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J. Biol. Chem., Vol. 276, Issue 32, 29969-29978, August 10, 2001

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Loss of Clumping Factor B Fibrinogen Binding Activity by Staphylococcus aureus Involves Cessation of Transcription, Shedding and Cleavage by Metalloprotease^*

Fionnuala M. McAleese, Evelyn J. Walsh, Magdalena Sieprawska^§, Jan Potempa^§, and Timothy J. Foster^¶

From the  Microbiology Department, Moyne Institute for Preventive Medicine, Trinity College, Dublin 2, Ireland and the ^§ Microbiology Department, Institute of Molecular Biology, Jagiellonian University, 31-120 Krakow, Poland

Received for publication, March 16, 2001, and in revised form, June 4, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The fibrinogen-binding protein clumping factor B (ClfB) of Staphylococcus aureus is present on the surface of cells from the early exponential phase of growth in greater amounts than on cells from late exponential phase and is barely detectable on cells from stationary phase. Expression of a clfB-lacZ fusion indicated that transcription stopped before the end of exponential phase. Mutations in the global regulators agr and sar had no effect on clfB transcription. The loss of ClfB protein from cells in stationary phase was due to expression ending before cells stopped growing, combined with shedding of some of the protein into the growth medium and dilution of those molecules remaining on the cell surface during the two to three cell division events leading to stationary phase. Two forms of the protein occurred on the cell surface, the smaller of which was generated by loss of a domain from the N terminus. The proportion of the smaller form increased as the cultures grew. The metalloprotease aureolysin was shown to be responsible for cleavage of ClfB. Cleavage was inhibited by EDTA and o-phenanthroline and did not occur in an aureolysin-deficient mutant. Purified aureolysin promoted cleavage of cell surface-located ClfB as well as the recombinant A domain of ClfB. Cleavage was detected at two sites, one located between residues Ser¹⁹⁷ and Leu¹⁹⁸ and the other between Ala¹⁹⁹ and Val²⁰⁰. The truncated form of ClfB did not bind fibrinogen.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Staphylococccus aureus is an important human pathogen causing both community-acquired and nosocomial infections (1, 2). Nosocomial infections associated with indwelling medical devices or surgical wounds can lead to more serious invasive diseases such as endocarditis, osteomyelitis, and septicemia. Infection is dependent upon the ability of the bacteria to adhere to the host extracellular matrix or to the layer of proteins coating implanted biomaterial such as catheters and prostheses (3, 4). S. aureus expresses distinct surface proteins, which can bind to host fibrinogen, fibronectin, collagen, and von Willebrand factor, thus enabling the bacteria to colonize and establish a focus of infection (3-6). These proteins belong to the MSCRAMM family of surface proteins (for microbial surface components recognizing adhesive matrix molecules) (4). In addition, fibronectin-binding proteins can also bind to fibrinogen and may promote bacterial adhesion to immobilized fibrinogen (7).

S. aureus expresses two related surface-associated fibrinogen-binding proteins, the clumping factors ClfA and ClfB (8-10). These proteins are covalently attached to the cell wall and mediate adherence of bacteria to immobilized fibrinogen, to blood clots, to conditioned biomaterial ex vivo, and to thrombi on damaged heart valves in a rat model of endocarditis (8, 11-14). The proteins also promote clumping of bacteria in the presence of soluble fibrinogen (8, 10).

In strain Newman, the clfB gene encodes a protein of 913 residues and was reported to migrate in SDS-PAGE¹ gels with an apparent molecular mass of 124 kDa (10). ClfA and ClfB have a domain organization characteristic of many surface proteins of Gram-positive bacteria (15) (Fig. 1). At the N terminus a signal sequence directs the protein across the cytoplasmic membrane. Region A is the surface-exposed ligand-binding domain (10). Region R is composed of serine-aspartate dipeptide repeats and functions as a stalk to display the ligand-binding A domain on the cell surface (16). The C terminus has features common to many cell wall-anchored proteins of Gram-positive bacteria, including an LPXTG motif (LPETG) responsible for covalently linking the protein to peptidoglycan, a hydrophobic domain, and positively charged residues at the extreme C terminus.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the structure of ClfB. S, signal sequence; A, ligand binding domain; R, serine-aspartate repeat region; W, wall-spanning region; M, membrane-spanning domain; +, positively charged residues at the extreme C terminus. The SLAVA motif is a metalloprotease cleavage site. The shaded area located between region A and region R represents a short proline-rich region.

The A regions of ClfA and ClfB have only 26% residue identity and promote binding to different parts of fibrinogen. ClfA binds to the extreme C terminus of the -chain (17, 18), whereas ClfB binds to the -chain (10).² Region A of ClfB contains a short motif SLAVA, a putative protease recognition sequence at which recombinant protein expressed in Escherichia coli is cleaved. A similar motif SLAAVA is present in ClfA (8).

In contrast to ClfA, which is present on cells at all stages of the growth cycle, ClfB is only present at a detectable and functional level on the surface of cells of strain Newman in the exponential phase of growth (10). This suggests that transcription of the clfB gene ceases during exponential phase and that the surface protein may be degraded by proteases or be shed into the growth medium. Transcription of the spa, fnb, and cna genes, encoding protein A, the fibronectin-binding proteins, and the collagen-binding adhesin, respectively, occurs only in exponential phase (19, 20), but in most cases sufficient protein remains on the surface of cells in the stationary phase to promote ligand binding.

The genes encoding surface proteins and extracellular proteins are subjected to control at the transcriptional level by the Agr and Sar global regulators (21, 22). The Sar protein represses transcription of the collagen binding protein independently of Agr (23), whereas expression of fibronectin binding protein A gene is activated by Sar in exponential phase and is repressed by Agr in stationary phase (20). Sar is also a repressor of the extracellular protease genes because sar mutants express much higher levels of several proteases (24).

