibrated with binding buffer (5 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9)). The cleared lysed cell supernatant was applied to the column and washed with binding buffer until the absorbance at 280 nm of the eluate was <0.001. Bound protein was eluted with a continuous linear gradient of imidazole (5-100 mM; total volume of 200 ml) in 0.5 M NaCl and 20 mM Tris-HCl (pH 7.9). Protein-containing fractions were dialyzed against 50 mM NaCl, 2 mM EDTA, and 20 mM Tris-HCl (pH 7.4) and applied to a Q-Sepharose column pre-equilibrated with the same buffer. Bound protein was eluted with a continuous linear gradient of NaCl (50-500 mM; total volume of 200 ml) in 20 mM Tris-HCl and 2 mM EDTA (pH 7.9). Eluted fractions were monitored by absorbance at 280 nm, and peak fractions were analyzed by SDS-polyacrylamide gel electrophoresis and Western immunoblotting. The glutathione S-transferase-Clf41-(221-559) fusion protein was purified by glutathione-Sepharose (Pharmacia Biotech Inc.) affinity chromatography and cleaved with bovine thrombin as described previously (3). The recombinant MSCRAMM fragment was isolated after passing the digest through a glutathione-Sepharose column, followed by ion-exchange chromatography on a Q-sepharose column as described above.

Analysis of Fibrinogen-binding Activity of Recombinant Proteins

Western Ligand Blot Assay-- Fibrinogen-binding proteins fractionated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane were detected using fibrinogen conjugated to horseradish peroxidase and enhanced chemiluminescence (Amersham Corp.) as described previously (3).

Bacterial Adherence Assay-- The adherence of S. aureus Newman cells expressing ClfA to microtiter wells coated with fibrinogen was assayed as described previously (6).

Enzyme-linked Immunosorbent Assay-- The ability of recombinant protein to bind to fibrinogen was analyzed using the enzyme-linked immunosorbent assay. Microtiter wells (Sarstedt, Inc.) were coated with 5 µg/ml fibrinogen (Kabi Pharmacia/Chromogenix) in coating solution (0.02% sodium carbonate buffer (pH 9.6)) for 18 h at room temperature. The plates were washed three times with PBS, 0.05% Tween 20, and 0.1% bovine serum albumin (PBS-TB). A solution of 2.5% bovine serum albumin and 0.05% Tween 20 in PBS was added to the wells to block any remaining protein-binding sites. After 1 h at 37 °C, the wells were washed again three times with PBS-TB, and purified recombinant protein in PBS was added and incubated for 2 h at 37 °C. The wells were again washed with PBS-TB and incubated with polyclonal antiserum raised against Clf41-(221-559), diluted 1:800, for 1 h. After further washing, 100 µl of horseradish peroxidase-labeled protein A (1:1000; Sigma) was added. Following incubation for 1 h at 37 °C and washing with PBS-TB, 100 µl of chromogenic substrate (580 µg/ml tetramethylbenzidine and 0.0001% H2O2 in 0.1 M sodium acetate buffer (pH 5.0)) was added per well and developed for 10 min, and the reaction was stopped by the addition of 50 µl of 2 M H2SO4. Plates were read at 450 nm in an enzyme-linked immunosorbent assay plate reader (Labsystems Multiskan Plus).

Fluorescence Polarization-- A fluorescence polarization assay was developed to determine the equilibrium constants for the interaction of the recombinant proteins with a synthetic peptide conjugated with a fluorescent probe. This peptide consisted of the 17 C-terminal residues of the gamma -chain of fibrinogen (i.e. GEGQQHHLGGAKQAGDV) and was synthesized by a solid-phase method on a p-benzyloxybenzyl alcohol resin using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and a Model 396 multiple peptide synthesizer (Advanced ChemTech Inc.). The peptide was labeled with fluorescein as follows. The peptide (1 mg in H20) was incubated with fluorescein succinimidyl ester (1 mg dissolved in 100 µl of dimethyl sulfoxide) at 37 °C for 60 min in the presence of coupling buffer (100 mM KH2PO4 (pH 7.0)). The reaction was quenched by the addition of 100 µl of 1 M Tris-HCl (pH 8.0), vortexed, and left at room temperature for 30 min. The fluoresceinated peptide was fractionated by reverse-phase chromatography on a Delta-Pak C18 Radial-Pak cartridge HPLC column connected to a Waters 486 multi-wavelength detector. The labeling procedure can yield three forms of fluoresceinated peptide with the probe attached to 1) the N-terminal amino group, 2) the amino group of the internal lysine residue, and 3) the amino groups in both positions. Fractionation by HPLC yielded two major and one minor fluorescein-containing peak. Substitutions at the internal lysine residue should yield a peptide resistant to trypsin digestion. The separated peptides were incubated with trypsin, followed by HPLC analysis. Only one of the original major peaks contained peptide susceptible to trypsin (data not shown). This peptide, which contains the fluorescein probe linked to the N-terminal amino group, was used in the fluorescence polarization studies. A scrambled peptide consisting of the 17 C-terminal residues of the gamma -chain in a random sequence (i.e. GHEHGLQGQGAVKDGAQ) was used as a control.

