From the 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
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ABSTRACT |
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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
IIb
3 recognize the same domain at the
extreme C terminus of the fibrinogen
-chain.
IIb
3 interaction with this domain is
known to occur in close proximity to a Ca2+-binding EF-hand
structure in the
-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
-chain peptide. Furthermore, Ca2+ (or Mn2+) could
inhibit the binding of the fibrinogen
-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
-chain of fibrinogen. Mutagenesis studies indicate
that the Ca2+-dependent inhibitory site is
located within the EF-hand motif at residues 310-321.
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INTRODUCTION |
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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 ()
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 2-,
2
-, and 2
-chains linked by disulfide bonds. This protein is
recognized by several integrins including the platelet integrin
IIb
3. Activation of platelets and
integrin
IIb
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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In earlier studies, Hawiger and co-workers (5, 6) showed that a
synthetic peptide mimicking the extreme C terminus of the fibrinogen
-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
-subunit of the platelet integrin
IIb
3 (8, 9). The
-chain-binding site
has been mapped to a region of the
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
-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
-subunit of
IIb
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
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
-chain peptide. Further biochemical characterization of
integrin
IIb
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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In this report, we propose a mode of interaction between ClfA and the
fibrinogen -chain peptide that exhibits some similarities to
IIb
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.
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EXPERIMENTAL PROCEDURES |
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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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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
-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 -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
-chain in a random sequence (i.e. GHEHGLQGQGAVKDGAQ) was
used as a control.
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(Eq. 1) |
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 M1 cm
1), were typically 20 µM in 10 mM Tris-HCl (pH 7.4). Molar
ellipticity is expressed in
degrees·cm2·mol
1.
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RESULTS |
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Effects of Cations on the Interaction between ClfA and the
Fibrinogen -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
-chain peptide. Since fibrinogen is known to be a
Ca2+-binding protein (23), we chose to use the
-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
-chain peptide effectively inhibits interaction between
ClfA and native fibrinogen (7).
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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
L
2 was dependent on Mg2+, and
monoclonal antibody 24 expression correlates with the ability to bind
ligand (24, 25).
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Interaction of ClfA Region A Mutants with the Fibrinogen -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
-chain peptide in a
fluorescence polarization assay (Fig.
7A).
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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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DISCUSSION |
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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 -chain (6, 7). Recently, this was confirmed
by showing that purified ClfA protein binds to the extreme C terminus
of the
-chain of fibrinogen and that a synthetic C-terminal
-chain peptide fully inhibits this interaction (7). The 12 residues at the C terminus of the fibrinogen
-chain also bind to the integrin receptor (
IIb
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
IIb
3 has been studied in some detail. The
-chain peptide of fibrinogen binds in a divalent
cation-dependent manner to a region of
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
IIb
3 and the fibrinogen
-chain peptide. Recently, Hu et al. (13) identified two classes of cation-binding sites in
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 -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
M
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 -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
-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
-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
IIb
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 -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
-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
IIb
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
-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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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.
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FOOTNOTES |
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* 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.
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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REFERENCES |
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