FIBRINOGEN BINDING PROTEIN

Abstract: The present invention relates to new fibrinogen binding proteins derived from Staphylococci, having a molecular weight of 19 kDa and/or 60 kDa, respectively.

Description: FIBRINOGEN BINDING PROTEIN DESCRIPTION Technical field The present invention relates to fibrinogen binding proteins. Further the invention relates to pharma¬ ceutical compositions and method for treatment. The object of the present invention is to obtain fibrinogen binding proteins. A further object is to obtain said protein by means of genetic engineering technique by using, e.g. a plas id comprising a nucleotide sequence coding for said protein. Background of the invention Clumping of Staphylococcus aureus in plasma has been suggested as a potential virulence factor. 1- 5 Several mechanisms can be responsible for this aggregation. A fibronectin-binding protein has been suggested to cause aggregation of staphylococci in fibronectin at the con- centration found in sera.5 r6 The presence of protein A causes staphylococci to aggregate in normal human sera, which frequently contain specific immunoglobulins directed against staphylococcal antigens .^ Due to a high cell sur¬ face hydrophobicity, many staphylococcal strains auto- regulate under isotonic conditions.8 It is believed that clumping of staphylococci in fibrinogen is caused by the so called clumping factor or fibrinogen-binding protein, situated on the staphylococcal cell surface. /9 Fibrinogen has also been suggested to mediate adhesion of S . aureus to cultured human endothelial cells 1 ^ anc j to catheters in vitro and in . vivo. 11 /12 j ^ as been disputed whether clumping factor is distinct from coagulase 1 or if it is a cell-bound form of coagulase.^, 14 staphylococcus aureus coagulases can be grouped into eight different serotypes 1 ^ and the existence of multiple molecular forms of coagulases SUBSTITUTESHEET has been suggested, 1 ^ although most investigators believe that lower molecular weight subspecies in coagulase pre¬ parations are due to proteolytic degradation of a larger protein. 17 Staphylococcal coagulases have been shown to in- duce polymerization of fibrinogen to fibrin by binding, and thereby activating, prothrombin. The coagulase-prothrombin complex causes the release of fibrinopeptides from fibrino¬ gen in a manner similar to that described for thrombin in physiological blood clotting. ^ Fibrinogen precipitation and network formation can also be induced non-enzymati- cally, e.g. by exposing fibrinogen to various highly posi¬ tively charged molecules like protamine, which interacts with specific negatively charged sites on the D-domain of fibrinogen.19 We have recently described staphylococcal compo¬ nents that interact with fibrinogen and which can be puri¬ fied from S. aureus culture supernatants.13 These are an 87 kDa coagulase and a 19 kDa fibrinogen-binding protein. The 87 and 19 kDa fibrinogen-binding proteins are essentially extracellular proteins, but can to some extent be found on the staphylococcal cell surface. Thus, these proteins can give rise to the clumping phenomenon both by inducing coa¬ gulation and by direct fibrinogen-binding. In this report we show that there are at least three distinct fibrinogen-binding proteins produced by S. aureus strain Newman, and that two of these proteins are coagulases. Results 5DS-PAGE analysis of fibrinogen binding-proteins produced at different times during staphylococcal cell growth Staphylococcus aureus strain Newman was grown in BHI or LB and samples were taken every hour for 14 h. Culture supernatants were applied onto fibrinogen-Sepharose SUBSTITUTESHEET and the eluted material was analysed on Coo assie blue- stained SDS-PAGE gels. Figure 1 shows fibrinogen-binding proteins from culture supernatants of staphylococci grown in LB under low aeration conditions. Under these condi- tions, an 87 kDa protein was produced in large amounts, mainly during the first 7 h and a 60 kDa protein appeared after 5-6 h and was produced in large amounts after 9 h of growth. Under high aeration conditions, the 87 kDa protein was produced in lower amounts and the switch to production of the 60 kDa protein accurred