S. aureus can secrete several proteases including the metalloprotease aureolysin, and several cysteine and serine proteases (25). The metalloprotease is expressed throughout the growth cycle, whereas the other proteases are expressed only in the stationary phase (25, 26). The functions of the proteases are poorly understood, but there is evidence that they can cleave both bacterial and host proteins. Metalloprotease activates the serine protease proenzyme by removing an N-terminal domain (27). It also activates prothrombin giving pseudocoagulase activity (28), and it cleaves mammalian plasma proteinase inhibitors (29, 30).

In this paper we have analyzed the transcriptional activity of the clfB gene promoter and the level of ClfB protein expressed on the bacterial cell surface at different stages of the growth cycle. The surface-associated fibrinogen-binding ClfB protein was cleaved to a smaller non-functional form by removal of an N-terminal domain. We provide evidence that ClfB is cleaved by the metalloprotease aureolysin. Cessation of transcription of clfB late in exponential phase, as well as a combination of inactivation of ClfB protein by protease, shedding of molecules into the growth medium, and dilution of the remaining functional surface protein as the cells enter stationary phase, explain the inability of bacteria from stationary phase to bind to fibrinogen in a ClfB-dependent manner.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Bacterial Strains and Growth Conditions-- The strains and plasmids used in this study are listed in Table I. E. coli was routinely grown on L-broth or agar and S. aureus on trypticase soy broth (Oxoid) or agar. For optimal expression of ClfB, bacteria were grown to an A₆₀₀ of 0.4 (exponential phase) or to 8-12 (stationary phase), in 50 ml of brain-heart infusion broth (BHI), in a 250-ml conical flask shaken at 200 rpm at 37 °C, as described previously (10). The following antibiotics were incorporated into the media when appropriate: ampicillin, 100 µg/ml; tetracycline, 2 µg/ml; kanamycin, 100 µg/ml; erythromycin, 5 or 10 µg/ml; chloramphenicol, 5 or 10 µg/ml. For zymography, cultures were grown in 20 ml of trypticase soy broth in 100-ml bottles with shaking at 160 rpm for 24 h.

DNA Manipulations-- DNA manipulations were performed using standard methods (31, 32). Enzymes were obtained from Roche Molecular Biochemicals or Promega and used as directed by the manufacturer. Genomic DNA was isolated from a 2-ml overnight culture of S. aureus using the AGTC^® bacterial genomic DNA purification kit (Edge BioSystems) adapted for use for staphylococci by the incorporation of lysostaphin (0.01 mg/ml final concentration; Ambicin L recombinant lysostaphin, Applied Microbiology). E. coli plasmid DNA was purified using a Wizard Mini-prep kit (Promega) and the Qiagen Plasmid Midi-prep kit (Qiagen Inc.).

Transduction and Electrotransformation-- Transduction in S. aureus was performed using bacteriophage 85 (33). Plasmids were transformed into E. coli cells made competent by CaCl₂ treatment (32) or electroporated into S. aureus strain RN4220 (33, 34).

Construction of clfB::lacZ and aur::lacZ Gene Fusions-- An 803-bp fragment from within the region A of clfB was amplified from pCU1-clfB⁺. The oligonucleotides were as follows: forward (5'-CGCGGATCCCCAGAACAATCGAACGATACAACG-3'), and reverse (5'-CGGGAATTCTTAATAATCTGTAAAGACAAATGTATACG-3'). Cleavage sites for BamHI or EcoRI (underlined) were incorporated in the 5' ends to facilitate directional cloning. PCR amplifications were carried out in a DNA thermal cycler (PerkinElmer Life Sciences). Reaction mixtures (100 µl) contained 250 µM dNTPs, 1.5 mM MgCl₂, 10 ng of pCU1-clfB⁺, 100 pM primers, and 5 units of Pfu polymerase in standard Promega Pfu reaction buffer. Amplification conditions were carried out with a 45-s denaturation step at 95 °C, a 45-s annealing step at 62 °C, and elongation at 72 °C for 2 min. The cycle was repeated 30 times, followed by incubation at 72 °C for 10 min. The single PCR product was purified using High Pure^TM PCR product purification kit (Roche Molecular Biochemicals) and cloned into pAZ106 (35), which carries a promoterless lacZ gene engineered for translation in Gram-positive bacteria. This yielded the `clfB'-lacZ plasmid pFMA4. This plasmid (10-50 µg DNA) was transformed into S. aureus RN4220 by electroporation, and erythromycin-resistant recombinants were selected. Since the plasmid cannot replicate in S. aureus, Em^r transformants can only occur if the plasmid integrates into the chromosome at the site of shared homology in clfB creating a clfB-lacZ transcriptional fusion. The clfB::lacZ fusion was subsequently transduced into Newman and 8325-4 wild type, agr, and sar strains using phage 85 and selecting for resistance to erythromycin (5 µg/ml). Strains bearing the clfB-lacZ fusion formed pale blue colonies when streaked on trypticase soy agar containing 5-bromo-4-chloro-3-indolyl--D-galactoside (40 µg/ml), indicating a functional lacZ gene.

The aur::lacZ chromosomal mutation was isolated by the same strategy. A 1148-bp PCR fragment internal to the aur gene was amplified with the following primers: forward (5'-CGCGGATCCCAAGATATGCATTTACAAGTATGG-3') and reverse (5'-CGGGAATTCCATCTACAAAGTATCCAAAAACATC-3') and cloned into pAZ106 forming pFMA5.