Recombinant protein in 10 mM Tris-HCl (pH 7.4) was incubated with 10 nM peptide in the dark at 20 °C for 3 h. Samples were then analyzed using a Beacon fluorescence polarization system. Binding curves were analyzed by nonlinear regression used to fit a binding function defined as in Equation 1,
&Dgr;P=<FR><NU>&Dgr;P<SUB><UP>max</UP></SUB> · [<UP>protein</UP>]</NU><DE>K<SUB>D</SUB>+[<UP>protein</UP>]</DE></FR> (Eq. 1)
where Delta P corresponds to the change in fluorescence polarization, Delta Pmax is the maximum fluorescence polarization change, and KD is the dissociation constant of the interaction. A single ligand-binding site was assumed in this analysis.

Analysis of Structure by Circular Dichroism Spectroscopy

The average secondary structures of recombinant proteins, in the presence and absence of metal ions, were monitored by CD spectroscopy on a Jasco J720 spectropolarimeter calibrated with 0.1% (w/v) 10-camphorsulfonic acid-d solution. CD spectra were measured at 25 °C in a 0.2-mm path length quartz cell, and six scans from 250 to 180 nm (far-UV) were generated and averaged. Protein concentrations, determined using an extinction coefficient at 280 nm (31,320 M-1 cm-1), were typically 20 µM in 10 mM Tris-HCl (pH 7.4). Molar ellipticity is expressed in degrees·cm2·mol-1.

    RESULTS
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Procedures
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References

Effects of Cations on the Interaction between ClfA and the Fibrinogen gamma -Chain Peptide (Residues 396-412)-- To examine a possible effect of divalent cations on the ClfA-fibrinogen interaction, a fluorescence polarization assay was developed using recombinant forms of ClfA encompassing full-length region A (Clf40, residues 40-559) or the smallest truncated protein that maintained fibrinogen-binding activity (Clf41, residues 221-559) and the fluorescently labeled C-terminal gamma -chain peptide. Since fibrinogen is known to be a Ca2+-binding protein (23), we chose to use the gamma -chain peptide as a ligand and assumed that the observed effects of metal ions would reflect interactions with ClfA. We have previously shown that the fibrinogen gamma -chain peptide effectively inhibits interaction between ClfA and native fibrinogen (7).

In the absence of added metal ions, Clf40-(40-559) bound the gamma -chain peptide with a dissociation constant of 20.8 ± 2.5 µM (Fig. 3). An unlabeled gamma -chain peptide inhibited the binding of the fluorescent peptide to ClfA in a concentration-dependent manner, whereas a scrambled version of the peptide had no effect at similar concentrations (Fig. 3, inset). Inclusion of Ca2+ (5 mM) significantly reduced the binding affinity for the peptide (KD > 200 µM) (Fig. 3). Incubation of Clf40-(40-559) with 5 mM Mn2+ caused complete loss of gamma -chain peptide-binding activity. On the other hand, Mg2+ or Na+ at similar concentrations had no effect on protein-peptide interaction (Fig. 4A). In addition, Mg2+ (5 mM) did not counteract the inhibitory effect of Ca2+ (data not shown). Incubation of Clf40-(40-559) with EDTA (5 mM) increased the affinity of MSCRAMM for the gamma -chain peptide by 2-fold (KD = 11.5 ± 0.6 µM) compared with non-EDTA-treated protein (KD = 20.8 ± 2.5 µM). It is likely that contaminating Ca2+ and Mn2+ in the assay solutions and protein preparations contribute to inhibition of the protein-peptide interaction. Inclusion of EDTA removes these ions. Furthermore, these data show that divalent cations are not required to promote gamma -chain peptide binding. Results similar to those described for Clf40-(40-559) were obtained when these experiments were repeated with the smaller construct, Clf41-(221-559). Thus, in the absence of added metal ions, Clf41-(221-559) bound the gamma -chain peptide with a dissociation constant of 15.0 ± 1.1 µM (Fig. 3), and the addition of 5 mM EDTA slightly reduced the KD to 11.1 ± 0.4 µM. When these experiments were repeated with Clf41-(221-559) without an N-terminal extension of histidine residues, identical results were obtained, indicating that the effects of cations on region A function are independent of the purification tag (data not shown).


