From the Department of Microbiology, Moyne Institute
of Preventive Medicine, Trinity College, Dublin 2, Ireland and the
¶ Center for Extracellular Matrix Biology and the Department of
Biochemistry and Biophysics, Institute of Biosciences and Technology,
Texas A&M University System Health Science Center,
Houston, Texas 77030-3303
Received for publication, August 31, 2000, and in revised form, October 20, 2000
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ABSTRACT |
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Clumping factor A (ClfA) is a cell
surface-associated protein of Staphylococcus aureus that
promotes binding of this pathogen to both soluble and immobilized
fibrinogen (Fg). Previous studies have localized the Fg-binding
activity of ClfA to residues 221-559 within the A region of this
protein. In addition, the C-terminal part of the A region (residues
484-550) has been implicated as being important for Fg binding. In
this study, we further investigate the involvement of this part of ClfA
in the interaction of this protein with Fg. Polyclonal antibodies
generated against a recombinant protein encompassing residues 500-559
of the A region inhibited the interaction of both S. aureus
and recombinant ClfA with immobilized Fg in a
dose-dependent manner. Using site-directed mutagenesis, two
adjacent residues, Glu526 and Val527, were
identified as being important for the activity of ClfA. S. aureus expressing ClfA containing either the E526A or V527S substitution exhibited a reduced ability to bind to soluble Fg and to
adhere to immobilized Fg. Furthermore, bacteria expressing ClfA containing both substitutions were almost completely defective in
Fg binding. The E526A and V527S substitutions were also introduced into
recombinant ClfA (rClfA-(221-559)) expressed in Escherichia coli. The single mutant rClfA-(221-559) proteins showed a
significant reduction in affinity for both immobilized Fg and a
synthetic fluorescein-labeled C-terminal Staphylococcus aureus is an important pathogen that
causes a wide spectrum of infections both in the community and in
hospitalized patients, ranging from skin abscesses to more serious
invasive diseases such as septic arthritis, osteomyelitis, and
endocarditis. It is also a major cause of surgical wound infection and
infections associated with indwelling medical devices (1). Primarily an extracellular pathogen, S. aureus colonizes the host by
adhering to components of the extracellular matrix. This process is
mediated by a family of cell surface-expressed proteins called
MSCRAMMs1 (2, 3). Several
MSCRAMMs of S. aureus have been identified, including those
that bind to collagen, bone sialoprotein, fibronectin, and fibrinogen
(Fg) (4-9).
Fg is a 340-kDa glycoprotein that is present at a concentration of ~9
µM in the blood. It is composed of six polypeptide chains (two A Clumping factor A (ClfA) was the first Fg-binding MSCRAMM of S. aureus to be identified and characterized in detail (see Fig. 1)
(6). This protein is the prototype of a family of staphylococcal surface proteins (Sdr protein family) characterized by the presence of
a unique serine-aspartate dipeptide repeat region (R region) (see Fig.
1) (6, 10, 11). ClfA has structural features that are common to many
other surface proteins expressed by Gram-positive bacteria. At the N
terminus is a signal sequence for Sec-dependent secretion
(see Fig. 1, S), whereas the C terminus contains an LPXTG motif, a hydrophobic membrane-spanning region
(M), and positively charged amino acid residues. The
LPXTG motif is the target of a transpeptidase (called
"sortase") that cleaves the motif between the threonine and glycine
residues and anchors the protein to the peptidoglycan cell wall (12,
13). The Fg-binding activity of ClfA has been localized to the
N-terminal A region of this protein (see Fig. 1) (14).
The binding site in Fg for ClfA has been localized to the C-terminal
end of the In a previous study, we sought to identify the Fg-binding site within
ClfA by constructing a series of recombinant truncates of the A region
of this protein (14). This analysis revealed that the smallest
recombinant truncate that retained Fg-binding activity was composed of
residues 221-550. Further truncation of either the N or C terminus of
this construct resulted in the loss of Fg-binding activity, suggesting
that the overall conformation of the protein is important in
maintaining the integrity of the Fg-binding site. However, it was also
noted that a non-Fg-binding truncate, composed of residues 332-550,
retained the ability to absorb out the clumping-blocking activity of
polyclonal antibodies (Abs) raised against the entire A region
(residues 40-559), whereas another truncate, composed of residues
221-484, did not (14). These observations raise the possibility that
the C-terminal part of the A region of ClfA (between residues 484 and
500) may contain at least part of the Fg-binding site of this protein.
In this study, we investigated the role of the C-terminal part of the A
region in the Fg-binding activity of ClfA. We found that polyclonal
antibodies raised against a recombinant ClfA truncate, composed of
residues 500-559 of the A region, blocked the interaction of both
S. aureus and recombinant ClfA with immobilized Fg. In addition, using site-directed mutagenesis, we identified two adjacent residues, Glu526 and Val527, within this part
of the A region that are important for the interaction of ClfA with
both soluble and immobilized Fg.
Bacterial Strains and Plasmids--
Escherichia coli
XL-1 Blue (Stratagene) was used for plasmid cloning and protein
expression. S. aureus strain RN4220 was the recipient used
for introducing plasmids into S. aureus by electroporation. Plasmids were subsequently transferred to DU5941, a mutant of S. aureus strain 8325-4 lacking expression of both ClfA and protein A
(strain 8325-4 clfA1::Tn917
Bacterial Growth Media and Antibiotics--
E. coli
strains harboring plasmids were routinely grown in L-broth or on L-agar
(26). S. aureus cultures were grown in trypticase soy broth
or on trypticase soy agar. Ampicillin (100 µg/ml) was used for the
selection of plasmids in E. coli, and chloramphenicol (10 µg/ml), erythromycin (3 µg/ml), or tetracycline (2 µg/ml) was
used for selection of plasmids or chromosomal markers in S. aureus.
Transformation and Transduction--
E. coli XL-1
Blue cells were made competent by CaCl2 treatment (27).
