Identification of Residues in the Staphylococcus aureus Fibrinogen-binding MSCRAMM Clumping Factor A (ClfA) That Are Important for Ligand Binding*

Orla M. HartfordDagger §, Elisabeth R. Wann§, Magnus Höök, and Timothy J. FosterDagger ||

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma -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

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 Aalpha -, two Bbeta -, and two gamma -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.

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 gamma -chain, a site that is also recognized by the platelet integrin alpha IIbbeta 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 alpha IIbbeta 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 gamma -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.

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Delta spa::TcR) (23). Strain DU5873, a mutant of strain Newman lacking expression of protein A (strain Newman Delta 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.

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.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Synthetic oligonucleotides for amplifying clfA gene fragments from S. aureus strain Newman genomic DNA and for site-directed mutagenesis of clfA
Restriction endonuclease sites are underlined.

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 gamma -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,


&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 change in fluorescence polarization, and KD is the dissociation equilibrium constant of the interaction. A single binding site was assumed in this analysis.

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, [Theta ]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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).



View larger version (20K):
[in this window]
[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 this window]
[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 (open circle ) 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.

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.



View larger version (19K):
[in this window]
[in a new window]
 
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 (open circle ) 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.

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 gamma -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 gamma -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).

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).


                              
View this table:
[in this window]
[in a new window]
 
Table II
Clumping titers of S. aureus strain DU5941 expressing mutant ClfA proteins
2-Fold serial dilutions of Fg were made in PBS starting at 1 mg/ml. Approximately 4 × 108 colony-forming units (in 20 µl) were added to 50 µl of each Fg dilution. After 5 min of vigorous shaking, the clumping titer was defined as the reciprocal of the highest dilution of Fg at which clumping was still visible.

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.



View larger version (25K):
[in this window]
[in a new window]
 
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.

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.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Binding of recombinant ClfA proteins to immobilized Fg. Purified wild-type rClfA-(221-559) protein (open circle ) and mutant rClfA-(221-559) proteins containing the substitutions E526A (), V527S (), E526A/V527S (black-square), and A528V/G532A (black-triangle) 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.

Interaction of Recombinant Mutant ClfA Proteins with the gamma -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 gamma -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 gamma -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 gamma -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 gamma -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.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6.   Binding of recombinant ClfA proteins to synthetic fluorescein-labeled C-terminal gamma -chain peptide. Increasing concentrations of the wild-type rClfA-(221-559) protein (open circle ) and the mutant rClfA-(221-559) proteins containing the E526A (), V527S (), E526A/V527S (black-square), and A528V/G532A (black-triangle) substitutions were incubated with the fluorescein-labeled gamma -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 gamma -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 gamma -chain peptide was too weak for a KD to be determined by this method. mP, millipolarization units.

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 beta -sheet and with a small alpha -helical component (data not shown).



View larger version (16K):
[in this window]
[in a new window]
 
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. [Theta ]MRW, mean residue ellipticity; deg, degrees.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma -chain, a region that extends as a flexible structure from the globular gamma -module (15-17, 20, 37). This site is also recognized by FnbpA and FnbpB, whereas ClfB binds to the alpha - and beta -chains of the Fg molecule (7, 31).3 Interestingly, the C terminus of the gamma -chain is also targeted by the platelet integrin alpha IIbbeta 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 alpha 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 alpha IIbbeta 3, these proteins do not share extensive amino acid identity.

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 alpha IIbbeta 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 gamma -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).

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 gamma -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 gamma -chain peptide. These results suggest that the adjacent residues Glu526 and Val527 are important for the Fg-binding activity of ClfA.

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 gamma -chain peptide and the structure of this complex has been solved (39).

As mentioned above, the A regions of FnbpA and FnbpB also bind to the C terminus of the gamma -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 gamma -chain. This possibility is currently under investigation in our laboratories.


