Purification of a bone sialoprotein-binding protein from Staphylococcus aureus

Eur. .I. Biochem. 222, 919-925 (1994) 0 FEBS 1994

Purification of a bone sialoprotein-binding protein from Staphylococcus aureus Aha YACOUB I, Per LINDAHL', Kristofer RUBIN', Mikael WENDEL?, Dick HEINEGARD' and Cecilia RYDEN'.' ' Department of Medical and Physiological Chemistry, University of Uppsala, Sweden

Department of Medical and Physiological Chemistry, University of Lund, Sweden Department of Infectious Diseases, University of Uppsala, Sweden

(Received January 12, 1994March 14, 1994) - EJB 94 0033/3

Bone sialoprotein (BSP) is selectively bound by Staphylococcus aureus cells isolated from patients suffering from infections of bone and joint tissues [RydCn C., Maxe, I., FranzCn, A., Ljungh, A., Heinegird, D. & Rubin, K. (1987) Lancet ZZ, 51.51. We now report on the purification of a cell- wall protein from Staphylococcus aureus, strain 024, that possesses affinity for bone sialoprotein. Staphylococcal cell-wall components with capacity to inhibit binding of '251-labeled BSP to staphylococcal cells were solubilized with LiCl (1.0 M, pH 5.0).

Preparative SDS/PAGE and protein-overlay experiments revealed that inhibitory activity present in LiCl extracts resided in a fraction of polypeptides with M , 75000-110000. Staphylococcal proteins solubilized with LiCl were chromatographed on a Mono-Q anion-exchange column. Inhibitory activity was eluted at 0.6-0.8 M NaCl and could be further purified by affinity chromatography on BSP-Sepharose. Elution of the affinity matrix with 0.1 M glycine, pH 3.0, specifically eluted inhibitory activity.

Analysis by SDS/PAGE revealed a single M, 97000 polypeptide in the eluate. The purified M, 97000 protein bound BSP in protein-overlay experiments. LiCl extracts from S. aureus, strain E514 or Staphylococcus epidermidis, strain 7686, both lacking the capacity to bind BSP did not contain the M, 97000 protein. Our data demonstrate the presence of a S. aureus cell-surface BSP-binding protein. This protein could be involved in bacterial tropism in osteomyelitis.

Bacterial adhesion and invasion are important events in the infectious process (Beachey, 1981 ; Finlay, 1990; Isberg, 1991). In vitro, bacterial cells adhere to nucleated mammalian cells (Thomas et al., 1988), platelets (Yeaman et al., 1992; Herrmann et al., 1993) and to immobilized components of the extracellular matrix (ECM) (Holderbaum et al., 198.5; Vercellotti et al., 198.5; Maxe et al., 1986; Herrmann et al., 1991 ; Switalski et al., 1993). Some of these adhesive processes have been shown to be mediated by specific adhesion receptors (Maxe et al., 1986; Herrmann et al., 1993; Switalski et al., 1993) and staphylococcal cells possess, at their cell surface, several proteins mediating specific interac- tions with ECM glycoproteins. These proteins include two fibronectin-binding proteins with molecular masses of 98 kDa and 108 kDa, respectively, (Flock et al., 1987; Signas et al., 1989; Jonsson et al., 1991) and a 13.5-kDa collagen- binding protein (Patti et al., 1992). The collagen-binding protein has been shown to be important for the adhesion of Staphylococci to cartilage in vitro (Switalski et al., 1993).