Southern Hybridization-- DNA hybridization was performed to check the integrity of the clfB::lacZ fusion. The 803-bp PCR fragment of the clfB A region described above was gel-purified and used as a template for PCR with the same primers using a PCR digoxigenin-labeling mix (Roche Molecular Biochemicals) to form a DIG-labeled clfB probe. Genomic DNA was digested to completion using XbaI and BamHI and processed for Southern hybridization (31, 32). Prehybridization, hybridization, and washing of the membrane were performed as described in the Roche Molecular Biochemicals DIG users' handbook. Filters were developed with anti-DIG-alkaline phosphatase conjugate and CSPD^® (Roche Molecular Biochemicals) and exposed to x-ray film at room temperature. Similarly, HindIII-cut genomic DNA was probed with a DIG-labeled aur gene fragment in order to verify the aur-lacZ mutant.

Measurement of -Galactosidase Activity-- Strains bearing the clfB-lacZ fusion were grown for 16 h in BHI (50 ml), washed twice in PBS, diluted 1:100 into 50 ml of pre-warmed BHI in 250-ml conical flasks, and grown at 37 °C at 200 rpm. Cells were assayed for -galactosidase activity using 4-methylumbelliferyl -D-galactopyranoside (MUG) as a substrate based on the method of Youngman (36). Briefly, cells (0.5 ml) were harvested by centrifugation (14,000 × g, 5 min) at intervals, the supernatant was removed, and pellets were snap-frozen at 70 °C. Cells were thawed and resuspended in 0.5 ml of ABT buffer (100 mM NaCl, 60 mM K₂HPO₄, 40 mM KH₂PO₄, 0.1% Triton 100). MUG (50 µl, 4 mg/ml) was added, and the cells were incubated at 25 °C for 60 min. The reaction was stopped by addition of 0.5 ml of 0.4 M Na₂CO₃. Samples were diluted 1:10 in ABTN (1 ml of ABT + 1 ml of 0.4 M Na₂CO₃), and 250 µl/well was added to a 96-well plate (Porvair). Dilutions were made depending on the levels of expression. -Galactosidase activity was determined by fluorescence using a Perkin Elmer LS50B luminescence spectrometer. A range of concentrations of 4-methylumbelliferone (Sigma) were used to generate a standard curve.

Expression and Purification of Recombinant Proteins-- Construction of pQE30 plasmids expressing recombinant domains of ClfB are described in Footnote 2. Recombinant ClfB proteins were expressed and purified as described previously for ClfA (18).

SDS-PAGE, Western Immunoblotting, and Zymography-- S. aureus cells were suspended to an A₆₀₀ of 40 in 30% raffinose plus 20 mM MgCl₂ in a final volume of 100 µl. To each sample, 12 µl of lysostaphin (2 mg/ml) and 8 µl of protease inhibitors (Complete mixture, Roche Molecular Biochemicals) were added and the suspension incubated at 37 °C for 20 min. Protoplasts were removed by centrifugation at 12,000 × g for 10 min. Supernatants containing wall-associated proteins were boiled for 5 min in an equal volume of final sample buffer (0.125 M Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) -mercaptoethanol, and 0.002% (w/v) bromphenol blue). SDS-PAGE was performed by standard methods (37). Gels were stained with Coomassie Blue or electrophoretically transferred to polyvinylidene difluoride Western blotting membranes (Roche Molecular Biochemicals) by the wet system (Bio-Rad). Membranes were incubated overnight at 4 °C in 10% blocking reagent (Marvel milk powder). Antibodies to rClfB44-196 were raised in young New Zealand White rabbits (2 kg) whose pre-immune sera showed no reaction with S. aureus wall-associated antigens in Western blots. Production of anti-rClfB region A (44) antibodies was described previously (10). Primary anti-ClfB A region antibody (10) was used at a dilution of 1:5,000 and the anti-rClfB44-196 antibody at a 1:500 dilution for a 1-h incubation at room temperature. Protein A-conjugated horseradish peroxidase (Sigma: a 1 mg/ml stock diluted 1:500) was used to detect bound antibody by incubation for 1 h at room temperature. Membranes were developed using LumiGLO chemiluminescent substrate (New England Biolabs), according to manufacturer's instructions and exposed to x-ray film.

For zymography analysis, samples of culture supernatants were treated with SDS buffer (4% SDS, 20% glycerol, 0.125 M Tris-HCl, pH 6.8, for 30 min at 37 °C and electrophoresed in 12% SDS-polyacrylamide gels with gelatin (0.1 mg/ml; Difco) incorporated into the gel (38).

Measurement of Protein in Growth Medium-- A volume of culture that contained the number of cells required to give an A₆₀₀ of 40 when concentrated to 1 ml was sampled at different time points. The supernatant was treated with 0.1 volume of 100% trichloroacetic acid for 1 h on ice. The samples were centrifuged for 20 min at 14,000 × g at 4 °C. The pellet was washed with 1 ml of acetone, dried at room temperature for 30 min, dissolved in 60 µl of final sample buffer, and boiled for 5 min. The bacterial cells were washed twice in PBS and resuspended in 30% raffinose, and cell wall proteins were released as described above. The solubilized wall proteins were precipitated in 1 ml of acetone and concentrated as above. 20 µl of both wall proteins and supernatant proteins were analyzed by SDS-PAGE.