after only 3 h resulting in a higher production of 60 kDa protein compared to when less air was supplied to the culture. Using a rich medium like BHI, and the same high aeration conditions, this switch again accurred after 7 h (data not shown). In all cultures, the 87 kDa protein was produced mainly during the exponen¬ tial growth phase and the 60 kDa protein mainly during the post-exponential growth phase. The switch from production of the 87 kDa protein to production of the 60 kDa protein reflected the nutritional status, rather than the optical density of the culture. A 19 kDa protein was produced constitutively during these 14 h of growth (Fig. 1). SDS-PAGE, affinity- and immuno-blot analysis of affinity purified proteins Staphylococcus aureus grown in BHI for 3-4 h pro¬ duced the 87 and 19 kDa proteins but no detectable 60 kDa protein. Such culture supernatants were applied onto fibri¬ nogen-Sepharose in order to purify the 87 and 19 kDa pro¬ teins. Similarly, culture supernatants from S. aureus grown in LB for 6-8 h, containing predominantly the 60 kDa pro¬ tein but also the 87 and 19 kDa proteins, were used to purify the 60 kDa protein. The crude material was first passed over fibrinogen-Sepharose, in order to eliminate the 87 and 19 kDa proteins, and the effluent (containing the 60 kDa protein which also bound to fibrinogen-Sepharose, but SUBSTITUTESHEET to a lower extent than the 87 and 19 kDa proteins) was applied onto prothrombin-Sepharose. The 87 and 19 kDa pro¬ teins did not bind to prothrombin-Sepharose. Eluted materi¬ al from affinity purifications was subjected to SDS-PAGE and affinity-blot analysis (Fig. 2). These blots were probed with fibrinogen or prothrombin, followed by rabbit antifibrinogen or rabbit antiprothrombin sera which had been pre-incubated with S. aureus culture supernatants in order to absorb naturally occuring antistaphylococcal anti- bodies. It could thus be shown that the 87 and 19 kDa pro¬ teins bound only to fibrinogen and not to prothrombin, while the 60 kDa protein bound both fibrinogen and pro¬ thrombin. Controls were performed by incubating filters with only pre-absorbed primary antibody, omitting fibrino- gen and prothrombin (data not shown). In these controls, no 87, 60 or 19 kDa proteins were detected. By using a dilu¬ tion series both of antigen and fibrinogen or prothrombin, it was shown that the binding reactions were specific and not the result if contaminating blood proteins in the fibrinogen and prothrombin preparations. For example, 10 ng/ml of fibrinogen could detect 0.1 ng of the 87 or 60 kDa proteins in these affinity-blots. When 10 ng/ml of pro¬ thrombin was used in these tests, 0.1 ng 60-kDa protein could be detected, while a concentration of 10 μg/ml of prothrombin could not detect a 1 ng 87-kDa band (data not shown) . The anti-19 serum recognized not only the 19 kDa protein but also the 87 kDa protein and a 35 kDa protein (Fig. 3). Furthermore, there was a close resemblance be- tween blots incubated with fibrinogen followed by anti¬ fibrinogen antibody and blots incubated with anti-19 serum. Antibodies to the 60 kDa protein seem to occur naturally among several mammalian species (e.g. rabbit, goat and man; data not shown). The anti-19 serum, as well as pre-im une serum from the same rabbit, showed some re- SUBSTITUTE SHEET activity towards this 60 kDa protein. However, pre-absorp- tion with 19 kDa protein completely abolished binding to the 19 and 35 kDa bands, but not to the 60 kDa band, while antiserum pre-absorbed with 60 kDa protein reacted with the 19 and 35 kDa bands but not with the 60 kDa band (Fig. 4). Peptide mapping Proteins were purified by a combination of affi¬ nity chromatography and preparative SDS-PAGE. The purity of these preparations was confirmed on silver stained SDS-PAGE gels (Fig. 5). Dimerisation of the 19 kDa protein into a 35 kDa protein could be detected on the silver stained gels. On affinity-blots, using fibrinogen and antifibrinogen antibodies, not only the 35 kDa dimer, but also bands of higher molecular weight were detected. Upon digestion with α-chymotrypsin, the dimerisation of the 19 kDa protein was disrupted, but the 19 kDa band