Studies in animal models have provided evidence for a pathophysiological importance of the staphylococcal 98-kDa and 108-kDa fibronectin-binding proteins in experimental endocarditis (Kuypers and Proctor, 1989; Schennings et al., 1993) and mastitis (Nelson et al., 1991). Furthermore, experi-

Correspondence to C. RydCn, Dept. of Medical and Physiologi- cal Chemistry, BMC Box 575, S-751 23 Uppsala, Sweden

Abbreviations. BSP, bone sialoprotein ; ECM, extracellular matrix.

mental data suggest a role of the 13.5-kDa collagen-binding protein in the development of septic arthritis in mice (Patti et al., 1994). Staphylococcal proteins with affinities for other components in the extracellular matrix, e.g. thrombospondin (Herrmann et al., 1991), vitronectin (Chhatwal et al., 1987; Paulsson et al., 1992), elastin (Park et al., 1991) and laminin (Lopez et al., 1985; Vercellotti et al., 198.5) have also been isolated from extracts of staphylococcal cells. Recently, an M , 60000 staphylococcal protein with lectin-like activity and with affinity for several ECM proteins has been isolated from Staphylococcus aureus, strain Newman (McGavin et al., 1993). Protein-overlay experiments showed similar proteins with varying M, to be synthesized by several strains of S. aureus, including an M , 72000 protein from S. aureus, strain 574 (McGavin et al., 1993).

Bone matrix is composed of an inorganic scaffold of hy- droxyapatite crystals. The organic constituents are dominated by collagen type I, but include several other proteins such as phosphoproteins (Linde et al., 1983), osteonectin (Termine et al., 1981), osteocalcin (Pan and Price, 1985), thrombospondin (Mosher, 1990), proteoglycan (FranzCn and HeinegArd, 1984; Herring and Kent, 1963) and sialoproteins (Fischer et al., 1983; FranzCn and HeinegArd, 1985; Oldberg et al., 1986, 1988a, b). Many of the ECM components present in bone tissue are also present in other types of connective tissues. However, a few constituents of the organic bone matrix, such as bone sialoprotein (BSP), have only been found in bone and dentin (FranzCn and Heinegird, 198.5; Oldberg et

920

al., 1988a). BSP has been isolated from human, bovine and rat bone, and the amino acid sequence of human BSP has been deduced from cDNA cloning (Oldberg et al., 3988a). The BSP core protein has a molecular mass of 33 600 Da and contains long stretches of acidic amino acid residues. BSP undergoes extensive post-translational modifications including glycosylation; 40-50% of the mass of the purified gly- coprotein is carbohydrate of which 13-14% is sialic acid. The binding of mammalian cells to immobilized BSP is mediated by an Arg-Gly-Asp sequence in the carboxy terminal part of BSP (Oldberg et al., 1988b).

Certain staphylococcal cells bind BSP and this interaction has been suggested to be of importance for staphylococcal tropism in osteomyelitis and septic arthritis (RydCn et al., 1987). The latter notion was based on an observed selectivity in ability to bind BSP among clinical isolates of Stuphylo- cocci. A high proportion of isolates from cases of septic arthritis and osteomyelitis bound BSP, whereas isolates from staphylococcal infections at other body locations usually did not (RydCn et al., 1987). Staphylococcal cells bind BSP in a specific reaction, having a dissociation constant around 10nM (RydCn et al., 1989). Furthermore, the putative staphylococcal BSP-binding protein recognized a binding site in the BSP core protein located at the amino terminus and distinct from the Arg-Gly-Asp binding site for mammalian cells (RydCn, et al., 1989).

In the present work, we report on the isolation and purification of an M, 97000 BSP-binding protein from staphylococcal cells, most likely from the cell walls. We conclude that this protein is novel and distinct from the ECM-binding proteins previously isolated from staphylococcal cells, based on differences in relative molecular masses, and the inability of other ECM components, such as fibronectin, fibrinogen, collagen or laminin to inhibit the binding of BSP by staphylococcal cells (RydCn et al., 1989).

MATERIALS AND METHODS

Reagents

Bovine serum albumin fraction V, ovalbumin grade VIII, DNAse type I and RNAse type A, benzamidine hydrochloride, phenylmethylsulfonyl fluoride and N-hydroxysuccini- midobiotin were all purchased from Sigma Chemical Co. Trypticase soy broth and agar base were from Oxoid Ltd. Horse radish peroxidase color-development reagent was from Bio-Rad. Percoll, Mono-Q and Mono-S columns were from Pharmacia AB. Streptavidin-peroxidase was from Amersham and "'1 was from New England Nuclear. Iodobeads were obtained from Pierce Chemical Co. Bone sialoprotein was purified from bovine bone as previously described (FranzCn and Heinegird, 1985) and radiolabeled to a specific activity of 2-4X lo7 cpndpg with Iz5I. Recombinant lysostaphin was purchased from Appl. Microbiol. Inc.