Adherence of Bacterial Cells to Immobilized Fibrinogen-- Adherence of S. aureus to immobilized fibrinogen was performed as described previously (39, 40). Briefly, microtiter plates were coated with 5 µg/ml fibrinogen (Calbiochem) in coating solution (0.02% sodium carbonate buffer, pH 9.6) and incubated overnight at 4 °C. Bovine serum albumin (2 mg/ml) was added, and the plates were incubated for 1 h at 37 °C. The plates were washed four times with PBS, and 100 µl of a bacterial cell suspension (1 × 10⁸ colony-forming units/ml) was added. The plates were incubated at 37 °C for 2 h. Plates were subsequently washed four times with PBS, and bound cells were fixed with formaldehyde (25% v/v) for 30 min and then stained with crystal violet (0.5% v/v) for 1 min. Absorbance was measured at 570 nm in an enzyme-linked immunosorbent assay plate reader (Labsystems Multiskan Plus).

Preparation of Concentrated Culture Supernatants-- Newman sar was grown in 50 ml of BHI in a 250-ml flask with shaking at 37 °C for 16 h at 200 rpm. Cells were pelleted by centrifugation at 10,000 × g for 10 min. The supernatants were concentrated 10-fold in a stirred cell ultrafiltration chamber (Amicon, Beverly, MA) equipped with a 10,000 molecular-mass cut-off membrane, and filter-sterilized. The supernatant was added to S. aureus cells, which had been grown to exponential phase, washed in PBS, and resuspended in one-fifth the volume of PBS. Chloramphenicol (20 µg/ml) was added to prevent growth and protein synthesis, and some samples contained a final concentration of 1 mM EDTA or 10 mM o-phenanthroline (Sigma). A 0.1 volume of the concentrated culture supernatant was added to the cells and incubated at 37 °C for 1.5 h.

Recombinant proteins rClfB44-542 and rClfB197-542 (1 mg/ml) were incubated with 80 µl of concentrated culture supernatant, with or without the addition of EDTA (1 mM), for 1.5 h at 37 °C. The rClfB197-542 sample was then passed through a Ni²⁺ activated Hi-trap column (5 ml, Amersham Pharmacia Biotech). Unbound protein (1-ml fractions) was collected, and bound protein was eluted with 500 mM NaCl in 20 mM Tris (pH 7.9).

Proteolytic Cleavage and N-terminal Sequencing of Recombinant ClfB-- Recombinant ClfB197-542 (1 mg/ml) was incubated with concentrated supernatant as described above and passed through a Ni²⁺-activated Hi-trap column. Unbound protein was collected and concentrated. Purified metalloprotease (0.9 µg) was added to 90 µg of rClfC44-542 or rClfB197-452 and incubated at 37 °C for 90 min. After SDS-PAGE, samples were transferred to polyvinylidene difluoride membranes using a Bio-Rad semidry blotter. N-terminal sequencing was performed by Edman degradation at the Department of Biochemistry, University of Cambridge (Cambridge, United Kingdom).

Protease Assay-- Protease activity of culture supernatants was measured using a protease assay kit (Calbiochem) with fluorescein thiocarbamoyl-casein as a substrate and measured by reading the absorbance at 492 nm.

Analysis of Fibrinogen Binding Activity of Recombinant Proteins-- The ability of recombinant proteins to bind to immobilized fibrinogen was analyzed using an enzyme-linked immunosorbent assay-like assay. Microtiter plates were coated with fibrinogen (10 µg/ml in PBS) overnight at 4 °C. The plates were washed three times with PBS and blocked with 5% bovine serum albumin for 2 h at 37 °C. After an additional three washes with PBS, recombinant protein (0.66-2 µmol) was added and incubated at 37 °C for 2 h. The wells were washed again and incubated with anti-ClfB region A antiserum diluted 1:5,000 in PBS, at 37 °C for 1 h. After further washing, horseradish peroxidase-labeled goat anti-rabbit IgG (Sigma) was added at a 1:2,000 dilution. Following incubation at 37 °C for 1 h and washing with PBS, 100 µl of chromogenic substrate (580 µg/ml tetramethylbenzidine and 0.0001% H₂O₂ in 0.1 M sodium acetate buffer (pH 5.2)) was added per well and developed for 10 min in the dark. The reaction was stopped by the addition of 2 M H₂SO₄ (50 µl/well). Plates were read at 450 nm.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression of ClfB on the Cell Surface-- It has been shown previously (10) that the ClfB protein of strain Newman is only present at detectable levels on the surface of cells growing in the exponential phase. Stationary phase cells lacked detectable ClfB protein and could not interact with fibrinogen in a ClfB-dependent manner. Using antibodies raised against purified recombinant region A of ClfB, we measured the ClfB protein present on the cell wall of strains Newman ClfA and 8325-4 ClfA at different stages of the growth cycle. Throughout the exponential phase of growth, strain Newman expressed a major immunoreactive protein of 150 kDa and a minor component of ~120 kDa (Fig. 2A), while strain 8325-4 expressed proteins of ~140 and 110 kDa (Fig. 2B) in proportions that varied according to the stage of growth. In Newman, the higher molecular mass protein predominated on both exponential and stationary phase cells, whereas, in 8325-4, the higher form predominated on cells from early exponential phase while only the smaller protein was detectable on cells from stationary phase. We postulate that this reduction in size of ClfB is due to a proteolytic cleavage event, which removes an N-terminal domain.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Time course of ClfB expression in Newman ClfA (A) and 8325-4 ClfA (B). The upper inset shows ClfB protein released from samples obtained from the first nine time points of the growth curve. Cells were adjusted to the same absorbance, and cell wall-associated proteins released by lysostaphin in the presence of raffinose. The same volume from each sample was prepared for SDS-PAGE. ClfB protein was detected by Western immunoblotting with anti-ClfB antibodies. The lower inset shows adherence to immobilized fibrinogen of bacterial cells from the same nine time points as in the upper inset. The arrow indicates the peak of transcription detected with clfB-lacZ in Fig. 3.