Electrocompetent S. aureus cells were prepared by the method
of Oskouian and Stewart (28). The pCU1-derived plasmids were initially
introduced into S. aureus strain RN4220 by electroporation (29) with a Gene Pulser II set at 2.3 kV, 25 microfarads, and 100 ohms
in a 0.2-cm cuvette and were subsequently transduced to strain DU5941
using phage 85 (30).
Manipulation of DNA--
DNA manipulations were performed by
standard procedures (27). Plasmid DNA for cloning and sequence analysis
was purified using the WizardTM Plus miniprep kit
(Promega). PCR-amplified DNA was purified using the
WizardTM PCR prep kit (Promega). DNA restriction and
modification enzymes were purchased from Roche Molecular Biochemicals.
Double-stranded plasmid DNA was sequenced by the dideoxy chain
termination method (27) using the Taq DyeDeoxy Terminator
Cycle sequencing kit and an automated sequencer (Applied Biosystems
Model 373A).
PCR Amplification of clfA Gene Fragments--
The PCR mixtures
contained 100 ng of template DNA, 100 pmol of forward and reverse
primer, 200 µM dNTP, ThermoPol reaction buffer (New
England Biolabs Inc.), and 1 unit of Vent® polymerase (New
England Biolabs Inc.). All reactions were carried out with a 1-min
denaturation step at 94 °C, a 1-min annealing step at 50-60 °C
(depending on the primer pair), and an extension time of 1 min/1
kilobase pair of DNA to be amplified. The standard cycle was repeated
for 30 cycles, followed by incubation at 72 °C for 10 min. PCR
amplifications were performed using a PerkinElmer Life Sciences DNA
thermocycler. Restriction enzyme sites were incorporated at the
5'-end of the primers to facilitate subsequent cloning of the PCR
products into the appropriate plasmid vector.
Site-directed Mutagenesis of clfA and Construction of the Shuttle
Plasmids Expressing Mutant ClfA Proteins--
A previously described
PCR method was used to introduce site-directed mutations into the
clfA gene (20). Briefly, the shuttle plasmid expressing the
mutant ClfA protein with the E526A substitution was constructed as
follows. Using S. aureus strain Newman genomic DNA as
template, a 915-base pair fragment of the clfA gene was amplified using the flanking primer F1 (covering the PstI
site in clfA) and the internal primer R2 (incorporating a
BglII site and the nucleotide mismatch required for the
desired mutation) (Table I). In a second
PCR, a 1135-base pair fragment of the clfA gene was
amplified using the internal primer F2 (incorporating a
BglII site) and the flanking primer R1 (incorporating a
HindIII site) (Table I). Primer R1 was complementary to
noncoding sequences 100 base pairs downstream from the clfA
stop codon in the chromosome. The two PCR products were then cleaved
with PstI and BglII or with BglII and
HindIII, as appropriate, and ligated together at the
BglII site. The mutant PstI-HindIII
clfA gene fragment was cloned into plasmid pCF77 (pCU1
carrying a copy of the wild-type clfA gene (23)), yielding
plasmid pClfA(E526A). This cloning reaction was facilitated by the
presence of a unique PstI site in the wild-type
clfA gene and involved replacing the wild-type PstI-HindIII clfA gene fragment in
pCF77 with the mutant PstI-HindIII clfA gene fragment. The pCF77-derived plasmids expressing
the ClfA proteins with the substitutions N525A, V527S, E526A/V527S, A528V/G532A, D537A, E546A, and E559A were generated in a similar fashion using the primers indicated in Table I. The DNA sequence of
each of the mutations was verified as described above.
Construction of Plasmids Expressing Mutant rClfA-(221-559)
Proteins--
The E526A, V527S, E526A/V527S, and A528V/G532A
substitutions were introduced into a recombinant protein composed of
residues 221-559 of ClfA, called rClfA-(221-559) (previously called
Clf41 (20)). To construct the plasmids expressing rClfA-(221-559) with
the E526A and A528V/G532A substitutions, a 1019-base pair fragment
(encoding residues 221-559) was amplified from the pCF77-derived plasmid carrying the mutant clfA gene of interest using
primers F1-A (incorporating a BamHI site) and R1-A
(incorporating a HindIII site) (Table I). The amplified DNA
was then cleaved with BamHI and HindIII and
ligated into the expression vector pQE30, which was also cleaved with
these enzymes. To construct the plasmids expressing rClfA-(221-559)
with the V527S and E526A/V527S substitutions, primers F2-A
(incorporating a BglII site) and R1-A were used (Table I).
The amplified DNA was then cleaved with BglII and
HindIII and ligated into pQE30, which was cleaved with
BamHI and HindIII. The DNA sequence of each of
the mutations was verified as described above. Construction of the
plasmid expressing wild-type rClfA-(221-559) has been described
previously (20). The rClfA-(221-559) proteins expressed from pQE30
contained an N-terminal extension of six histidine residues (His tag),
facilitating purification by immobilized metal chelate affinity chromatography.
Construction of Plasmid Expressing rGST-C1fA-(500-559)
Protein--
DNA encoding residues 500-559 of the A region of ClfA
was amplified by PCR with primers F10 (incorporating a BamHI
site) and R10 (incorporating a HindIII site) (Table I) using
S. aureus strain Newman genomic DNA as template. The
amplified product was cloned into plasmid pGEX-KG and cleaved with
BamHI and HindIII, yielding plasmid
pGST-ClfA-(500-559). The recombinant fusion protein expressed was
called rGST-ClfA-(500-559).
Expression and Purification of Recombinant ClfA
Proteins--
Cells harboring the pQE30-derived plasmids were grown,
and bacterial cell lysates were prepared as described previously (20). The fusion proteins containing an N-terminal His tag were purified by
immobilized metal chelate affinity chromatography as described previously (31). Cells harboring plasmid pGST-ClfA-(500-559) were grown, and the rGST-ClfA-(500-559) protein was purified on a
glutathione-Sepharose column as described previously (7).