was left intact. This pro¬ tease did not have any apparent effect on the 87 kDa pro¬ tein, whereas the fibrinogen-binding ability of the 60 kDa protein was completely lost after treatment with α-chymo¬ trypsin. On the contrary, treatment of these proteins with staphylococcal V8 protease only partly digested the 60 kDa protein while the 87-kDa protein was digested into low molecular weight peptides (Fig. 5). NH2~terminal seguence analysis Analyses of NH2-terminal sequences revealed that the 87 kDa protein was related to previously described coa¬ gulases, while the 19 kDa protein had a unique NH2~terminal sequence. The NH2-terminal sequence of the 60 kDa coagulase was blocked (Table 1). Coagulase test Coagulase tests were performed with proteins purified by a combination of affinity chromatography and SUBSTITUTESHEET preparative SDS-PAGE. These preparations did not contain contaminations of other staphylococcal proteins as shown on silver stained SDS-PAGE gels (Fig. 5). The 87 and 60 kDa proteins coagulated rabbit plasma, while the 19 and 35 kDa proteins produced a precipitate or a weak coagulase reac¬ tion in these Table 1 NH2~terminal sequence analysis Staphylococcal strain NH2~terminal sequence S. aureus BB IVTKD YSKES RV EN SKYGT S. aureus 213 IVTKD YSKES RVNEK SKKGA S. aureus 8325-4 IVTKD YSGKS QVNAG SKΝGT S. aureus Newman 87 kDa IVTKD YSGKS QVΝAG SKΝGT S. aureus Newman 60 kDa S. aureus Newman 19 kDa SEGYG PREKK PVSIΝ HΝIVE S. aureus Newman 35 kDa M-Y- P-EKK PV- Table 2 Coagulase test tests. Dilution series of the 87 and 60 kDa proteins showed that there was a dose-response relationship. To produce a positive reaction, 25 ng of the 60 kDa protein was re- SUBSTITUTESHEET quired, while only 1 ng of the 87 kDa protein was needed to coagulate rabbit plasma within 24 h. The activities of the 87 and 60 kDa coagulases were not affected by the addition of the protease inhibitors NEM, EDTA, aprotinin and PMSF, to the rabbit plasma. In the presence of heparin, higher concentrations of both coagulases were needed for a posi¬ tive reaction. Rabbit plasma containing the protease inhi¬ bitor DFP was not clotted by either coagulase (Table 2). Identification of the 19 kDa fibrinogen-binding (fib) pro¬ tein from strain FDA 486. S. aureus strain FDA 486, which was the strain from which the library was obtained, was shown to express the 19 kDa fib protein. Affinity purified material from staphylococcal culture supernatants were analyzed in Western blots after SDS-PAGE separation (Fig. 1). The 19 kDa fibrinogen-binding protein expressed by the FDA 486 strain bound fibrinogen and the anti-fib serum, comparable to the protein purified from strain Newman. Cloning of the fib gene in E. coli. A genomic library containing DNA from S. aureus strain FDA 486 was screened with the anti-fib serum. A clone designated λfib-50 was isolated. This clone expressed a fibrinogen-binding protein of approximately 16 kDa, which bound anti-fib serum in a Western blot experiment. The fib gene was further subcloned into a pBluescript SK+vector (Fig. 2). Digestion of λfib-50 with HindiII generated 3 fragments containing staphylococcal DNA. One of these, con- tained by pBfiblll expressed the fibrinogen-binding protein (Fig. 1). The 2.4 kb insert in the pBfiblll plasmid was isolated and digested with Xbal, resulting in two fragments of 1.7 and 0.7 kb respectively. These fragments were sub¬ cloned into the pBluescript SK+ vector as well as the M13mpl8 and M13mpl9 vectors (Fig. 2). Of the resulting SUBSTITUTESHEET plasmids the pBfibT was found to express a fibrinogen- binding protein which was slightly larger than the recombi- nant protein produced by the pBfiblll plasmid (Fig. 1). The invention further comprises a microorganism containing at least one hybrid-DNA-molecule according to above. The plasmid pBfiblll in an E. coli XL has been de¬ posited at the Deutsche Sammlung von ikroorganismen (DSM), and has thereby obtained deposition number DSM Seguencing of the fib gene from S. aureus FDA 486. The fib gene contained in the Ml3 constructs was sequenced by the Sanger dideoxy-chain termination method. The pBfibT plasmid was found to contain an open reading frame of 309 bp. The