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Fig. 3.   Effect of Ca2+ and Mn2+ on the interaction between region A of ClfA and the fibrinogen gamma -chain peptide. The binding of the gamma -chain peptide to purified Clf40-(40-559) was measured in the absence of metal ions (×) and in the presence of 5 mM Ca2+ (black-square------black-square) or 5 mM Mn2+ (black-square- - -black-square). The interaction of the fibrinogen gamma -chain peptide with Clf41-(221-559) (bullet ) is shown for comparison. Several other binding curves were obtained at different concentrations of Ca2+, all of which approached the same level of binding at saturation. These have been omitted for clarity. These binding studies were performed under equilibrium conditions as described under "Experimental Procedures." Equation 1 was used to calculate the binding curves. Each binding assay was repeated at least three times, each yielding a similar result. Inset, binding of the fluorescein-labeled fibrinogen gamma -chain peptide (10 nM) to Clf40-(40-559) (40 µM) was assessed in the presence of increasing concentrations of unlabeled gamma -chain peptide (bullet ) and scrambled gamma -chain peptide (square ). Data are presented as means ± S.E. of three independent experiments.


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Fig. 4.   Effects of divalent cations on the interaction of Clf40(40-559) with the fibrinogen gamma -chain peptide (A) and S. aureus cell adherence to native fibrinogen (B). Binding of the fluorescent gamma -chain peptide to Clf40-(40-559) in A or bacterial adherence to fibrinogen in B was measured as a function of Ca2+ (bullet ) or Mg2+ (square ). These assays were performed at least three times, each yielding a similar result.

The interaction of Clf40-(40-559) with a set amount of fluoresceinated gamma -chain peptide across a concentration range of Ca2+ and Mg2+ was also measured. With Mg2+, the binding of the gamma -chain peptide to Clf40-(40-559) was unaffected across the entire concentration range tested. In contrast, concentrations of Ca2+ above 2 mM inhibited the interaction (Fig. 4A). At concentrations greater than 5 mM, very little binding of the gamma -chain peptide to region A was observed. Very similar results were obtained when the effects of different cations on S. aureus cell adhesion to immobilized fibrinogen were measured (Fig. 4B). Concentrations of Ca2+ above 2 mM inhibited bacterial adhesion to fibrinogen, whereas Mg2+ did not affect cell adhesion throughout the concentration range analyzed. These results indicate the existence of an inhibitory Ca2+-binding site in region A of ClfA with an apparent KD of 2.5 mM, a value similar to the physiological concentration of Ca2+ (~2.5 mM) present in normal human sera. Thus, under some physiological conditions, this site may be occupied. Inclusion of Mg2+ had no effect on Ca2+-induced inhibition (data not shown), suggesting that Mg2+ is unable to compete with Ca2+ for binding to the inhibitory binding site and that there is a certain degree of specificity for Ca2+.

Effect of Ca2+ Ions on the Structure of Region A: Circular Dichroism Analysis-- Circular dichroism spectroscopy was used to investigate if the binding of Ca2+ ions affects the secondary structures of the Clf40-(40-559) and Clf41-(221-559) proteins. Cation binding by integrins has been shown to be associated with conformational changes. For example, expression of the epitope recognized by monoclonal antibody 24 on alpha Lbeta 2 was dependent on Mg2+, and monoclonal antibody 24 expression correlates with the ability to bind ligand (24, 25).