Anti-rGST-ClfA-(500-559) Polyclonal Antibodies--
Polyclonal
Abs to rGST-ClfA-(500-559) were prepared by immunizing a New Zealand
White rabbit subcutaneously with 50 µg of the recombinant protein
emulsified with an equal volume of Freund's complete adjuvant. The
rabbit was boosted twice over a period of 1 month with the same amount
of antigen in Freund's incomplete adjuvant. The immunoglobulins were
precipitated with 25% ammonium sulfate, and IgG was purified by
affinity chromatography on a protein A-Sepharose 4B column (Amersham
Pharmacia Biotech).
Western Immunoblot and Western Ligand Affinity Blot
Assays--
SDS-polyacrylamide gel electrophoresis was performed by
standard methods (32). Proteins were visualized on gels by Coomassie Brilliant Blue R-250 staining. S. aureus cell wall proteins
were prepared from stabilized protoplasts by digestion with lysostaphin (Ambicin L recombinant lysostaphin, Applied Microbiology) as
described previously (23). For the Western immunoblot assay, released cell wall-associated proteins were transferred to polyvinylidene difluoride membranes (Roche Molecular Biochemicals) using a semidry system (Bio-Rad) as described previously (23). Remaining
protein-binding sites were blocked by incubating the membranes in 5%
(w/v) nonfat dry milk in Tris-buffered saline (TBS; 10 mM
Tris-HCl and 150 mM NaCl, pH 7.4) for 18 h at 4 °C.
The ClfA proteins were detected with rabbit anti-rClfA-(40-559)
polyclonal Abs (diluted 1:1000 in blocking reagent), followed by
horseradish peroxidase (HRP)-conjugated protein A (diluted 1:500 in
blocking reagent; Sigma). Bound protein A was detected by enhanced
chemiluminescence (New England Biolabs Inc.). For the Western ligand
affinity blot assay, the recombinant ClfA proteins were transferred to
a polyvinylidene difluoride membrane, and the membranes were incubated
with blocking reagent as described above. The membranes were then
incubated with HRP-conjugated human Fg (10 µg/ml in blocking
reagent), and bound protein was visualized by enhanced
chemiluminescence. Human Fg (Calbiochem) was conjugated to HRP
according to the manufacturer's instructions (Pierce).
Bacterial Cell Immunoblot Assay--
Bacterial cell immunoblot
assays were performed as described previously (33) using S. aureus cultures grown in trypticase soy broth for 15 h at
37 °C with aeration.
Bacterial Cell Clumping Assay--
S. aureus strains
were grown in trypticase soy broth for 15 h at 37 °C with
aeration, harvested by centrifugation at 3000 × g for
10 min, and washed with phosphate-buffered saline (PBS; Oxoid Ltd.). A
suspension of ~4 × 108 colony-forming units in a
20-µl volume was added to 50 µl of 2-fold serial dilutions of human
Fg (starting at 1 mg/ml) in the wells of a microtiter plate. The
reciprocal of the highest dilution of Fg giving clumping after 5 min
was defined as the titer.
Bacterial Adherence Assay--
S. aureus strains were
grown in trypticase soy broth for 15 h at 37 °C with aeration,
harvested by centrifugation at 3000 × g, and washed
with PBS. For the inhibition of bacterial adherence by the
anti-rGST-ClfA-(500-559) polyclonal Abs, 2-fold serial dilutions of
purified IgG in PBS were preincubated with strain DU5873 cells
(~5 × 107 colony-forming units) with shaking for
2 h at room temperature. Strain DU5873 (a protein A-deficient
mutant of strain Newman) was used in this assay to prevent the
nonimmune reaction between IgG and protein A. The cells were then
transferred to wells in a microtiter plate (Sarstedt, Inc.) coated with
human Fg (500 ng/well), and bacterial adherence was measured using
crystal violet staining as described previously (23). Polyclonal Abs
raised against a recombinant form of the Fg-binding region of ClfB
(rGST-ClfB-(45-542)) were used as a control in this assay (7).
Measurement of the relative adherence of strain DU5941 (~1 × 108 colony-forming units), expressing wild-type and mutant
ClfA proteins, to immobilized human Fg was also performed using crystal
violet staining as described previously (23).
Enzyme-linked Immunosorbent Assay--
For the Ab inhibition
studies, a recombinant His-tagged protein composed of the entire A
region of ClfA, called rClfA-(40-559), was used (previously called
Clf40 (20)) (see Fig. 1). The wells of microtiter plates were coated
with 1 µg of human Fg (Enzyme Research Laboratories) for 18 h at
4 °C. After washing with TBS, the wells were blocked with 5% (w/v)
bovine serum albumin (BSA) in TBS for 2 h at room temperature and
then washed again with TBS containing 0.05% Tween 20 (TBS-T). The
rClfA-(40-559) protein (10 nM) was preincubated with
increasing concentrations of the polyclonal Abs in TBS containing 0.1%
BSA for 1 h at room temperature. The samples were then added to
the Fg-coated wells for 1 h at room temperature. The wells were
washed with TBS-T, and bound protein was detected with an anti-His tag
monoclonal antibody (diluted 1:3000 in TBS-T containing 0.1% BSA;
CLONTECH), followed by goat anti-mouse alkaline
phosphatase-conjugated polyclonal Abs (diluted 1:2000 in TBS-T
containing 0.1% BSA; Bio-Rad). Finally, bound alkaline
phosphatase-conjugated Abs were detected by the addition of
p-nitrophenyl phosphate (Sigma) in 1 M
diethanolamine and 0.5 mM MgCl2, pH 9.0, at
room temperature for 30-60 min. The plates were read at 405 nm.