Bacterial strains

S. aureus, strain 024 was originally isolated from a patient suffering from acute osteomyelitis (RydCn et al., 1989). S. aureus, strain E514 was isolated from a patient suffering from acute endocarditis (Rydtn et al., 1989). Staphylococcus epidermidis, strain 7686, isolated from a peritoneal catheter, was kindly donated by G. Pulverer, University of Cologne, Germany, and was used as a control in binding experiments.

Bacteria were stored at -70"C, subcultured on blood agar (5% horse erythrocytes) and grown in trypticase soy broth.

Binding of '*'I-BSP to staphylococcal cells

Binding of "'I-BSP to staphylococcal cells, and detection of inhibitory activity was quantified as described previously (RydCn et al., 1989). In brief, staphylococcal cells were grown for 10-12 hours in trypticase soy broth, harvested cells were washed twice in ice-cold phosphate-buffered saline (NaCUP,; 0.1 3 M NaC1, 10 mM sodium phosphate, pH 7.4) and resuspended in binding-buffer (137 mM NaC1, 5 mM KCI, 0.7 mM MgS0,,1.2 mM CaCl,, 10 mM Hepes, pH 7.4, 0.1% ovalbumin) to a concentration of 10'" bacteria/ ml. '251-labeled BSP (105cpm) was added to the bacteria in a total incubation volume of 0.5 ml. Fractions of LiC1-extracts of S. aureus to be tested for their content of inhibitory activity, were dialyzed against binding buffer and samples were supplemented with ovalbumin to a final concentration of 0.1%.

Solubilization of bacteria

Staphylococcal cells were extracted with LiCl by incu- bating washed bacteria in 1.0 M LiCI, pH 5.0, under vigorous shaking for 2 hours at 45°C. Intact bacteria were sedimented by centrifugation at 1000OXg and the supernatant was dialyzed against NaCUP, When lysostaphin was used to digest bacteria, the bacterial pellet was washed twice and resuspended in NaCI/P, containing 5 mM benzamidine, 10 mM EDTA and 2 mM phenylmethylsulfonyl fluoride. Recombi- nant lysostaphin was added in the presence of DNase and RNase as previously described (RydCn et al., 1983) and the solution was incubated at 37°C under vigourous shaking for 60 min. The lysate was centrifuged and the supernatant dialyzed against NaClP,.

Ion-exchange and affinity chromatography

Non-immune rabbit IgG and BSP were coupled to CNBr- activated Sepharose-CL (Pharmacia) by the method recom- mended by the manufacturer. Mono-Q and Mono-S columns were adapted to a Pharmacia FPLC system. In the purification of BSP-binding proteins, LiCl extracts of Staphylococci, dialyzed against NaCIP,, were routinely passed over IgG- Sepharose to remove protein A. Samples were applied to the Mono-S column in 50 mM sodium actetate, pH 5.

Determination of protein concentration

binding method (Read and Northcote, 1981). Protein was quantified by the Coomassie blue G dye-

Polyacrylamide gel electrophoresis

The BSP-binding proteins from Staphylococci were subjected to SDS/PAGE. The separation gels consisted of 12% acrylamide, and the stacking gels of 3% acrylamide. The samples were dissolved in sample buffer (4% SDS, 4% sucrose, 0.0625 M Tris/hydrochloride, pH 8.8) and boiled for 3 min prior to electrophoresis. Protein bands were visualized by Coomassie brilliant blue or by silver staining (Wray et al., 1981).

921

0 10 15

Time (hours) B

0 5 10 15

Time (hours)

Fig.1. Binding of 1Z51-labeled BSP to S. aureus strain 024 (0) and S. epidermidids strain 7686 (0) as a function of cultivation time. (A) Growth curve for the two strains used for binding studies. (B) Staphylococcal cells were harvested at the indicated time points, washed and incubated with radiolabeled BSP for 90 min at 4°C. Binding was calculated as described in Materials and Methods.