The CD spectra of Clf41-(221-559) were dominated by a large minimum at 215 nm. Inclusion of Ca2+ and Mn2+ had a reproducible effect on the protein far-UV CD spectra at ~200 nm (Fig. 5). This effect on secondary structure was dependent on the concentration of divalent cation and on the particular cation used. Ca2+ altered the CD spectra of Clf41-(221-559) by reducing the signal at 200 nm in a concentration-dependent manner (Fig. 5). The presence of Mn2+ resulted in a qualitatively similar change, although the effect was more pronounced (data not shown). Identical results were obtained with Clf41-(221-559) without an N-terminal extension of six histidine residues, indicating that the effects of Ca2+ and Mn2+ on the secondary structure of region A are independent of the purification tag (data not shown).


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Fig. 5.   Structural analysis of Clf41-(221-559) showing the effect of increasing Ca2+ concentrations on the far-UV CD spectrum. The Ca2+ concentrations are (from bottom to top at 200 nm) are 0, 1, 2.5, 5, and 7.5 mM. Increasing concentrations of Mn2+ resulted in qualitatively similar changes. Comparable cation concentration-dependent effects were also observed with Clf40-(40-559).

The CD spectra of Clf40-(40-559) were dominated by a large minimum at 200 nm, suggesting differences in secondary structure compared with Clf41-(221-559). The spectroscopic differences observed reflect the effect of the additional 180 amino acid residues present in Clf40-(40-559). However, deconvolution of the spectra using SELCON (26) indicated only minor differences in the percentage alpha -helix and beta -sheet of each construct (estimated alpha -helix/beta -sheet is 15/35% for Clf40-(40-559) and 15/40% for Clf41-(221-559)). As with Clf41-(221-559), the addition of Ca2+ (and Mn2+) to Clf40-(40-559) altered the far-UV spectra at 200 nm in a concentration-dependent manner (Fig. 6, upper panel). Cation-induced changes in the far-UV spectra of Clf40-(40-559) and Clf41-(221-559) were completely reversible by the addition of EDTA (10 mM).


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Fig. 6.   Structural analysis of site-directed and deletion mutants of region A. Far-UV CD spectra of the wild-type (Clf40-(40-559)) and mutant proteins without metal ions (bullet ) and in the presence of 5 mM Ca2+ (black-triangle) were generated as described under "Experimental Procedures."

The effect of Ca2+ and Mn2+ on secondary structure correlates with the effect that these metal ions have on the ability of the protein to bind the C-terminal gamma -chain peptide. These data indicate that when Ca2+ and Mn2+ bind to region A of ClfA, alterations to the secondary structure of the protein occur, with Mn2+ having the largest effect. These structural alterations may be responsible for the inhibition of ligand binding.

Interaction of ClfA Region A Mutants with the Fibrinogen gamma -Chain Peptide-- The role of the putative Ca2+-binding EF-hand at residues 310-321 in ligand binding was investigated using deletion mutagenesis and site-directed mutagenesis. Deletion of amino acids 310-321 from Clf40-(40-559) and Clf41-(221-559) resulted in complete loss of fibrinogen-binding activity. These mutant proteins did not bind fibrinogen in a Western ligand blot assay (data not shown). They also failed to bind to the fibrinogen C-terminal gamma -chain peptide in a fluorescence polarization assay (Fig. 7A).


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Fig. 7.   Functional analysis of site-directed and deletion mutants of region A of ClfA. A, the interaction of the fluorescently labeled fibrinogen gamma -chain peptide (10 nM) with the wild-type (Clf40-(40-559)) and mutant proteins was measured under equilibrium conditions. Equation 1 was used to calculate the binding curves. B, shown is the effect of Ca2+ (5 mM) on the interaction of the fluorescently labeled fibrinogen gamma -chain peptide (10 nM) with the wild-type and mutant proteins. Experiments in which Ca2+ was not included are shown for comparison. open circle , Clf40-(40-559); bullet , Clf51-(Delta 310-321); triangle , Clf68 (D310A/D312A/T318A/D321A); square , Clf69 (D310A); black-square, Clf94 (D310A/D312A).

The EF-hand motif was further investigated by mutating putative cation-coordinating residues in Clf40-(40-559) and Clf41-(221-559) and characterizing the interactions of these mutant proteins with the fluorescently labeled C-terminal gamma -chain peptide. The amino acid residues investigated were replaced with alanine, a residue that was not expected to interfere with existing secondary structure, but would be unable to coordinate cations. The corresponding base changes were made using a novel PCR mutagenesis technique as described under "Experimental Procedures." The recombinant proteins containing the mutations were purified to homogeneity by metal ion chromatography and anion exchange chromatography. Structural analysis of the isolated mutant proteins by CD spectroscopy indicated differences in secondary structure resulting from the introduction of mutations (Fig. 6). Interestingly, the observed effect of Ca2+ on structure was significantly less in the mutants as compared with the wild-type protein (Fig. 6).