The Fg-binding activity of the purified wild-type and mutant
rClfA-(221-559) proteins was analyzed by enzyme-linked immunosorbent assay. The wells in microtiter plates were coated with human Fg (100 ng/well) in PBS for 15 h at 4 °C. The wells were then washed with PBS containing 0.05% Tween 20 (PBS-T) and blocked with 5% (w/v)
BSA in PBS-T at 37 °C for 3 h. After washing the wells with PBS-T, purified recombinant proteins in PBS were added, and the plates
were incubated for 2 h at 37 °C. The wells were then washed with PBS-T, and anti-rClfA-(40-559) polyclonal Abs (diluted 1:2500 in
PBS) were added for 1 h at 37 °C. Following further washing with PBS-T, HRP-conjugated goat anti-rabbit polyclonal Abs (diluted 1:2000 in PBS; Dako Corp.) were added for 1 h at 37 °C.
Finally, bound HRP-conjugated Abs were detected by the addition of 580 mg/ml tetramethylbenzidine and 0.0001% H2O2 in
0.1 M sodium acetate buffer, pH 5.0, at room temperature
for 10 min. The reaction was stopped by the addition of 2 M
H2SO4, and the plates were read at 450 nm.
Fluorescence Polarization--
A peptide composed of the 17 C-terminal residues of the Circular Dichroism Spectroscopy--
Far-UV CD data were
collected with a Jasco J720 spectropolarimeter calibrated with
d10-camphorsulfonic acid employing a band pass
of 1 nm and integrated for 4 s at 0.2-nm intervals. All samples were in 1 mM Tris-HCl, pH 7.4. CD spectra were recorded at
room temperature in cylindrical 0.5-mm path length cuvettes. Twenty scans were averaged for each spectrum, and the contribution from buffer
was subtracted in each case. The mean residue ellipticity, [ Antibodies to the C-terminal Part of the A Region Inhibit the
Interaction of ClfA with Immobilized Fg--
To investigate the role
of the C-terminal part of the A region in the Fg-binding activity of
ClfA, a recombinant GST fusion protein encompassing residues 500-559
of ClfA, rGST-ClfA-(500-559) (Fig. 1),
was expressed and purified on a glutathione-Sepharose column. This
protein failed to bind to immobilized Fg in an enzyme-linked immunosorbent assay (data not shown). Polyclonal Abs were raised against rGST-ClfA-(500-559) and were found to bind to nondenatured rClfA-(221-559) (the minimum Fg-binding truncate of ClfA) (Fig. 1)
(20) on a dot blot (data not shown). The purified
anti-rGST-ClfA-(500-559) polyclonal Abs inhibited the adherence of
S. aureus strain DU5876 to immobilized Fg in a
dose-dependent manner, whereas anti-rGST-ClfB-(45-542) polyclonal Abs had no effect (Fig. 2). As
expected, purified anti-rClfA-(40-559) polyclonal Abs (raised against
a recombinant form of the entire A region of ClfA) (Fig. 1) (20) also
had a potent inhibitory effect on bacterial adherence in this assay
(data not shown).
The anti-rGST-ClfA-(500-559) polyclonal Abs also inhibited the
interaction of rClfA-(40-559) with immobilized Fg in a
dose-dependent manner (Fig.
3). In fact, the inhibitory activity of
these Abs was comparable to that of purified anti-rClfA-(40-559)
polyclonal Abs, whereas preimmune Abs had no inhibitory effect in this
assay (Fig. 3). These results suggest that the region of ClfA spanning residues 500-559 is important for the Fg-binding activity of this protein.
Identification of Residues within the C-terminal Part of the A
Region That Are Important for the Fg-binding Activity of ClfA--
The
role of residues within the C-terminal part of the A region of ClfA in
ligand binding was investigated by a site-directed mutagenesis
approach. Previous studies revealed that rClfA-(221-559) can bind to a
synthetic peptide representing the 17 C-terminal residues of the
To analyze the Fg-binding activity of the mutant ClfA proteins, the
strains were tested for their ability to clump in the presence of
soluble Fg. The bacteria were mixed with 2-fold serial dilutions of Fg
(starting at 1 mg/ml), and the clumping titer was taken as the
reciprocal of the highest dilution of Fg at which cell clumping was
still visible. In this assay, strain Newman, which carries a single
chromosomal copy of the clfA gene, had a clumping titer of
1024, whereas strain DU5941(pCU1), a ClfA-negative mutant of strain
8325-4 carrying the "empty" shuttle plasmid pCU1, failed to clump
(Table II). The clumping titer of
strain DU5941(pCF77), which expresses the wild-type ClfA protein from a
pCU1-derived plasmid, was 2-fold lower than that of wild-type strain
Newman (Table II). Strain DU5941(pClfA(E526A)) displayed a
16-fold reduction in clumping titer compared with strain DU5941(pCF77)
in this assay (Table II). However, the clumping titers of strains
DU5941(pClfA(D537A)), DU5941(pClfA(E546A)), and
DU5941(pClfA(E559A)) were identical to that of strain
DU5941(pCF77).
As Glu526 appeared to be important for the Fg-binding
activity of cell surface-expressed ClfA, we examined the effect of
mutating other residues in the vicinity of Glu526
(i.e. Asn525, Val527,
Ala528, and Gly532) (Fig. 1) on Fg binding.
Strain DU5941(pClfA(V527S)) had a 8-16-fold lower clumping titer than
strain DU5941(pCF77). However, strains DU5941(pClfA(N525A))
and DU5941(pClfA(A528V/G532A)) had clumping titers identical to that of
strain DU5941(pCF77). The most dramatic effect on cell clumping was
observed for strain DU5941(pClfA(E526A/V527S)), which exhibited a
512-fold reduction in clumping titer compared with strain
DU5941(pCF77) (Table II). These results suggest that Glu526 and Val527 are important for the
interaction of cell surface-expressed ClfA with soluble Fg.