Preparative polyacrylamide gel electrophoresis

Preparative SDS/PAGE was performed essentially as described previously (Rubin et al., 1986). In brief, LiCl extracts from S. aureus, strain 024 cells were electrophoresed and the gels were sliced. The proteins were electrophoretically eluted from the gel slices overnight in an electroelution appa- ratus. The various fractions, corresponding to polypeptides with different M, values, were dialyzed and analyzed for their capacity to inhibit the binding of '251-labeled BSP to staphylococcal cells. The M , limits of the various gel slices were estimated from their relative distances on the gel, and related to migration of standard proteins run on the same gel.

t

-1 0 1 -3 -2

Log (LiCI concentrationlhl)

Fig. 2. '251-labeled BSP binding to staphylococcal cells incubated with different concentrations of lithium chloride. Binding of radiolabeled BSP to staphylococci in buffer alone was 2.93 ng/109 cells.

Protein-overlay technique Lysate from staphylococcal cells and the purified BSP-

binding proteins were separated on SDSPAGE and electrophoretically transferred to nitrocellulose sheets (membrane filters, BA 83 0.2 pm, Schleicher and Schuell), as described previously (Maxe et al., 1986; Blobel and Dobberstein, 1975). After protein transfer, the sheets were incubated in NaCl/P, supplemented with 5 % bovine serum albumin at 20°C for 1 hour to inhibit non-specific binding. The sheets were incubated with biotinylated BSP followed by streptavidin-peroxidase conjugate and bound enzyme was visualized by the addition of horse radish peroxidase color-development reagent. Biotin-labeled standard was used as described earlier (Maxe et al., 1986). Sheets with lysate or BSP-binding protein were also incubated with 125T-labeled BSP (lo5 c p d m l overnight at 4°C under slow agitation), transferred to X-ray film, exposed overnight and subsequently developed. In some experiments, sheets were blocked in a buffer containing 2.0% ovalbumin, replacing the bovine serum albumin prior to incubation with '251-BSP.

RESULTS Binding of BSP to staphylococcal cells as a function of growth phase

The capacity of S. uureus, strain 024 to bind 'Z51-labeled BSP was low at early phases of growth, increased to maxi- mum at late log phase, remained high in the early stationary phase, then started to decline (Fig. 1). Based on these results, bacteria were harvested after 10-12 hours of cultivation to ascertain the maximal yield of BSP-binding protein. The same cultivation time was used when extracts from S. aureus, strain E514 and S. epiderrnidis, strain 7686 were prepared.

Solubilization of BSP-binding components from staphylococcal cells

The effect of treatment with various concentrations of LiCl on the capacity of S. aureus, strain 024 to bind l2'1-

922

100 I- 7 1

-6 -5 -4 -3 -2 -1 0

Log (protein concentration g . L-1)

Fig. 3. Binding of lZ5I-Iabeled BSP to S. aureus strain 024 in the presence of lysates and purified BSP-binding protein. Inhibitors used were lysates from lithium-chloride-treated Staphylococci (M) and lysostaphin-treated Staphylococci (O), purified BSP-binding protein (A) from BSP-Sepharose and material collected after passage of lysate on BSP-Sepharose (O), all from S. u u i e u ~ strain 0 2 4 and lithium chloride lysate from S. epidermidis strain 7686 (0). The initial concentrations of inhibitor used corresponded to 0.56 mg/ml for lysates and 1.8 pg/ml for the purified BSP-binding protein.

labeled BSP was determined. LiCl in concentrations above 0.5 M significantly decreased the capacity of the staphylococcal cells to bind '"I-BSP (Fig. 2).