pBfibJ vector contained a putative TGGAGGA ribosomal binding site situated 15-9 base pairs upstream from the ATG start codon. Furhter upstream puta¬ tive promoter sequences were identified. Computer assisted analysis revealed an open reading frame of 495 bp corres¬ ponding to 165 amino acids including a signal sequence of 29 amino acids. The first 23 amino acids in the mature pro¬ tein were identical to the sequence obtained by NH2~termi- nal analysis of the purified native protein. The predicted molecular mass of the fib protein is 15.9 kb. The complete nucleotide and deduced amino acid sequence is shown in Fig. 3. Seguencing of the fib gene from S. aureus Newman. Based on the DNA sequence obtained from the fib gene cloned from S. aureus FDA 486, primers were produced (Fig. 2). These primers were used to ampify the fib gene, both from strain FDA 486 and from strain Newman, using the polymerase chain reaction method. The resulting fragments were sequenced using fluorescent base terminators on an automatic sequenator. A comparison between the fib genes from these staphylococcal strains is shown in Fig. 4. The SUBSTITUTE SHEET sequence from the cloned fib gene from S. aureus FDA 486 was confirmed using the same sequencing strategy. A com¬ parison between the deduced amino acid sequences of the fib proteins from the two strains is shown in Fig. 5. Computer assisted analysis of the fib gene seguence. Using either the nucleotide or the amino acid sequence as a probe, no close similarity to any protein or nucleotide sequence in the University of Wisconsin Genetics Computer Group database was found. The protein showing the closest resemblance was coagulase from S. aureus. A sequence of 22 amino acids repeated twice with a spacing of 9 amino acids, located in the NH2~terminal part of the mature protein showed homology to the COOH-terminal part of coagulase, where several 27 amino acids long repeats are situated (Fig. 6). Discussion We have previously described a 87 kDa fibrinogen- binding protein which exerts coagulase activity and is pro¬ duced by S. aureus in culture supernatants .13 we have suggested that this 87 kDa coagulase and a 19 kDa fibrino¬ gen-binding protein, both of which are present on the cell surface, are involved in the clumping of S. aureus in fibrinogen. In this study we show that S. aureus strain Newman has two different types of coagulase secreted in a sequential manner during cell growth (Fig. 1). The 87 kDa coagulase was produced early during growth and was later replaced by the 60 kDa coagulase. The rate at which this switch occurred varied with growth rate and type of media used, i.e. under low aeration conditions or in a rich medi¬ um this switch was postponed (data not shown). This suggests that the presence of some environmental factor(s) induces the production of the 87 kDa protein and suppresses 60 kDa protein production. It is likely that the 87 kDa SUBSTITUTE SHEET coagulase is negatively regulated by the agr locus together with protein A. 0 It was concluded from the results of the analyses by SDS-PAGE and i munoblotting of proteins purified by affinity chromatography that both the 60 and 87 kDa pro¬ teins bound fibrinogen, but only the 60 kDa protein bound prothrombin (Fig. 2). This is contradictory to our previous results where the 87 kDa protein was shown to bind pro¬ thrombin.13 in these earlier experiments, 10 μg/ml pro- thrombin was used. This was unfortunate, as we have since shown that contamination with 1 ng/ml fibrinogen can detect a band of 100 ng of fibrinogen-binding protein in immuno- blot experiments. When antigens were diluted to 1 or 0.1 ng per band and ligands were used at 10 ng/ml, background due to contamination in these preparations was eliminated (data not shown) . Thus the following nucleotide sequence is present in the gene coding for said protein: GAGCGAAGGA TACGGTCCAA GAGAAAAGAA ACCAGTGAGT ATTAATCACA ATATCGTAGA GTACAATGAT GGTACTTTTA AATATCAATC TAGACCAAAA TTTAACTCAA CACCTAAATA TATTAAATTC AAACATGACT ATAATATTTT AGAATTTAAC GATGGTACAT TCGAATATGG TGCACGTCCA CAATTTAATA AACCAGCAGC GAAAACTGAT GCAACTATTA AAAAAGAACA AAAATTGATT CAAGCTCAAA ATCTTGTGAG AGAATTTGAA AAAACACATA CTGTCAGTGC