Interaction of Cell Surface-expressed Mutant ClfA Proteins with
Immobilized Fg--
The ability of strain DU5941 expressing the
wild-type and mutant ClfA proteins to adhere to immobilized Fg was
analyzed in the wells of a microtiter plate. As anticipated, strain
DU5941(pCU1), which lacks the clfA gene, failed to adhere
significantly to immobilized Fg in this assay (Fig.
4). An ~60% reduction in adherence was observed for both strains DU5941(pClfA(E526A)) and DU5941(pClfA(V527S)) compared with strain DU5941(pCF77). Furthermore, the adherence of
strain DU5941(pClfA(E526A/V527S)) was reduced by 90% compared with
strain DU5941(pCF77). In contrast, strain DU5941 expressing the mutant
ClfA proteins with the substitutions N525A, A528V/G532A, D537A, E546A,
and E559A adhered to immobilized Fg at levels similar to strain
DU5941(pCF77) (Fig. 4). Thus, Glu526 and Val527
also appear to be important for the interaction of cell
surface-expressed ClfA with immobilized Fg.
Interaction of Recombinant Mutant ClfA Proteins with Intact
Fg--
To examine the effects of the E526A, V527S, E526A/V527S, and
A528V/G532A substitutions on ClfA more closely, they were introduced into the minimum Fg-binding recombinant truncate of ClfA,
rClfA-(221-559) (Fig. 1). The purity of the isolated recombinant
His-tagged proteins was verified by SDS-polyacrylamide gel
electrophoresis (data not shown). The ability of each protein to bind
to soluble HRP-conjugated Fg was analyzed in a Western ligand affinity
blot assay. The wild-type protein and the mutant proteins containing
the E526A, V527S, or A528V/G532A substitution bound to soluble Fg in
this assay (data not shown). However, the mutant protein containing the
E526A/V527S substitution failed to bind to soluble Fg (data not shown).
The ability of the wild-type and mutant rClfA-(221-559) proteins to
bind to immobilized Fg was analyzed in an enzyme-linked immunosorbent
assay. Increasing concentrations of the soluble recombinant proteins
were incubated in Fg-coated wells, and bound protein was detected with
anti-rClfA-(40-559) polyclonal Abs, followed by HRP-conjugated goat
anti-rabbit polyclonal Abs. The wild-type protein and the mutant
proteins containing the E526A, V527S, and A528V/G532A substitution
bound to immobilized Fg in a dose-dependent manner in this
assay (Fig. 5). The mutant protein containing the A528V/G532A substitution bound to Fg at a level similar
to the wild-type protein. However, the mutant proteins containing the
E526A and V527S substitutions exhibited a reduced level of binding
compared with the wild-type protein. In fact, when an ~10
nM concentration of each protein was used, the binding of
these two mutant proteins was only 35% of that of the wild-type protein (Fig. 5). Furthermore, the mutant protein containing the E526A/V527S substitution failed to bind significantly to immobilized Fg
in this assay.
Interaction of Recombinant Mutant ClfA Proteins with the Analysis of the Secondary Structures of the Recombinant Mutant ClfA
Proteins by Far-UV CD Spectroscopy--
It is possible that the
introduction of the E526A, V527S, or E526A/V527S substitution into
rClfA-(221-559) altered the secondary structure of the protein, which
might account for the reduction in Fg-binding activity observed for the
mutant proteins. To address this possibility, the secondary structures
of the wild-type and mutant rClfA-(221-559) proteins were analyzed by
far-UV CD spectroscopy. Analysis of the wild-type protein gave a
spectrum with a maximum at 190 nm and a minimum at 215 nm (Fig.
7, A-D, solid
lines), consistent with previous studies (20). The spectrum of
each of the mutant proteins looked almost identical to that of the wild-type protein (Fig. 7, A-D, broken lines).
However, small changes in the intensity of the signals obtained at 190 and ~200 nm were observed for the mutant proteins containing the
E526A, E526A/V527S, and A528V/G532A substitutions compared with the
wild-type protein, the difference being most pronounced for the E526A
substitution (Fig. 7A). Nonetheless, deconvolution of the
spectra revealed that the wild-type and mutant proteins had very
similar secondary structural compositions, dominated by a S. aureus is predominantly an extracellular pathogen
that colonizes the host by adhering to components of the extracellular matrix. This process is mediated by cell surface-expressed protein adhesins termed MSCRAMMs (2, 3). The ability of S. aureus to
adhere to immobilized Fg and fibrin is an important factor in promoting
wound infection, foreign body infection, and endocarditis (35, 36). As
such, the inhibition of this interaction in vivo represents
a viable target for the design of novel agents to prevent S. aureus infections.
To date, four Fg-binding MSCRAMMs have been identified on the surface
of S. aureus, namely ClfA, ClfB, FnbpA, and FnbpB (6-7, 31).3 The Fg-binding A
regions of these proteins share ~20-25% amino acid identity. ClfA
is the prototype Fg-binding MSCRAMM of S. aureus. The
binding site in Fg for this protein has been localized to the extreme C
terminus of the It is conceivable that knowledge of the Fg-binding site of ClfA could
provide valuable insight into the binding site of not only the Fnbp
proteins, but also the platelet integrin
Previously, the C-terminal part of the A region was implicated as being
important for the Fg-binding activity of ClfA (14). It was noted that
an N-terminal truncate of the minimum Fg-binding region of ClfA failed
to bind to Fg, but was capable of absorbing out the inhibitory activity
of polyclonal Abs raised against the entire A region (14). However,
deletion of residues 484-550 from the C terminus of the A region
resulted in a truncate that was not capable of binding to Fg or of
absorbing out the inhibitory Abs. Assuming that the inhibitory Abs
recognize epitopes that overlap or are close to the Fg-binding site,
these observations raise the possibility that at least part of the
Fg-binding site of ClfA is formed by residues 484-550.