    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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Waldvogel, F. A. (1995) in Principles and Practice of Infectious Diseases (Mandel, G. L. , Bennett, J. E. , and Dolin, R., eds), 4th Ed. , pp. 1754-1777, Churchill-Livingstone, Inc., New York
2. Patti, J. M., Allen, B. L., McGavin, M. J., and Höök, M. (1994) Annu. Rev. Microbiol. 48, 585-617[CrossRef][Medline] [Order article via Infotrieve]
3. Foster, T. J., and Höök, M. (1998) Trends Microbiol. 6, 461-501[CrossRef][Medline] [Order article via Infotrieve]
4. Switalski, L. M., Speziale, P., and Höök, M. (1989) J. Biol. Chem. 264, 21080-21086[Abstract/Free Full Text]
5. Tung, H., Guss, B., Hellman, U., Persson, L., Rubin, K., and Ryden, C. (2000) Biochem. J. 345, 611-619[CrossRef][Medline] [Order article via Infotrieve]
6. McDevitt, D., François, P., Vaudaux, P., and Foster, T. J. (1994) Mol. Microbiol. 11, 237-248[Medline] [Order article via Infotrieve]
7. Ní Eidhin, D., Perkins, S., François, P., Vaudaux, P., Höök, M., and Foster, T. J. (1998) Mol. Microbiol. 30, 245-257[CrossRef][Medline] [Order article via Infotrieve]
8. Signas, C., Raucci, G., Jönsson, K., Lindgren, P. E., Anantharamaiah, G. M., Höök, M., and Lindberg, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 699-703[Abstract]
9. Jönsson, K., Signas, C., Müller, H. P., and Lindberg, M. (1991) Eur. J. Biochem. 202, 1041-1048[Abstract]
10. Josefsson, E., McCrea, K. W., Ní, Eidhin, D., O'Connell, D., Cox, J., Höök, M., and Foster, T. J. (1998) Microbiology (Read.) 144, 3387-3395[Abstract]
11. McCrea, K. W., Hartford, O., Davis, S., Ní, Eidhin, D., Lina, G., Speziale, P., Foster, T. J., and Höök, M. (2000) Microbiology (Read.) 146, 1535-1546[Abstract/Free Full Text]
12. Schneewind, O., Fowler, A., and Faull, K. F. (1995) Science 268, 103-106[Medline] [Order article via Infotrieve]
13. Mazmanian, S. K., Liu, G., Ton-That, H., and Schneewind, O. (1999) Science 285, 760-763[Abstract/Free Full Text]
14. McDevitt, D., François, P., Vaudaux, P., and Foster, T. J. (1995) Mol. Microbiol. 16, 895-907[Medline] [Order article via Infotrieve]
15. Hawiger, J., Timmons, S., Strong, D. D., Cottrell, B. A., Riley, M., and Doolittle, R. F. (1982) Biochemistry 21, 1407-1413[Medline] [Order article via Infotrieve]
16. Strong, D. D., Laudano, A. P., Hawiger, J., and Doolittle, R. F. (1982) Biochemistry 21, 1414-1420[Medline] [Order article via Infotrieve]
17. McDevitt, D., Nanavaty, T., House-Pompeo, K., Bell, E., Turner, N., McIntire, L., Foster, T. J., and Höök, M. (1997) Eur. J. Biochem. 247, 416-424[Abstract]
18. Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D. D., and Dolittle, R. F. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2068-2071[Abstract]
19. Kloczewiak, M., Timmons, S., Lukas, T., and Hawiger, J. (1984) Biochemistry 23, 1767-1774[Medline] [Order article via Infotrieve]
20. O'Connell, D. P., Nanavaty, T., McDevitt, D., Gurusiddappa, S., Höök, M., and Foster, T. J. (1998) J. Biol. Chem. 273, 6821-6829[Abstract/Free Full Text]
21. Kirchhofer, D., Gailit, J., Ruoslahti, E., Grzesiak, J., and Pierschbacher, M. D. (1990) J. Biol. Chem. 265, 18525-18530[Abstract/Free Full Text]
22. Smith, J. W., Piotrowicz, R. S., and Mathis, D. (1994) J. Biol. Chem. 269, 960-967[Abstract/Free Full Text]
23. Hartford, O., François, P., Vaudaux, P., and Foster, T. J. (1997) Mol. Microbiol. 25, 1065-1076[Medline] [Order article via Infotrieve]
24. Augustin, J., and Gotz, F. (1990) FEMS Microbiol. Lett. 54, 203-207[Medline] [Order article via Infotrieve]
25. Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192, 262-267[Medline] [Order article via Infotrieve]
26. Miller, J. H. (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
27. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
28. Oskouian, B., and Stewart, G. C. (1990) J. Bacteriol. 172, 3804-3812[Medline] [Order article via Infotrieve]
29. Schenck, S., and Laddaga, R. A. (1992) FEMS Microbiol. Lett. 73, 133-138[Medline] [Order article via Infotrieve]
30. Foster, T. J. (1998) Methods Microbiol. 27, 433-454
31. Wann, E. R., Gurusiddappa, S., and Höök, M. (2000) J. Biol. Chem. 275, 13863-13871[Abstract/Free Full Text]
32. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
33. Hartford, O., McDevitt, D., and Foster, T. J. (1999) Microbiology (Read.) 145, 2497-2505[Abstract/Free Full Text]
34. Rich, R. L., Demeler, B., Ashby, K., Deivanayagam, C. C. S., Petrich, J. W., Patti, J. M., Narayana, S. V. L., and Höök, M. (1998) Biochemistry 37, 15423-15433[CrossRef][Medline] [Order article via Infotrieve]
35. Vaudaux, P., Pittet, D., Haeberli, A., Lerch, P. G., Morgenthaler, J. J., Proctor, R. A., Waldvogel, F. A., and Lew, D. P. (1993) J. Infect. Dis. 167, 633-641[Medline] [Order article via Infotrieve]
36. Moreillon, P., Entenza, J. M., Franciolo, P., McDevitt, D., Foster, T. J., François, P., and Vaudaux, P. (1995) Infect. Immun. 63, 4738-4743[Abstract]
37. Spraggon, G., Applegate, D., Everse, S. J., Zhang, J. Z., Veerapandian, L., Redman, C., Doolittle, R. F., and Grieninger, G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9099-9104[Abstract/Free Full Text]
38. Gulino, D., Boudignon, C., Zhang, L., Concord, E., Rabiet, M.-J., and Marguerie, G. (1992) J. Biol. Chem. 267, 1001-1007[Abstract/Free Full Text]
39. Deivanayagam, C. C. S., Perkins, S., Danthuluri, S., Owens, R. T., Bice, T., Nanavathy, T., Foster, T. J., Höök, M., and Narayana, S. V. L. (1999) Acta Crystallogr. 55, 554-556[CrossRef]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.