ACACAGAAAA GCACAAAAGG CAGTCAACTT AGTTTCGTTT GAATACAAAG TGAAGAAAAT GGTCTTACAA GAGCGAATTG ATAATGTATT AAAACAAGGA TTAGTGAG whereby this nucleotide sequence encodes for the following protein starting at nucleotide 243: (In Fig. 3 nucleotides 156-242 encode a signal peptide. ) SEGYGPREKK PVSINHNIVE YNDGTFKYQS RPKFNSTPKY IKFKHDYNIL EFNDGTFEYG ARPQFNKPAA KTDATIKKEQ KLIQAQNLVR EFEKTHTVSA HRKAQKAVNL VSFEYKVKKM VLQERIDNVL KQGLVR SUBSTITUTE SHEET Although antisera to the 19 kDa protein re¬ cognized the 87 kDa protein (Fig. 3), pre-absorbtion with 19 kDa protein, which could eliminate the binding to the 19 kDa protein, could not completely abolish this binding to the 87 kDa protein. In addition, antisera to the 87 kDa protein did not specifically recognize the 19 kDa protein (data not shown) . The immunological cross-reactivity could be due to structural similarities in the fibrinogen-binding sites of these proteins. Antisera to the 19 kDa protein also recognized the 35 kDa protein (Fig. 3). We have pre¬ viously shown that the 19 kDa protein spontaneously forms 35 kDa dimers (not reducible with 2-mercapto ethanol) and to a lesser extent higher molecular weight bands that seem to be trimers and tetramers of this protein.13 Minor bands in the preparation could thus be due to further aggregation of the 19 kDa protein or to degradation of the 87 kDa pro¬ tein (Fig. 3). By pre-absorbing the rabbit anti-19 serum with either 19 or 60 kDa proteins, it was shown that there were no shared antigenic epitopes between the 60 kDa pro- tein and 19 kDa protein (Fig. 4). It is likely that anti¬ bodies against the 60 kDa protein are present in the most normal rabbit sera. This reactivity is not due to unspeci- fic binding to immunoglobulins . The purified 60 kDa protein did not bind control antibodies in immunoblots, and was thus shown not to contain protein A activity. Peptide mapping analysis suggested that the 87, 60 and 19 kDa proteins are not closely related (Fig. 5). It was shown that digestion with α-chymotrypsin and staphylococcal V8 protease gave different peptide banding patterns with the three different proteins, and that the 60 kDa protein completely lost its ability to bind fibrinogen upon digestion with α-chymotrypsin, whereas the 87 and 19 kDa proteins were unaffected. Analyses of NH2-terminal sequences suggested that the 87 kDa coagulase is identical to the coagulase from SUBSTITUTE SHEET strain 8325-4 (Table 1). This is in agreement with the fact that these strains produce coagulases of the same serotype. The NH2~terminal sequence of the 19 kDa protein was not homologous to any of the previously described coagulases. Computer-assisted analysis revealed that this NH2~terminal sequence is a new, unique sequence.21 The strong similarity between this sequence and the NH2~terminal sequence of the 35 kDa protein further strengthens the evidence that the 35 kDa protein is a di er of the 19 kDa protein. The 60 kDa coagulase seems to represent a third type of fibrinogen- binding protein from S. aureus . In the coagulase test, it was shown that highly purified preparations of both the 87 and 60 kDa proteins exerted coagulase activity (Table 2). There was a clear dose-response relationship when dilution series of the 87 and 60 kDa proteins were subjected to coagulase tests (data not shown). It appears that the 87 kDa coagulase had a higher specific activity than the 60 kDa coagulase; how¬ ever, the treatment of these proteins during purification could have influenced their coagulase activities (for example the 60 kDa coagulase seemed to be more sensitive than the 87 kDa coagulase to exposure to the acetic acid used in the elution step) . These clotting reactions were due to true coagulase reactivity, since no inhibition could be achieved by addition of protease inhibitors .2 ,23 Addi¬ tion of heparin influenced the clotting of both coagulases, such that a higher concentration of coagulase was needed for positive tests. Since heparin inhibits physiological blood clotting, we suggest that this negative effect is not due to direct interaction of heparin with staphylococcal coagulases. DFP, which is a powerful protease inhibitor, has previously been shown to inhibit both thrombin- and coagulase-mediated plasma clotting.2 Addition of DFP to rabbit plasma inhibited clotting by both coagulases. SUBSTITUTE SHEET The amino acid sequence of a 43 kDa fragment from the S. aureus 213 (serotype II) coagulase has been deter¬ mined. 