In this study, we further investigated the involvement of the
C-terminal part of the A region of ClfA in Fg binding. Polyclonal Abs
were raised against a recombinant protein encompassing residues 500-559 of ClfA (rGST-ClfA-(500-559)). It was found that these Abs
could inhibit the interaction of both S. aureus and soluble recombinant ClfA with immobilized Fg (Figs. 2 and 3, respectively). These observations support the notion that at least part of the Fg-binding site may be contained within residues 500-559 of ClfA. However, we observed that the rGST-ClfA-(500-559) protein was unable
to absorb out the inhibitory activity of Abs raised against the entire
A region (data not shown), suggesting that other parts of the A region
may also participate in forming the Fg-binding site.
To identify specific residues within the C-terminal part of the A
region of ClfA that may be involved in Fg binding, a site-directed mutagenesis approach was employed. Expression of the mutant ClfA proteins on the surface of S. aureus revealed that only two
of the substitutions, E526A and V527S, reduced the ability of the bacteria to bind to soluble and immobilized Fg (Table II and Fig. 4,
respectively). In fact, the bacteria that expressed the mutant ClfA
protein containing the E526A/V527S substitution were almost completely
defective in Fg binding. The importance of these two residues for the
activity of ClfA was further investigated in the context of a
recombinant protein encompassing the minimum Fg-binding region
(residues 221-559) of this MSCRAMM. The recombinant ClfA protein
containing the E526A or V527S substitution exhibited a reduced ability
to bind to both immobilized Fg and a synthetic fluorescein-labeled
C-terminal The question arises as to whether Glu526 and
Val527 are directly involved in binding to Fg or whether
they are important for maintaining the conformation of the
ligand-binding site in ClfA. Analysis of the secondary structures of
the recombinant mutant ClfA proteins by far-UV CD spectroscopy revealed
that the spectra of these proteins were almost identical to that of the
wild-type ClfA protein. This suggests that introduction of the E526A
and V527S substitutions into recombinant ClfA had not resulted in
dramatic alterations in the secondary structure of the protein.
However, the possibility that changes in tertiary structure had
occurred in the mutant proteins cannot be ruled out at this point. The
direct involvement of Glu526 and Val527 in
ligand binding can be verified when recombinant ClfA has been co-crystallized with the As mentioned above, the A regions of FnbpA and FnbpB also bind to the C
terminus of the -chain peptide compared
with the wild-type protein, whereas the double mutant rClfA-(221-559)
protein was almost completely defective in binding to either species. Substitution of Glu526 and/or Val527 did not
appear to alter the secondary structure of rClfA-(221-559) as
determined by far-UV circular dichroism spectroscopy. These data
suggest that the C terminus of the A region may contain at least part
of the Fg-binding site of ClfA and that Glu526 and
Val527 may be involved in ligand recognition.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-, two B
-, and two
-chains) that are arranged in a
symmetrical dimeric structure. A key player in hemostasis, Fg mediates
platelet adherence and aggregation at sites of injury. In addition, it is cleaved by thrombin to form fibrin, which is the major component of
blood clots. Fg is also one of the main proteins deposited on implanted biomaterials.
-chain, a site that is also recognized by the platelet
integrin
IIb
3 (15-19). Indeed,
recombinant ClfA is a potent inhibitor of both Fg-mediated platelet
aggregation and adherence of platelets to immobilized Fg in
vitro (17). As for
IIb
3, the binding
of ClfA to Fg is regulated by divalent cations, including
Ca2+ and Mn2+ (20-22). Both of these cations
inhibit ClfA-mediated clumping of S. aureus in the presence
of soluble Fg and the interaction of recombinant ClfA with a synthetic
fluorescein-labeled C-terminal
-chain peptide (20). Consistent with
this, ClfA has a putative EF-hand motif (residues 310-321) within the
A region that is required both for Ca2+ regulation and
ligand binding (see Fig. 1) (20). Overlapping this putative EF-hand
motif is another motif (YTFTDYV) that occurs in the same position in
the A regions of the other members of the Sdr protein family and also
in the A regions of the fibronectin-binding MSCRAMMs (FnbpA and FnbpB)
of S. aureus (see Fig. 1) (10). However, the function of
this motif is currently unknown.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
spa::TcR) (23). Strain DU5873, a
mutant of strain Newman lacking expression of protein A (strain Newman
spa::TcR) (14) was used for the
antibody inhibition studies. The shuttle plasmid pCU1 (24), which
confers resistance to chloramphenicol in S. aureus and
ampicillin in E. coli, was used to express the wild-type and
mutant ClfA proteins in strain DU5941. Plasmids pQE30 (QIAGEN Inc.) and
pGEX-KG (25) were used for recombinant protein expression.
Synthetic oligonucleotides for amplifying clfA gene fragments from S. aureus strain Newman genomic DNA and for site-directed mutagenesis
of clfA
-chain of Fg was synthesized and labeled
with fluorescein at the N terminus as described previously (20).
Increasing concentrations of the wild-type and mutant rClfA-(221-559)
proteins in 10 mM HEPES, 150 mM NaCl, and 3.4 mM EDTA, pH 7.4, were incubated with 10 nM
labeled peptide for 3 h at room temperature in the dark. Polarization measurements were taken with a Model LS50B luminescence spectrometer using FLWinLab software (both from PerkinElmer Life Sciences). Binding data were analyzed by nonlinear regression used to
fit a binding function as defined by Equation 1,
where
(Eq. 1)
P corresponds to the change in fluorescence
polarization,
Pmax is the maximum change in
fluorescence polarization, and KD is the
dissociation equilibrium constant of the interaction. A single binding
site was assumed in this analysis.