The effects of the mutations on the interaction of Clf40-(40-559) with the fibrinogen gamma -chain peptide are shown in Fig. 7A. Similar effects were observed when the mutations were introduced into the smaller construct (Clf41-(221-559) (data not shown). Thus, it is apparent that substitutions within the putative EF-hand affect peptide-binding affinity. Substitution of four residues (D310A/D312A/T318A/D321A) exhibited the most dramatic decrease. Because of the very low affinity of this quadruple mutant for the peptide, the dissociation constant could not be measured accurately. The D310A/D312A double mutant bound the peptide over three times more weakly than the wild-type protein (KD = 66.5 ± 7.4 µM). Mutation of the first aspartate in the EF-hand (D310A) had no effect on gamma -chain peptide-binding activity.

The effects of Ca2+ on the interaction of the D310A single mutant and the D310A/D312A double mutant with the gamma -chain peptide were also measured. As shown in Fig. 7B, the degree of inhibition induced by the inclusion of Ca2+ is significantly less with the mutants compared with the wild-type protein. These results show that the introduced mutations have (a) reduced the ability of region A of ClfA to bind to the fibrinogen gamma -chain peptide and (b) reduced the ability of Ca2+ to inhibit this interaction. One interpretation of these data is that the inhibitory Ca2+-binding site and the fibrinogen gamma -chain peptide-binding site share contact points within amino acid sequence 310-321.

Interaction of Region A Mutants with Fibrinogen-- An enzyme-linked immunosorbent assay was developed (as described under "Experimental Procedures") to assess the effects of the mutations on the interaction of Clf41-(221-559) with native fibrinogen (Fig. 8). Deletion of the EF-hand motif or substitution of four residues within it had the severest effect on fibrinogen-binding ability. The D310A/D312A double mutant also displayed significant reduction in the ability to bind fibrinogen. The D310A single mutant reduced binding to 60% of the wild type. These results further implicate amino acids 310-321 of region A as playing an important part in the ligand-binding function of ClfA.


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Fig. 8.   Interaction of region A mutants with native fibrinogen. Purified wild-type (W.T) and mutant proteins were incubated with immobilized fibrinogen followed by anti-Clf41-(221-559) antibodies. Binding of the primary antibody was detected using horseradish peroxidase-labeled protein A and quantitated by absorbance at 450 nm. Binding of Clf41-(221-559) to fibrinogen was assigned a value of 100. Introduction of mutations or deletions did not significantly affect the ability of Clf41-(221-559) to bind the polyclonal antiserum raised against Clf41-(221-559) (as judged by quantitative Western blot analysis; data not shown). Data are expressed as means ± S.E. of two independent experiments.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The adhesion of microorganisms to host tissues is the critical first step in the series of events that lead to clinically manifested infections. It has become evident that eukaryotic adhesive ECM components that support adhesion of host cells also serve as ligands for pathogenic microorganisms (1). Fibrinogen, the blood plasma coagulation protein, is also found in the ECM and plays important roles in wound healing. During coagulation, fibrinogen is proteolytically converted to fibrin, which forms the structure of the blood clot. In addition, fibrinogen is the major blood protein deposited on implanted biomaterial (27). Immobilized fibrin/fibrinogen in a blood clot, in the ECM, or present on the surface of biomaterial can serve as a substrate for the adherence of S. aureus cells (28, 29).