5 τh e coagulase activity is contained in the NH2- terminal region of this molecule, and the prothrombin- binding capacity resides further downstream within this region.26 Coagulases from S. aureus BB (serotype I), S. aureus 213 and S. aureus 8325-4 (serotype III) have recent¬ ly been expressed in E. coli.27~29 ι j > h e amino acid sequences of these recombinant coagulases reveal a quite strong homo- logy (>90%) in the C-terminal halves of the molecules, while the NH2~terminal regions show only approximately 50% homology.30 The gene clones encoding these coagulases do not contain sequences that correspond to the NH2~terminal sequence of the 19 kDa protein. The 87 kDa coagulase from strain Newman (serotype III) seems to be identical to the coagulase from S. aureus 8325-4. Coagulases of serotypes I and II have been shown to bind human prothrombin.17 r18 However, it has not been established if the coagulase from strain 8325-4 binds prothrombin. It is interesting, how- ever, that a coagulase from strain Newman, a strain of human origin, does not bind human prothrombin, although our preliminary results indicate that prothrombin is required for its function.13 it is possible that the 60 kDa coagu¬ lase, which has a strong affinity for prothrombin and a somewhat weaker affinity for fibrinogen (as compared to the 87 kDa coagulase), has a different mode of action than the 87 kDa coagulase. The ability of the 19 kDa fibrinogen- binding protein to oligomerize, to precipitate fibrinogen in plasma and to some extent coagulate plasma, suggests that this protein could affect fibrinogen in the same manner as paracoagulating substances have been suggested to In conclusion, S. aureus strain Newman produces two distinct fibrinogen-binding coagulases . These are pro- duced in a sequential manner during growth and have dif- SUBSTITUTE SHEET ferent binding and antigenic properties. A third fibrino¬ gen-binding protein is a 19 kDa protein, which spon¬ taneously forms di ers and larger aggregates. The role of coagulases and clumping factors (or fibrinogen-binding pro- teins as we suggest is the correct designation for these proteins) in staphylococcal virulence and pathogenicity has not yet been established. However, in our preliminary study, 90% of 40 S. aureus isolates from wound infections had coagulase activity, and among these >60% produced the 87 kDa protein. It is notable that coagulases are produced in large amounts by S. aureus and in such a fashion that there is always one type of coagulase present in the cul¬ ture medium. The fact that these proteins interact specifi¬ cally with host proteins makes coagulases interesting sub- jects for further study. An E. coli clone expressing the 19 kDa protein has been isolated and sequence determination is in progress in our laboratory. Binding of staphylococci to fibrinogen on coated coverslips or on catheters has been described (Cheung and Fischetti, 1990; Cheung et al., 1991; Herrmann et al., 1988; Kuusela et al., 1985; Mohammad et al., 1988; Vaudaux et al., 1989). It is also a well known fact that most Staphylococcus aureus clump in the presence of fibrinogen. It has been suggested that this clumping reaction involves a small peptide on the C00H-terminal part of the gamma chain on the fibrinogen molecule (Strong et al . , 1982). On the other hand, the fibrinogen-binding component of the staphylococci remained elusive for a long time. It was suggested that a fibrinogen-binding protein would be attached to the staphylococcal cell surface (Duthie, 1954; Espersen, 1987; Jeljaszewicz et al. , 1983). We have identi¬ fied 3 different fibrinogen-binding proteins from Staphy¬ lococcus aureus , all of which can be found on the staphy¬ lococcal cell surface (Boden and Flock, 1992). However, these proteins cannot be described as cell surface proteins SUBSTITUTE SHEET because they are mainly expressed extracellularly. In addi¬ tion one of the