]MRW, was expressed in
degrees·cm2/dmol. Quantification of secondary structural
components was performed using the deconvolution programs SELCON and
VARSLC1. These programs were previously found to be reliable for the
analysis of the collagen-binding MSCRAMM of S. aureus
(34).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Schematic representation of ClfA of S. aureus. S, signal peptide; A,
Fg-binding region (residues 40-559); R, serine-aspartate
dipeptide repeat region (residues 560-876); W, cell
wall-spanning region; M, membrane-spanning region; +,
positively charged tail. The positions of the cell wall-anchoring LPDTG
motif, the putative EF-hand motif, and the conserved motif are
indicated. The recombinant ClfA proteins used in this study are also
illustrated, and the amino acid residues contained within each protein
are indicated in parentheses. The sequence of amino acids 500-559 of
ClfA is also shown, and the residues that were substituted in this
study are underlined.
View larger version (17K):
[in a new window]
Fig. 2.
Inhibition of S. aureus
adherence to immobilized Fg using anti-rGST-ClfA-(500-559)
polyclonal Abs. Strain DU5873 (~5 × 107
colony-forming units) was preincubated with increasing concentrations
of purified anti-rGST-ClfA-(500-559) polyclonal Abs ( ) for 2 h
at room temperature and then added to wells coated with human Fg (500 ng/well). Anti-rGST-ClfB-(45-542) polyclonal Abs (
) were included
as a control. Bacterial adherence was measured using crystal violet
staining as described previously (23). Values are representative of one
experiment. This experiment was performed twice with similar
results.
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Fig. 3.
Inhibition of recombinant ClfA binding to
immobilized Fg using anti-rGST-ClfA-(500-599) polyclonal Abs.
rClfA-(40-559) was preincubated with increasing concentrations of
purified anti-rGST-ClfA-(500-559) polyclonal Abs ( ),
anti-rClfA-(40-559) polyclonal Abs (
), or preimmune Abs (
) for
1 h at room temperature and then added to wells coated with human
Fg (1 µg/well). After incubation for 1 h at room temperature,
bound protein was detected with an anti-His tag monoclonal antibody and
alkaline phosphatase-conjugated goat anti-mouse polyclonal Abs,
followed by development with p-nitrophenyl phosphate
substrate. The plates were read at 405 nm. Values are the means ± S.D. of triplicate wells and are representative of one experiment. This
experiment was performed three times with similar results.
-chain of Fg
(395GEGQQHHLGGAKQAGDV411) and that
modification of the lysine residue (Lys406) in this peptide
inhibits this interaction
(20).2 This raised the
possibility that a complementary acidic residue(s) within the A region
of ClfA may be involved in binding to the
-chain peptide and thus to
intact Fg. To investigate this possibility, we substituted several
acidic residues (Glu526, Asp537,
Glu546, and Glu559) within the C-terminal part
of the A region of ClfA with alanine (Fig. 1). The wild-type and mutant
ClfA proteins were expressed on the surface of S. aureus
strain DU5941 using the multicopy plasmid pCU1. Expression of the
ClfA proteins was verified by Western immunoblot analysis of cell
wall extracts using anti-rClfA-(40-559) polyclonal Abs. As
anticipated, a protein of ~185 kDa was observed in each case (data
not shown). The expression level of each of the ClfA proteins was
compared in a bacterial cell immunoblot assay. Dilutions of the cell
suspensions were pipetted onto nitrocellulose membranes and probed with
anti-rClfA-(40-559) polyclonal Abs, and the highest dilution of cells
at which a positive immunoreaction was still visible was determined in
each case. No difference was observed for strain DU5941 expressing the
wild-type or mutant ClfA proteins in this assay, suggesting that the
proteins are expressed at similar levels on the cell surface (data not shown).
Clumping titers of S. aureus strain DU5941 expressing mutant ClfA
proteins
View larger version (25K):
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Fig. 4.
Bacterial adherence to immobilized Fg.
Suspensions (~1 × 108 colony-forming units) of
S. aureus strain DU5941 expressing wild-type and mutant ClfA
proteins from pCU1 were added to wells coated with human Fg (500 ng/well). Bacterial adherence was measured using crystal violet
staining as described previously (23). Strain DU5941(pCU1), which lacks
expression of ClfA, was included in the assay as a negative control.
Values are the means ± S.D. of triplicate wells and are
representative of one experiment. This experiment was performed twice
with similar results.
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Fig. 5.
Binding of recombinant ClfA proteins to
immobilized Fg. Purified wild-type rClfA-(221-559) protein ( )
and mutant rClfA-(221-559) proteins containing the substitutions E526A
(
), V527S (
), E526A/V527S (
), and A528V/G532A (
)
were incubated in wells coated with human Fg (100 ng/well). Bound
protein was detected by the addition of anti-rClfA-(40-559) polyclonal
Abs and HRP-conjugated goat anti-rabbit polyclonal Abs, followed by a
chromogenic substrate. The plates were read at 450 nm. Background
binding to the blocking agent (5% (w/v) BSA) was subtracted from the
values obtained for the Fg-coated wells. Values are representative of
one experiment. This experiment was performed three times with similar
results.
-Chain
Peptide--
We investigated the ability of the mutant
rClfA-(221-559) proteins to interact with a synthetic
fluorescein-labeled peptide representing the 17 C-terminal residues of
the
-chain of Fg by fluorescence polarization. In this assay, the
wild-type protein bound to the peptide with a KD of
8.5 ± 2.5 µM (Fig. 6), an apparent affinity similar to that
previously reported for this interaction (20). The mutant protein
containing the A528V/G532A substitution bound to the
-chain peptide
with a similar apparent affinity as the wild-type protein
(KD = 4.5 ± 1.4 µM). However,
the mutant proteins containing the E526A and V527S substitutions bound
to the
-chain peptide with an ~10-fold lower apparent affinity (KD = 57.5 ± 10.1 and 52.2 ± 16.2 µM, respectively). The mutant protein containing the
E526A/V527S substitution bound to the
-chain peptide too weakly for
a KD to be determined by this method (Fig. 6).