The purification of putative BSP-binding components from staphylococcal cells was monitored with an assay in which the capacity of various fractions of staphylococcal cell-wall extracts to inhibit the binding of IZ5I-BSP to intact staphylococcal cells was determined. This activity is referred to as inhibitory activity. The presumption that BSP-binding components from the staphylococcal cell walls were solubilized by LiCl was supported by the finding that LiC1-extracted material from S. uureus, strain 024 cells contained inhibitory activity (Fig. 3). Optimal extraction was obtained with 1 .0M LiCl (data not shown). LiCl extracts from S. aureus, strain 514 or S. epidermidis, strain 7686 cells did not possess inhibitory activity (data not shown). Lysostaphin digestion of S. aureus, strain 024 cells also yielded a lysate possessing inhibitory activity in the BSP-binding assay (Fig. 3).

Purification of BSP-binding components from S. aureus cells

We have earlier shown that protein A does not interfere with the binding of BSP to staphylococcal cells (RydCn et al., 1989). In accordance, LiCl extracts from S. aureus, strain 024 cells retained all inhibitory capacity after passage through an IgG-Sepharose column (data not shown).

Recently, proteins with affinity for several ECM glycoproteins, including BSP, were purified from S. aweus strains Newman and strain 574 by cation exchange chromatography on Mono-S columns (McGavin et al., 1993). However, no inhibitory activity, assayed according to the current protocol with ovalbumin in the incubation buffers, was retained on Mono-S columns (data not shown). LiCl extracts were se-

quentially passed through IgG-Sepharose and Mono-S columns and the flow-through was dialyzed against 10 mM sodium borate, pH 8.0, and applied to an anion-exchange Mono-Q column. The column was eluted by a NaCl gradient (0-1.0 M). The flow-through from the Mono-Q column was devoid of inhibitory activity. Retained inhibitory activity could be eluted at 0.6-0.8 M NaCl together with a substan- tial portion of the protein that had bound to the Mono-Q matrix (Fig. 4). The fractions eluted at 0.6-0.8 M NaCl were pooled, dialyzed against 137 mM NaCI, 5 mM KCI, 0.7 mM MgS04, 1.2 mM CaCI,, 10 mM Hepes, pH 7.4, and applied to a BSP-Sepharose column. The BSP affinity matrix retained all detectable inhibitory activity. Elution with 0.5 M NaCl released only a minor part of the inhibitory activity, but a significant amount of the protein that had bound to the column (Fig. 5). The majority of the inhibitory activity, but only minor parts of the protein, was eluted with a low-pH buffer (0.1 M glycine, pH 3.0; Fig. 5).

M, of the BSP-binding component from S. aureus

Analysis by SDS/PAGE of material eluted at pH 3.0 from the BSP-Sepharose columns and visualization of proteins by silver staining revealed the presence of a single protein band with an apparent M , of 97000 (Fig. 6, lane B). The ability of this protein to bind BSP was investigated by protein-overlay experiments. The M , 97000 protein electrophoretically transferred to nitrocellulose sheets, bound both biotinylated BSP (Fig. 6, lane C) and '251-labeled BSP (Fig. 6, lane D). In bacterial extracts prepared from cells of S. aureus, strain E514 or S. epidermidis, strain 7686, no M , 97000 BSP-binding protein was detected by the protein-overlay technique (data not shown).

Protein-overlay analysis of unfractionated lysates of S. aureus, strain 024 cells revealed the presence of additional BSP-binding proteins with M, values of 70000, 47000, 30000 and about 22000 (Fig. 6, lane E). Binding of BSP to the Mr 70000, 47000, 30000 and 22000 but not to the Mr 97 000 band was abolished after incubation of the nitrocellulose membranes with 2.0% ovalbumin (Fig. 6, lane F). In contrast, incubations of the membranes with an excess of unlabeled BSP abolished binding to the M, 97000 protein, but only marginally decreased binding to the M, 70000, 47000, 30000 and 22000 bands (data not shown). Lyso- staphin lysate of S. uureus, strain 024 cells subjected to protein-overlay analysis revealed the Mr 97000 band, as well as several additional bands of lower molecular mass (Fig. 6, lane G).