The difference in sizes of the two immunoreactive forms of ClfB expressed by Newman and 8325-4 is probably due to the different lengths of the R regions. The region R-encoding part of the Newman clfB gene is 832 bp, whereas that of 8325-4 is 660 bp. In both strains the level of ClfB protein was highest on cells in the mid-exponential phase of growth, decreased as cells entered stationary phase (Fig. 2), and was virtually undetectable on cells from late stationary phase. Similarly, ClfB-promoted adherence was highest for cells from early exponential phase and declined as cells progressed to stationary phase. With both strains, loss of binding correlated with a reduction in the level of the ClfB protein (Fig. 2). It should be noted that ClfB is the only factor promoting adherence to fibrinogen in these experiments. The strains carry a mutation in the clfA gene, and expression of the fibronectin-binding proteins FnBPA and FnBPB (which can also bind fibrinogen; Ref. 7) occurs at such low levels that they do not contribute significantly. This is indicated by very low adherence of a ClfA ClfB mutant of Newman and 8325-4 (A₆₀₀ < 0.05, data not shown).

Shedding and Dilution Contribute to Loss of ClfB from Cells in Stationary Phase-- The reduction in ClfB protein detected on stationary phase cells could be explained in part by dilution of the protein present on cells if transcription (and translation) stops in exponential phase. To test this hypothesis, cells from a mid-exponential phase culture of strain Newman (when the 150-kDa ClfB protein is maximally expressed) were concentrated to an A₆₀₀ of 40. Serial 2-fold dilutions were made, and the ClfB protein was then released by lysostaphin. These samples were compared by Western immunoblotting with ClfB protein released from stationary phase Newman cells concentrated to an A₆₀₀ of 40. After transcription of clfB stopped during exponential phase at an A₆₀₀ of ~1.0 (Fig. 3), three additional doublings occurred before the cells reached late stationary phase (A₆₀₀ = 10-12, 4 × 10⁹ cells/ml) (Fig. 2). After three doubling dilutions, the same amount of ClfB protein was detected in the sample from the exponential phase cells as from stationary phase cells (data not shown). Thus, the reduction of ClfB protein on stationary phase cells of strain Newman and the corresponding reduction in fibrinogen binding activity can be explained by dilution of the protein among daughter cells after expression has stopped.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Transcription of clfB detected with a clfB::lacZ reporter fusion. Transcription of clfB was monitored throughout the growth cycle of strains Newman clfB::lacZ (A), 8325-4 clfB::lacZ (B), Newman agr clfB::lacZ (C), and Newman sar clfB::lacZ (D) by measuring -galactosidase activity using MUG as the substrate. Each experiment was carried out five times with similar results. , A600 of bacterial culture; , -galactosidase activity (MUG units).

In addition to the dilution effect, we were able to detect ClfB protein in the culture supernatant after concentration by trichloracetic acid precipitation (data not shown). With strain Newman, we have estimated that 25% of the ClfB protein present on exponential phase cells is shed from the cell surface, possibly due to the action of peptidoglycan-degrading enzymes during cell division.

Transcription of clfB Stops in Late Exponential Phase-- Expression of the clfB gene during the growth cycle of S. aureus strains 8325-4 and Newman was monitored with a transcriptional fusion formed between the clfB promoter and a promoterless lacZ gene (clfB::lacZ). For strain Newman, -galactosidase activity reached a maximum of 580 units in late exponential phase and decreased rapidly as the cells entered stationary phase (Fig. 3A). This suggests that -galactosidase from Escherichia coli is unstable in the cytoplasm of S. aureus because the loss of activity is greater than can be explained by dilution. A similar trend was observed with strain 8325-4 although the maximum -galactosidase activity was much lower (82 units; Fig. 3B). This suggests that the clfB promoter in 8325-4 is weaker than in Newman or that the level of any regulatory molecules involved in controlling clfB differs in the two strains. The peak of transcription of clfB coincides with protein expression and adherence as shown in Fig. 2. Cessation of transcription (and hence translation) in late exponential phase can explain in part the loss of ClfB protein from cells in stationary phase.

As a control, a lacZ transcriptional fusion to the protein A gene (spa::lacZ) (41) was transduced into strain Newman. The level of -galactosidase was highest in mid- to late exponential phase and considerably lower in cells from stationary phase, which agrees with previously reported expression of spa in 8325-4 (41, 42). However, the maximum level was 10-fold higher than with the clfB::lacZ fusion. The clfB promoter therefore appears to be considerably weaker than that of the protein A gene. Overexpression of clfB from a multicopy plasmid, however, does enable the detection of ClfB in stationary phase (data not shown). This suggests that the presence of protein A and absence of ClfB on the cell surface in stationary phase, considering that their respective genes are only transcribed in exponential phase, is not due to differences in turnover but rather the amount of these proteins present, which is subject to dilution after transcription has stopped.

Effect of agr and sar on clfB Expression-- To investigate if the global transcriptional regulators agr (21, 22) regulate transcription of clfB, the clfB::lacZ fusion was combined with sar or agr mutations in strain Newman. The level of transcription of clfB-lacZ in the sar mutant was consistently 1.25-fold higher than the wild type, but this was not considered to be significant. Neither the sar mutation nor the agr mutation in strain Newman had any effect on the maximum level of -galactosidase activity or on the timing of peak transcription levels in exponential phase cells, and neither enhanced transcription of clfB in stationary phase (Fig. 3, C and D). Similar trends were observed for 8325-4 sar and agr mutants (data not shown).