S. aureus has long been known to form clumps in the presence of blood plasma. Hawiger et al. (5) identified fibrinogen as the plasma protein responsible for this phenomenon. Further study revealed that the domain of fibrinogen that interacts with the fibrinogen receptor of S. aureus is located at the carboxyl-terminal of the gamma -chain (6, 7). Recently, this was confirmed by showing that purified ClfA protein binds to the extreme C terminus of the gamma -chain of fibrinogen and that a synthetic C-terminal gamma -chain peptide fully inhibits this interaction (7). The 12 residues at the C terminus of the fibrinogen gamma -chain also bind to the integrin receptor (alpha IIbbeta 3) on the surface of platelets, resulting in aggregation in vitro and the formation of platelet-fibrin thrombi (9, 18-20, 30). These residues mediate initial contact with nonstimulated platelets and on activation are sufficient to promote stable adhesion to fibrinogen (21). The mode of interaction between fibrinogen and alpha IIbbeta 3 has been studied in some detail. The gamma -chain peptide of fibrinogen binds in a divalent cation-dependent manner to a region of alpha IIb corresponding to an EF-hand-like sequence (10, 12). A requirement of divalent cations for ligand binding to integrins is often observed. However, inhibition of integrin-ligand binding by Ca2+ has also been reported (13, 25, 32-34), although there is no evidence to indicate that Ca2+ inhibits the interaction between alpha IIbbeta 3 and the fibrinogen gamma -chain peptide. Recently, Hu et al. (13) identified two classes of cation-binding sites in beta 3-containing integrins: sites that, when occupied by Ca2+, promote ligand binding and sites that inhibit ligand binding. The location of the inhibitory cation-binding site(s) has not been identified.

Analysis of the primary structure of region A of ClfA identified a potential divalent cation-binding EF-hand motif at residues 310-321, which differs from the EF-hand consensus at only one residue, a non-cation-coordinating site (Fig. 2A). Secondary structure analysis of the primary sequence of ClfA predicts this putative EF-hand to be flanked by alpha -helices, which are required for correct EF-hand conformation (22). The proposed cation-binding EF-hand motif lies within the minimum 329-residue segment of region A that retains fibrinogen-binding activity (residues 221-550) (3). Also present within the fibrinogen-binding domain is a putative MIDAS motif, a cation-binding sequence contained within the I-domain of integrins (35). Analysis of this motif by site-directed mutagenesis indicated that it plays a role in ClfA binding to fibrinogen.2 The I-domain of an integrin-like protein from Candida albicans, which has homology to the I-domain of the leukocyte integrin alpha Mbeta 2, also has homology to the fibrinogen-binding domain of ClfA (36).

We have shown that Ca2+ plays a role in the interaction between ClfA and fibrinogen. Preliminary experiments showed that Ca2+ at high concentrations prevented clumping of S. aureus cells in the presence of fibrinogen. In addition, clumping of bacteria (due to the interaction of ClfA and fibrinogen) could be reversed by the addition of Ca2+ (data not shown). These effects were prevented by the addition of EGTA or EDTA. In the studies reported in this work, we have used purified components and a quantitative binding assay. Ca2+ dramatically inhibits the interaction between the gamma -chain peptide and region A of ClfA. Mn2+ is a more potent inhibitor than Ca2+, suggesting that Mn2+ has a higher affinity for the inhibitory cation-binding site. Alternatively, Mn2+ may bind to a different cation-binding site. The protein-peptide interaction is not affected by Mg2+ or monovalent cations (Figs. 3 and 4A). Ca2+ also inhibited the adherence of S. aureus cells to immobilized fibrinogen (Fig. 4B) at concentrations similar to those that affect the gamma -chain peptide interaction. These observations are consistent with the presence of an inhibitory site(s) in ClfA that allows Ca2+ and Mn2+ (but not Mg2+) to bind. When Clf40-(40-559) and Clf41-(221-559) were incubated with the concentrations of Ca2+ that inhibited ligand binding, distinct differences in secondary structure were evident (Figs. 5 and 6). Thus, Ca2+ binding to an inhibitory binding site(s) in region A of ClfA induces a conformational change in the ligand-binding site that apparently results in inhibition of the ClfA-ligand interaction. It is noted that inclusion of EDTA/EGTA slightly increased the affinity of the fibrinogen gamma -chain peptide for ClfA, indicating that divalent cations are not required to promote peptide binding. This feature represents a significant difference from the observed metal ion dependence of the alpha IIbbeta 3-fibrinogen interaction (12).