identified fibrinogen-binding proteins was found to be coagulase, a well known extracellular staphy¬ lococcal protein. The other fibrinogen-binding proteins were a second type of coagulase of 60 kDa and a 19 kDa fibrinogen-binding protein without coagulase activity. In this report we describe the cloning, expression and se¬ quencing of this 19 kDa protein. Expression of recombinant protein from the λ cloned isolated and the subclones of this was investigated (Fig. 1). The λ clone and the pBfiblll subclone both ex¬ pressed a 15 kDa protein which bound fibrinogen and the anti-fib serum, whereas strain pBfibT expressed a protein of approximately 18 kDa. This was due to the fact that this construct contained a fusion product between the β-gal pro¬ tein and the fib protein, lacking the first 33 amino acids. All recombinant proteins seemed to bind fibrinogen to a lesser extent than the native protein from S. aureus, since the binding of fibrinogen gave a weaker response than the binding of anti-fib serum in immunoblots . The opposite was true for the native protein. The nucleotide sequence of the intact fib gene revealed an open reading frame of 165 amino acids, in¬ cluding a signal sequence of 29 amino acids (Fig. 3). The signal sequence had the characteristics of a typical signal sequence (Pugsley, 1989), such as a net positive charged region spanning the first 8 residues at the NH2-terminus, a central core of 5 hydrophobic and 9 neutral residues with a strong probability of forming an α-helix, a turn-inducing alanine 6 residues downstream from the cleavage site, alanines at position -3 and -1 before the cleavage site and glutamic acid at position +2 in the mature protein, creating a net positive charge difference between the NH2- terminus of the signal peptide and the NH2~terminus of the mature polypeptide. The first 23 amino acids of the mature SUBSTITUTE SHEET protein were completely homologous to the sequence obtained by NH2-terminal sequence analysis of purified fib protein (Boden and Flock, 1992). In addition, putative promote sequences and ribosomal binding site could be identified. However, there were no obvious transcription termination sequences and instead a second ribosomal binding site was identified 150 nucleotides downstream of the fib gene. This second Shine-Dalgarno sequence was followed by a start codon and a signal sequence which was homologous to the signal peptide of the fib gene. This putative peptide was not homologous to any previously described protein. One could speculate however, that these proteins are coded for by the same polycistronic transcript and thus regulated in the same way. According to our previous findings the fib protein seems to be constitutively expressed in S. aureus Newman (Bodέn and Flock, 1992). Since the previously studied protein was purified from S. aureus strain Newman the fib gene from this strain was also sequenced. Comparison between the two sequences show a remarkable resemblance (Fig. 5). The only differen¬ ces being two amino acids in the signal sequence and the conservative change of the basic amino acid arginine for a likewise basic lysine at the very COOH-terminal end. On the nucleotide level there is one additional conservative nucleotide change apart from the three changes on amino acid level. Downstreams of the structural gene there are major differences between the two strains in a stretch of approximately 80 nucleotides. Closer to the putative second peptide in this transcript further downstreams the simi- larity is again higher, indicating that there is a simi¬ larity between the two strains also regarding this second peptide. Computer assisted analysis of the deduced amino acid sequence suggested that there was a high probability for formation of an α-helix in the signal sequence as well SUBSTITUTE SHEET as in the last 60 to 70 residues of the COOH-terminus ( ) . In addition the sequence NST at position 64-66, indicates that there is a glycosylation site. A glycosylation of the native protein could account for the discrepancy between the size of the native and the recombinant protein. When comparing the amino acid sequence of the fib protein to other known sequences in