Fig. 4 (A and B) compares the level of ClfB protein released from the same number of 8325-4 and Newman cells isolated from early exponential phase. Strain Newman expressed 2.8-fold more ClfB protein than 8325-4, consistent with the transcription data. The agr mutation had no effect on the level of protein in either strain, but, surprisingly, there was an apparent 2-3-fold increase in ClfB protein in the sar mutants. This could be explained by a higher affinity of the antibodies for the truncated form of the protein, as sar had no effect on transcription of the clfB gene. The effect of the sar mutation on processing of ClfB is discussed in the next section.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Expression and activity of ClfB protein by regulatory mutants. A, strains were grown to mid-exponential phase (A600 = 0.4) and samples were concentrated to an A600 of 40 and prepared for Western immunoblotting with anti-ClfB antibodies. B, the amount of protein in each track was measured by densitometry. Lane 1, Newman wild type; lane 2, Newman agr; lane 3, Newman sar; lane 4, 8325-4 wild type; lane 5, 8325-4 agr; lane 6, 8325-4 sar. C, adherence of sar mutants to immobilized fibrinogen. These results are representative of three experiments.

Proteolytic Cleavage of ClfB Results in Loss of an N-terminal Domain and Loss of Fibrinogen Binding Activity-- It has been reported that several extracellular proteases are greatly overexpressed in a sar mutant of S. aureus 8325-4 (24). Here, 8325-4 sar and Newman sar expressed 2.5- and 5-fold higher protease levels than the respective wild-type strains as measured using fluorescein thiocarbamoyl-casein as a substrate (data not shown). Fig. 4 demonstrates the effects of this elevated protease activity on the processing of ClfB by Newman clfA sar and 8325-4 clfA sar. In the Newman sar mutant, both the 150- and the 120-kDa immunoreactive proteins were detected (Fig. 4A), whereas the 150-kDa moiety predominated on the Sar⁺ strain, as described above (Fig. 2A). The proportion of the 120-kDa protein compared with the larger form in the Newman sar mutant increased as the culture grew, and only this truncate was detected on stationary phase cells (data not shown). In the 8325-4 sar mutant, only the 110-kDa truncated form of ClfB was detected, even at the earliest stage of the growth cycle (Fig. 4A). These results are consistent with the sar mutants expressing elevated levels of the protease(s) that cleave the N terminus of ClfB and the 8325-4 sar mutant having more proteolytic activity than the Newman sar mutant.

Proteolytic cleavage of the wall-associated ClfB protein resulted in loss of about 30 kDa from the N terminus. To determine if the truncated form of ClfB remaining on the cell surface retained fibrinogen binding activity, the ability of the sar clfA mutants to adhere to immobilized fibrinogen was measured. Compared with wild-type Newman, the sar mutant adhered to immobilized fibrinogen at the same level as the wild type (Fig. 4C). In contrast, the 8325-4 sar mutant, which was totally devoid of the higher form of ClfB, adhered very weakly to immobilized fibrinogen (Fig. 4C). It can be concluded that loss of the N-terminal moiety of wall-associated ClfB in 8325-4 resulted in loss of fibrinogen binding activity. The residual adherence to fibrinogen could be due to trace amounts of the full-length form of ClfB or to the fibrinogen binding activity of FnBPA and FnBPB.

Polyclonal antibodies generated against rClfB44-196 were used in Western blotting experiments to examine ClfB expressed on the bacterial surface. The anti-rClfB44-196 antibodies reacted with the 150- and 140-kDa full-length forms of ClfB from strains Newman and 8325-4, respectively, but did not recognize the truncated 120- or 110-kDa truncated forms (data not shown). In contrast, polyclonal antibodies directed against full-length region A (10) reacted with both forms of ClfB expressed by strains Newman and 8325 (Fig. 4A). This indicates that the N-terminal domain is lost in the truncated forms of ClfB.

To investigate if proteolysis and loss of ClfB occurred in clinical isolates, five strains that caused endocarditis and five that caused bone or joint infections were analyzed. In nine of the strains, extensive breakdown of ClfB was observed on cells from early exponential phase whereas in early stationary phase most strains were devoid of detectable ClfB or retained very low levels of the lower molecular weight form (data not shown). It can be concluded that expression and processing of ClfB by 8325-4 is typical of the majority of wild-type strains. Variation in the size of ClfB proteins observed in these strains is likely to be due to differences in the length of the region R repeats, as is the case for the difference in the size of the Newman and 8325-4 proteins.

ClfB Is Cleaved and Inactivated by Metalloprotease-- To identify the protease(s) involved in cleavage of ClfB, strain Newman cells from early exponential phase cultures expressing predominantly full-length ClfB were incubated with a concentrated supernatant from a stationary phase culture of Newman sar, which expresses high levels of proteases. Chloramphenicol (20 µg/ml) was added to the exponential phase cells to inhibit bacterial growth and to prevent further synthesis of ClfB protein. The supernatant converted the 150-kDa form of ClfB to the 120-kDa moiety (Fig. 5A). Addition of EDTA or o-phenanthroline (both chelators of divalent cations) prevented cleavage, suggesting that a metalloprotease was responsible. Prolonged incubation of the cells with the supernatant did not result in any further degradation of ClfB, which indicates that the protein remaining on the cell surface after loss of the N-terminal domain is very resistant to staphylococcal proteases and that proteolysis is probably not responsible for reduction of ClfB on stationary phase cells (Fig. 5B).

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Effects of concentrated culture supernatants on ClfB. A, exponential phase cells of S. aureus Newman were incubated in the presence of chloramphenicol with concentrated stationary phase culture supernatants as follows: lane 1, no supernatant; lane 2, supernatant from Newman sar; lane 3, supernatant from Newman sar with 1 mM EDTA; lane 4, supernatant from Newman sar with 10 mM o-phenanthroline; lane 5, supernatant from Newman sar aur. B, exponential phase cells of S. aureus strain Newman were incubated in the presence of chloramphenicol with concentrated stationary phase culture supernatant of S. aureus Newman sar for up to 20 h.