Deletion mutagenesis and site-directed mutagenesis were used to identify the Ca2+-dependent inhibitory site. Deletion of the EF-hand (residues 310-321) or substitution of four of the putative cation-coordinating residues resulted in almost complete loss of both fibrinogen-binding activity and gamma -chain peptide-binding activity (Figs. 7A and 8). A single substitution (D310A) and a double substitution (D310A/D312A) caused a significant reduction in ligand-binding affinity (Fig. 8). In addition, the inclusion of Ca2+ with these mutant proteins only marginally affected their binding to the gamma -chain peptide (Fig. 7B). The proteins may have retained some ability to bind Ca2+, explaining the small reduction in activity in the presence of the ion. Incomplete EF-hand motifs in other proteins can chelate Ca2+, for example, the platelet integrin alpha IIbbeta 3 (31, 37). The reduced inhibitory effect of Ca2+ with the D310A and D310A/D312A mutant proteins compared with the wild type indicates that the ion interacts with the inhibitory site less effectively. This conclusion is supported by the observation that Ca2+-induced changes in secondary structure were significantly less in the mutants compared with the wild-type protein (Fig. 6). Taken together, these results implicate the EF-hand motif at residues 310-321 as an inhibitory Ca2+-binding site. Substitution of four cation-coordinating residues or deletion of the EF-hand motif should result in complete loss of cation-binding ability, but as these proteins have no fibrinogen-binding activity, inhibition of function by Ca2+ and Mn2+ could not be assessed.

In summary, these findings indicate that changes in amino acid sequence 310-321 of region A compromise the protein's ability to bind fibrinogen and to bind Ca2+ at an inhibitory cation-binding site. There are several possible explanations for this observation. One interpretation is that the inhibitory binding site and the fibrinogen gamma -chain peptide-binding site share contact points within sequence 310-321. A model can be proposed in which Ca2+, at millimolar concentrations, binds to an inhibitory binding site within region A of ClfA that overlaps with the ligand-binding site (Fig. 9A). Occupation of this Ca2+-binding site directly interferes with ligand binding. In an alternative model, the inhibitory site and the fibrinogen-binding site are physically distinct (Fig. 9B). Interaction of Ca2+ with the inhibitory site induces a conformational change in the ligand-binding site that affects the affinity for fibrinogen. Further work is needed to differentiate between these models.


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Fig. 9.   Model depicting the interaction of region A of ClfA, the C terminus of the gamma -chain of fibrinogen, and Ca2+. Occupation of the inhibitory site(s) by Ca2+ prevents fibrinogen binding by inducing a conformational change in the ligand-binding site. The competitive inhibition of ligand binding by Ca2+ indicates either that ligand (L) and Ca2+ compete for the same site on ClfA (A) or that ligand and Ca2+ bind to distinct sites that are mutually exclusive (B).

Does Ca2+ have a role in the regulation of ClfA activity in vivo? The IC50 determined for the interaction of S. aureus cells with fibrinogen is similar to the total concentration of Ca2+ present in normal human sera, although the concentration of ionized Ca2+ is lower (maintained between 1.0 and 1.3 mM) (40). Even at these lower concentrations, Ca2+ may regulate ligand-binding function in a subset of ClfA molecules bound to the cell wall of S. aureus. Indeed, the concentration of Ca2+ in the local environment of ClfA may vary significantly due to the protein's acidic aspartate-serine repeat sequence and the extent of the acidic cell wall and capsule, both of which associate with divalent cations. Thus, the ability of Ca2+ to affect ClfA function may be a direct consequence of the protein's microenvironment on the cell surface, an environment that changes during in vivo growth. One can speculate that Ca2+-dependent regulation of ClfA activity prevents all of the receptors on intravascular S. aureus cells from being occupied by soluble fibrinogen, thus allowing the bacteria (under the right conditions) to adhere to solid-phase fibrinogen or fibrin clots. In addition, this process may allow cells to detach from the initial vegetation, allowing microbial proliferation.

We have shown that ClfA contains a class of Ca2+-binding sites that, when occupied, regulate fibrinogen-binding activity. Regulation of integrin-ligand binding by divalent cations is well documented, and this phenomenon represents an intriguing similarity between eukaryotic and prokaryotic adherence proteins.

    FOOTNOTES

* This work was supported by Wellcome Trust Grant 041823 (to T. J. F) and by National Institutes of Health Grant AI20624 and grants from the Markey Foundation and the Neva and Wesley West Foundation (to M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Microbiology Dept., SmithKline Beecham Pharmaceuticals, Collegeville, PA 19426-0989.

par To whom correspondence should be addressed. Tel.: 353-1-6082014; Fax: 353-1-6799294; E-mail: tfoster{at}mail.tcd.ie.

1 The abbreviations used are: ECM, extracellular matrix; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography.

2 D. P. O'Connell, T. Nanavaty, M. Höök, and T. J. Foster, manuscript in preparation.

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Abstract
Introduction
Procedures
Results
Discussion
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