LiCl extracts of S. uureus, strain 024 cells were fraction- ated according to M , by preparative SDS/PAGE. Inhibitory activity was detected in fractions corresponding to M , rang- ing over 75000-110000 (Fig. 7). Little or no inhibitory activity was detected in fractions representing other M , regions.

DISCUSSION

Here we have reported on the isolation of an M, 97000 protein from S. amreus, strain 024 that binds BSP. The BSP- binding protein was purified by Mono-Q anion-exchange chromatography and affinity chromatography on BSP-Sepha- rose from LiC1-extracts of staphylococcal cells. The major part of the inhibitory activity, present in LiCl extracts of staphylococcal cells. resided in the Mr 97000 protein, based on experiments employing preparative SDS/PAGE and a pro-

923

100

30

50

10

20

D 1

Fraction number

Fig. 4. Ion-exchange chromatography of extracts from S. uureus strain 024 on a mono-Q column in an FPLC-system. The column was eluted with a linear gradient of NaCl (0-1.0 M in 0.5 M borate, pH 8.0). Eluted material was used as inhibitor in the binding of '*'I- BSP to staphylococcal cells (A). The amount of protein in the fractions is indicated by relative absorbancy measurements (0).

1

0 10 20 30 40

Fraction number

Fig. 5. Affinity chromatography on BSP-Sepharose of extracts from S. uureus strain 024 cells. After applying extract to the column, the column was washed with 0.5 M NaCl (indicated by the arrow) and subsequently tightly bound protein was specifically eluted by 0.1 M glycine, pH 3.0, as indicated. Protein concentrations of fractions were measured by the Coomassie blue G dye-binding method (0) and activity was measured as inhibitory capacity of the eluted fractions (A).

tein-overlay technique. All significant inhibitory activity mi- grated in the M, range 75000-110000 in SDS/PAGE, as detected by preparative electrophoresis, and the protein-overlay experiments revealed that the M, 97000 polypeptide was the

Fig.6. SDSPAGE (12% acrylamide) and protein overlay of purified BSP-binding protein@) from S. aureus strain 024. The gel was stained with silver nitrate. Molecular-mass standards are indicated (low molecular mass, BioRad). Lane A, LiCl extract of S. aureus strain 024; lane B, affinity-purified BSP-binding protein from S. uureus strain 024; lane C, visualization of protein bands transferred from a slab gel to nitrocellulose and incubated with biotinylated BSP and subsequently developed with avidin-peroxidase followed by horse radish peroxidase color-development reagent; lane D, same as lane C, but incubated with '251-Iabeled BSP overnight and bound protein identified by autoradiography; lane E, extract of S. aureus strain 0 2 4 incubated with 1Z51-labeled BSP; lane F, same as lane E, but incubated with 2% ovalbumin prior to incubation with 'Z51-labeled BSP; lane C , lysostaphin lysate from S. uureus strain 0 2 4 incubated with "'I-labeled BSP.

only polypeptide band with specific affinity for BSP in this Mr region.

Extraction with LiCl did not disrupt the staphylococcal cells, as ascertained by the fact that cells extracted by LiCl were able to grow in culture even after extraction (data not shown). These data suggest that LiCl extraction predomi- nantly releases proteins from the cell surface, and it is thus

924

M, x i0 -3

> 110

75-110

65-75

55-65

40-55

33-40

25-33

< 25

0 25 50

Tnhihitorv activitv (%)

Fig. 7. Binding of '2sI-Iabeled BSP to staphylococcal cells in the presence of electroeluted material from extracts of S. uureus, strain 024. Gels from SDSPAGE, cut at indicated levels, were electroeluted in the presence of SDS. Eluted material was dialyzed and used as inhibitor in the binding of "'I-BSP to staphylococcal cells. The indicated approximate M, values were estimated from relative distances on the gel. Binding in buffer alone was set to 100% equal to 0% inhibition.

likely that the M, 97 000 BSP-binding protein described herein is present on the staphylococcal cell surface, although the possibility that the protein could be excreted from an intracellular location without disrupting the cells during the LiCl extraction could not be excluded.