In order to determine if the metalloprotease aureolysin was responsible for cleaving ClfB, the purified enzyme was incubated with exponential phase S. aureus Newman cells. It rapidly converted the 150-kDa ClfB to the 120-kDa form (data not shown). Also, the supernatant of a Newman sar aur mutant carrying an insertion in the aureolysin gene failed to cleave ClfB (Fig. 5A, lane 5).

Zymographic analysis of culture supernatants from S. aureus strains revealed that 8325-4 sar produced ~30-fold more aureolysin than Newman sar, which explains the more profound cleavage of ClfB by the former strain (data not shown). This experiment also confirmed the lack of metalloprotease activity in the supernatants of 8325-4 sar aur and Newman sar aur mutants (Fig. 6 shows data for Newman sar and Newman sar aur). Interestingly, two other proteolytic activities were missing from the supernatants of the sar aur mutants in addition to aureolysin. One of these was the V8 serine protease, which was inactivated by preincubation of the supernatant with a specific serine protease inhibitor, dichloroisocoumarin. Neither of these two proteases contributed to cleavage of ClfB because pretreatment of culture supernatant with EDTA or o-phenanthroline eliminated this activity and prolonged incubation of ClfB with protease-rich supernatant resulted in no further truncation of the protein.

View larger version (93K): [in this window] [in a new window]   Fig. 6.   Gelatin zymography of S. aureus Newman culture supernatants. Supernatants were diluted 2-fold in SDS-PAGE sample buffer, incubated at 37 °C for 30 min, and electrophoresed in 12% polyacrylamide gels incorporating gelatin. 15 µl of supernatant was loaded per well. Lane 1, culture supernatant of Newman sar; lane 2, culture supernatant of Newman sar preincubated with 1 mM dichloroisocoumarin (DIC); lane 3, culture supernatant of Newman sar incubated with 1 mM o-phenanthroline (OF); lane 4, culture supernatant of Newman sar aur. Proteinase bands were developed by incubation of the gel for 3 h at 37 °C in 20 mM Tris-HCl, 0.5 mM CaCl2, pH 7.8. Arrows indicate the positions of the serine protease (V8) and the metalloprotease (Aur).

Finally, the full-length recombinant ClfB A domain (rClfB44-542) and the fibrinogen-binding truncate lacking residues 44-196 but retaining the SLAVA motif, rClfB197-542, were incubated with the purified metalloprotease. The ~70-kDa rClfB44-542 protein was rapidly converted to a ~40-kDa truncate (Fig. 7), whereas rClfB197-542 lost its N-terminal His tag and was not retained on a Ni²⁺ chelate column (data not shown). The truncate derived from rClfB44-542 failed to react both with anti-rClfB44-196 antibodies and the probe for the N-terminal His tag, but it did react with polyclonal antibodies antibodies (data not shown). N-terminal sequencing of the 40-kDa moiety showed it to be a C-terminal truncate that had been cleaved between Ser¹⁹⁷ and Leu¹⁹⁸ or between Ala¹⁹⁹ and Val²⁰⁰. Furthermore, the truncate was totally devoid of fibrinogen binding activity (Fig. 7). We could not detect the intact N-terminal domain after cleavage of rClfB44-542, suggesting that the metalloprotease or other proteases cleave at several sites within that region, generating peptides that are too small to be resolved by SDS-PAGE.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Binding of rClfB44-542 to immobilized fibrinogen. Plates coated with 10 µg/ml fibrinogen were incubated with rClfB44-542 or with the metalloprotease-treated sample (60 min at 37 °C) (, no protease; , incubated with metalloprotease). Inset, a Coomassie Blue-stained SDS-PAGE gel showing the effect of metalloprotease on recombinant rClfB44-542. Lane 1, rClfB44-542 incubated at 37 °C in PBS buffer; lane 2, rClfB44-542 incubated at 37 °C with metalloprotease.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The work described in this paper provides an explanation for an earlier observation that the ClfB protein is only present in a detectable and functional form on cells from the exponential phase of growth (10). We have shown that this is a more complex phenomenon than originally thought. It involves cessation of transcription of the clfB gene toward the end of exponential phase and proteolytic cleavage of surface-bound ClfB to remove an N-terminal domain causing loss of fibrinogen binding activity. Shedding of full-length and truncated ClfB into the growth medium and dilution of molecules remaining on the surface during the last three cell division events leading to stationary phase also contribute to the loss of ClfB from the cell surface in stationary phase.

ClfB expression and activity was found to reach a maximum in mid-late exponential phase and declined steadily as cultures progressed into stationary phase. By late stationary phase, there was only a trace amount of ClfB present on the cell surface and ClfB-mediated adherence to fibrinogen was very low. A clfB-lacZ reporter fusion was used to study transcription of clfB. Maximum reporter activity was observed in late exponential phase cells (A₆₀₀ = 1-1.5), coinciding with maximum protein expression, and declined rapidly thereafter. Neither the agr nor the sar global regulators had any significant effect on clfB transcription, in contrast to other surface protein genes. Genes encoding protein A (spa) and fibronectin-binding protein (fnbA) are negatively regulated by agr (20, 42), and the gene encoding the collagen binding protein cna is negatively regulated by sarA independently of agr (23). Expression of clfB-lacZ was lower in strain 8325-4 compared with Newman. This correlated with lower levels of ClfB protein in the former strain.