Thus, as for cell surface-expressed ClfA, Glu526 and
Val527 appear to be important for the interaction of
soluble recombinant ClfA with Fg.
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Fig. 6.
Binding of recombinant ClfA proteins to
synthetic fluorescein-labeled C-terminal
-chain peptide. Increasing
concentrations of the wild-type rClfA-(221-559) protein (
) and the
mutant rClfA-(221-559) proteins containing the E526A (
), V527S
(
), E526A/V527S (
), and A528V/G532A (
) substitutions were
incubated with the fluorescein-labeled
-chain peptide (10 nM) at room temperature for 3 h in the dark. The
interaction of each protein with the peptide was measured under
equilibrium conditions. Values are the means of duplicate reactions and
are representative of one experiment. This experiment was performed
three times with similar results. Equation 1 was used to fit the
binding data. From the three experiments, the KD
values for the interaction of the wild-type rClfA-(221-559) protein
and the mutant rClfA-(221-559) proteins containing the A528V/G532A,
E526A, and V527S substitutions with the
-chain peptide were
calculated to be 8.5 ± 2.5, 4.5 ± 1.4, 57.5 ± 10.1, and 52.2 ± 16.2 µM, respectively. The binding of
the mutant rClfA-(221-559) protein containing the E526A/V527S
substitution to the
-chain peptide was too weak for a
KD to be determined by this method. mP,
millipolarization units.
-sheet and
with a small
-helical component (data not shown).
View larger version (16K):
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Fig. 7.
Structural analysis of recombinant ClfA
proteins. Far-UV CD spectra of the wild-type and mutant
rClfA-(221-559) proteins were generated as described under
"Experimental Procedures." In each panel, the solid line
represents the wild-type protein, and the broken line
represents the mutant protein. A, E526A; B,
V527S; C, E526A/V527S; D, A528V/G532A.
[ ]MRW, mean residue ellipticity;
deg, degrees.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain, a region that extends as a flexible
structure from the globular
-module (15-17, 20, 37). This site is
also recognized by FnbpA and FnbpB, whereas ClfB binds to the
- and
-chains of the Fg molecule (7, 31).3 Interestingly, the
C terminus of the
-chain is also targeted by the platelet integrin
IIb
3 (18, 19). Further extending the
similarity between ClfA and the mammalian integrin, the Fg-binding activity of both of these adhesins is regulated by extracellular Ca2+ and Mn2+ (20-22). Consistent with this,
the
IIb-subunit of the platelet integrin contains four
Ca2+-binding EF-hand motifs, and a single putative
EF-hand motif has been identified within the A region of ClfA
(20, 38). However, despite the functional similarity between ClfA and
IIb
3, these proteins do not share
extensive amino acid identity.
IIb
3. In a previous study, we localized
the Fg-binding activity of ClfA to a stretch of ~330 amino acids
(residues 221-550) within the A 330 region of this protein
(14). Deletion of residues at the N or C terminus of this minimum
Fg-binding truncate resulted in the loss of Fg-binding activity,
suggesting that the conformation of the protein is important for
maintaining the integrity of the binding site (14). In a more recent
study, we focused on the putative EF-hand motif at residues 310-321
within the minimum Fg-binding region of ClfA (Fig. 1) (20).
Site-directed mutagenesis of this motif revealed that it is required
not only for the Ca2+ regulation of the activity of ClfA,
but also for the Fg-binding activity per se of this protein
(20). However, the far-UV CD spectra of the mutant ClfA proteins were
significantly different from that of wild-type ClfA, suggesting that
the mutations had resulted in alterations in the secondary structure of
the protein (20). As such, it is not clear whether the putative EF-hand motif represents a common binding site for Ca2+ and the
-chain of Fg or whether the binding of Ca2+ to this
motif regulates the conformation (and activity) of a distinct binding
site within ClfA. Consistent with the second possibility, the binding
of Ca2+ to the A region of ClfA results in structural
changes, as determined by far-UV CD spectroscopy (20).
-chain peptide (Figs. 5 and 6, respectively).
Furthermore, the recombinant ClfA protein containing the E526A/V527S
substitution was completely defective in binding to immobilized Fg and
to the
-chain peptide. These results suggest that the adjacent
residues Glu526 and Val527 are important for
the Fg-binding activity of ClfA.
-chain peptide and the structure of this
complex has been solved (39).
-chain of Fg (31).3 Like ClfA,
preliminary studies have revealed that the C-terminal part of the A
regions of the Fnbp proteins is also important for Fg
binding.3 Alignment of the amino acid sequences of the
three proteins revealed that Val527 in ClfA is also present
in FnbpB and is replaced by leucine in FnbpA, which is a conservative
substitution. However, Glu526 in ClfA is not conserved in
the Fnbp proteins, being replaced by glycine in both proteins. Whether
these residues are important for the Fg-binding activity of the Fnbp
proteins remains to be determined. It is conceivable that differences
in the residues involved in Fg binding in ClfA, FnbpA, and FnbpB could
reflect differences in the specificity of these proteins for residues in the
-chain. This possibility is currently under investigation in
our laboratories.
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FOOTNOTES |
---|
* This work was supported by Wellcome Trust Grant 052320 (to T. J. F), National Institutes of Health Grant AI20624 (to M. H.), and Inhibitex Inc.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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
353-1-6082014; Fax: 353-1-6799294; E-mail: tfoster@tcd.ie.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M007979200
2 D. P. O'Connell, S. Gurusiddappa, T. J. Foster, and M. Höök, unpublished observations.
3 E. R. Wann and M. Höök, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: MSCRAMMs, microbial surface components recognizing adhesive matrix molecules; Fg, fibrinogen; ClfA, clumping factor A; rClfA, recombinant ClfA; Abs, antibodies; PCR, polymerase chain reaction; GST, glutathione S-transferase; TBS, Tris-buffered saline; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
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