The BSP-binding Mr 97000 protein from S. aureus, strain 024 appears to be distinct from other, previously identified, staphylococcal proteins with affinity for ECM glycoproteins. This notion is based on differences in M, values, but also on the fact that the binding of BSP to staphylococcal cells is not inhibited by other matrix or plasma glycoproteins, such as fibrinogen, fibronectin and collagen (Rydkn et al., 1989). Staphylococcal protein(s) with M , values 60000 and 72000, possessing affinity for several ECM glycoproteins, including BSP, has recently been characterized (McGavin et al., 1993). The M , 60000 protein was reported to possess lectin-like activity, and most likely binds carbohydrate structures present on many matrix glycoproteins. The properties of the M, 97 000 BSP-binding protein are different from those of the M, 60000 and 72000 proteins. Thus, in contrast to the Mr 60000 and 72000 proteins, that bound to a Mono-S column (cation exchanger), the M, 97000 protein was retained on a Mono-Q column (anion exchanger), suggesting differences in isoelectric points for the respective proteins. Furthermore, the purified M, 60000 and 72000 proteins possessed stimulatory activity on the binding of BSP to staphylococcal cells, whereas the M, 97000 protein was purified using an assay determining inhibitory activity. It, therefore, seems reason- able to conclude that the presently described M, 97000 BSP- binding protein is unrelated to the M, 60000 and 72000 BSP- binding proteins described by McGavin et al. (1993). Unfrac- tionated LiCl extracts contained polypeptides with M, values 97000, 70000, 47000, 30000 and 22000 that bound BSP. The binding of lZ5I-BSP to all of these polypetides, except the M , 97000 polypeptide, was inhibited by the presence of

ovalbumin in the incubation buffer. In contrast, unlabeled BSP only inhibited binding to the M, 97000 protein. Lyso- staphin lysate from S. aureus, strain 024 cells also inhibited binding of radiolabeled BSP to staphylococcal cells, and protein overlay also revealed M, 97000 protein in such prepara- tions. Several additional bands of lower molecular mass were also detected and, in some experiments, degradation of the M, 97000 protein could not be excluded, whereafter extraction with LiCl was preferred for further purification of the BSP-binding protein.

The reason that inhibitory activity, but not stimulatory activity, was detected in the present study may be that ovalbumin rich in carbohydrate, was present in the binding assay used to detect activity. Ovalbumin contains oligosaccharides that may block lectin-like activity present in the staphylococcal extracts. In the studies by McGavin et al. (1993), bovine serum albumin was used as a scavenger protein in binding assays. Since bovine serum albumin lacks carbohydrate, it is possible that the stimulatory lectin-like activity (Mr 60000 and 72000 proteins) was allowed to dominate over the inhibitory activity (Mr 97000 protein). Theses data are in agreement with the finding that staphylococcal cells recog- nize a site present in the core protein of BSP independently of substituents to the protein (RydCn et al., 1989).

BSP is found only in bone, and the interaction between BSP and staphylococcal cells could be important for the targeting of Staphylococci to bone in osteomyelitis, and may explain the maintenance of bacteria in the bone tissue. In a recent study using a murine model for staphylococcal-in- duced septic arthritis, it was demonstrated that the collagen- binding adhesin is a virulence factor for septic arthritis (Patti et al., 1994). The novel M, 97000 BSP-binding protein described in the present communication may represent another virulence factor and take part in the specific localization of Staphylococci to bone tissue, since all staphylococcal cells tested so far leading to septic arthritis in mice possess BSP- binding properties (Bremell et al., 1991; Abdelnour et al., 1993).

We are indebted to Eva Andersson for excellent technical assis- tance and to Magnus Hook and Joseph Patti for valuable discussions. The study was supported by grants from the Swedish Medical Re- search Council, The Swedish Society of Medicine, Gustaf V:s 80- Brs jond, Satra Brunns research fund and Axel and Margret Ax:son Johnsnns Stifielse.

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Purification of a bone sialoprotein-binding protein from Staphylococcus aureus