The size of the full-length ClfB protein is different in strain Newman (150 kDa) compared with 8325-4 (140 kDa) reflecting a difference in the length of the repeat region R. In both Newman and 8325-4, full-length ClfB was cleaved to truncated forms of ~120 or 110 kDa, respectively. Cleavage occurred earlier in the growth cycle of 8325-4 and to a greater extent than with strain Newman. This reflects differences in the level of metalloprotease expressed by the two strains.

SarA is a major repressor of transcription of several protease genes (30). The level of several extracellular proteases is higher in a sarA mutant of 8325-4 compared with the wild type. Since SarA had no effect on transcription of the clfB gene, we used sarA mutants to investigate the effect of proteases on cell wall-associated ClfB. In Newman sarA, the truncated form of ClfB appeared earlier in the growth cycle and was present at an increased level compared with the wild-type strain. In 8325-4 sarA, only the truncated form of ClfB could be detected, even at the earliest part of the growth cycle. These cells adhered very poorly to immobilized fibrinogen, indicating that the truncated form of ClfB had lost function.

The recombinant ClfB protein is particularly sensitive to cleavage at a putative proteolytic cleavage site, SLAVA. Cleavage of rClfB at this site by a putative E. coli metalloprotease resulted in the loss of the ability of the protein to bind to fibrinogen.² Region A of ClfA has a similar protease recognition site, SLAAVA (9), although in this case cleavage does not result in loss of fibrinogen binding activity. Thus, it was hypothesized that the SLAVA motif could be the site at which proteolysis occurs in ClfB protein expressed on the staphylococcal cell surface. S. aureus Newman cells expressing full-length ClfB protein were incubated with a concentrated stationary phase culture supernatant from a protease-overexpressing strain, Newman sar. Complete cleavage of the 150-kDa ClfB protein to the truncated form occurred. Cleavage was inhibited by EDTA and o-phenanthroline, suggesting involvement of a metalloprotease. Similarly, incubation of recombinant rClfB44-542 and rClfB197-542 with the Newman sar culture supernatant resulted in proteolytic cleavage at the SLAVA motif and in loss of fibrinogen binding activity. Cleavage occurred between Ser¹⁹⁷ and Leu¹⁹⁸, or between Ala¹⁹⁹ and Val²⁰⁰ within the SLAVA motif and was inhibited by EDTA and o-phenanthroline. The metalloprotease of S. aureus is known to cleave at the N-terminal side of hydrophobic residues including leucine and valine (43) and is inhibited by EDTA and o-phenanthroline. It is produced throughout the growth cycle, in contrast to the other staphylococcal proteases, which are expressed predominantly in stationary phase (21, 22). -Galactosidase assays using the aur-lacZ fusion showed that aur is transcribed from early exponential phase (data not shown). Although the metalloprotease mediates activation of the serine protease proenzyme (Ref. 23; Fig. 6), it seems unlikely that serine protease or any other protease activated by the metalloprotease is responsible for modifying ClfB, as cleavage occurs in exponential phase and the ClfB cleaving activity of stationary phase culture supernatants is completely inhibited by EDTA. This work demonstrates that the metalloprotease has a central role in modifying a surface protein of S. aureus. It cleaves the N terminus of ClfB, resulting in loss of fibrinogen-binding activity and is possibly also responsible for processing of ClfA.

After transcription of the clfB gene and translation of the ClfB protein stops in late-exponential phase, the fate of the ClfB protein remaining on the cell surface was considered. It was observed that the concentration of the protein per cell, and also the ability of bacteria to adhere to fibrinogen, decreased as the bacteria entered stationary phase. The majority of the remaining protein on the cell surface (in 8325-4 and all clinical strains examined) was degraded to the inactive truncate. However, there was no indication that further degradation of the protein occurred, even after prolonged incubation of non-growing exponential cells with concentrated culture supernatant. Some full-length ClfB protein was found in the culture supernatant, but the amount detected did not explain the reduction in ClfB protein on stationary phase cells. We propose that the apparent loss of ClfB is in part explained by a dilution effect, as the cells continue to grow and divide after transcription (and translation) has stopped. Thus, a combination of shedding by autolysis and dilution of ClfB molecules on the surface of daughter cells can explain the reduction of ClfB on the cell surface in stationary phase.

The biological significance of the truncation of ClfB during the growth cycle is unclear. However, we argue that it must be of importance because it occurs in most clinical isolates. The loss of an N-terminal domain from the surface-associated protein may release biologically active peptide fragments. The intact domain was not detected in culture supernatants or when purified rClfB44-552 was cleaved with purified aureolysin, indicating that this cleavage product is broken down further. Alternatively, cleavage of ClfB may promote the detachment of bacterial cells from colonized sites and facilitate the spread of infection within the host.

	ACKNOWLEDGEMENTS

We acknowledge helpful discussions and comments by S. Perkins and M. Höök.

	FOOTNOTES

^* This work was supported by the Health Research Board of Ireland and Wellcome Trust Grant 052320 (to T. J. F.) and by Grant 6 P04A 083 20 from the Committee of Scientific Research (Komitet Badan Naukonych, Warsaw, Poland).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.

^¶ To whom correspondence should be addressed. Tel.: 353-1-6082014; Fax: 353-1-6799294; E-mail: tfoster@tcd.ie.

Published, JBC Papers in Press, June 8, 2001, DOI 10.1074/jbc.M102389200

² S. Perkins, E. Walsh, T. J. Foster, and M. Höök, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; BHI, brain-heart infusion broth; bp, base pair(s); PCR, polymerase chain reaction; PBS, phosphate-buffered saline; DIG, digoxigenin; MUG, 4-methylumbelliferyl -D-galactopyranoside.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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