the database of the University of Wisconsin Genetics Computer Group, the protein with the highest similarity is coagulase from S. aureus. Coagulase from three different serorypes has been cloned and sequenced (Bodέn and Flock, 1992; (Kaida et al., 1989; (Kaida et al., 1987; (Phoni daeng et al., 1990). All of these contain repeated sequences of 27 residues in the COOH-terminus. The coagulase from strain Newman has been cloned (), but the sequence has not yet been published. A construct containing the 177 most NH2~terminal amino acids of the Newman coagulase was shown to bind fibrinogen (McDevitt et al., 1992). In the fib protein a sequence of 22 amino acids, repeated twice with a spacing of 9 amino acids, located in the NH2~terminal part of the mature pro¬ tein showed homology to the COOH-terminal part of coagulase (Fig. 6 ) . We suggest that this region is responsible for the fibrinogen binding. Materials and methods Bacterial strains and culture conditions. Staphylococcus aureus Newman was kindly provided by M. Lindberg, Swedish University of Agricultural Sciences, Upp- sala, Sweden. Staphylococci were grown overnight in Brain Heart Infusion (BHI) medium (Difco Laboratories, Detroit, MI) or in Luria-Bertani (LB) medium. After centrifugation, the bacterial pellet was resuspended in 20 culture volumes of freshly prepared BHI or LB and grown at 37°C with SUBSTITUTE SHEET constant shaking in Ehrlenmeyer flasks (low aeration) or in indented flasks (high aeration). Affinity chromatography. Staphylococcal proteins were affinity purified as described previously. 0 Briefly, fibrinogen-Sepharose and prothrombin-Sepharose were pre¬ pared by coupling human fibrinogen (IMCO, Stockholm, Sweden) or human prothrombin (Sigma Chemical Co, St. Louis, MO) to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden), by the procedure recommended by the manufacturer. The Sepharose was equilibrated with phosphate-buffered saline (PBS;145 mM NaCl, 10 mM phosphate, pH 7.4) con¬ taining 0.05% Nonidet P-40. Staphylococcal culture super¬ natants supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM EDTA and 0.05% Nonidet P-40 were applied. The absorbed material was eluted with 0.7% acetic acid containing 0.05% Nonidet P-40. The eluted material (eluate) was concentrated in Centricon microconcentrators (Amicon, Danvers, MA) or by acetone precipitation. SDS-PAGE, affinity- and immuno-blotting. SDS-PAGE and subsequent diffusion blotting was performed using the PhastSystem (Pharmacia) as described previously.10 Nitro¬ cellulose filters were incubated for 1 h at room tempera- ture with human fibrinogen or human prothrombin at con¬ centrations between 1 ng/ml and 10 μg/ml in PBS supple¬ mented with 0.05% Tween 20. Primary antibodies [rabbit anti (human)fibrinogen (Dakopatts, Glostrup, Denmark), rabbit anti(human)prothrombin (Dakopatts), and rabbit anti-19 kDa protein] were diluted 1:1000 and incubated with the filters for 2 h. The rabbit anti-19 kDa protein antibodies (anti-19 serum) were obtained by subcutaneous immunization of rabbits with a highly purified 19 kDa protein preparation emulsified in complete Freund's adjuvant. In order to eliminate naturally occurring antistaphylococcal antibodies SUBSTITUTE SHEET in rabbit antifibrinogen or rabbit antiprothrombin anti¬ sera, these were pre-absorbed with staphylococcal culture supernatants from cells grown in LB for 6 h. Undiluted antisera was added to 10 volumes of culture supernatant and incubated at room temperature for 1 h or at 4°C for 4 h be¬ fore diluting the antibody to the appropriate concentra¬ tion. The anti-19 serum was absorbed with 19 or 60 kDa pro¬ teins purified from preparative gels. The gel slices were homogenised in PBS containing 0.1% Nonidet P-40 before being added in a 10-fold excess to the antisera and in¬ cubated as described above. Alkaline phosphatase (ALP) con¬ jugated goat anti-rabbit immunoglobulin G antibodies (Sigma) were diluted 1:1000 and incubated with the filters for 1 h. The ALP reaction was developed in 100 mM Tris hydrochloride (pH 8.0) containing 10 mM MgCl2, 0.02 mg α- naphtylphosphate per ml (E. Merck AG, Darmstadt, Germany) and 0.02 mg Fast Blue (Merck) per ml for 10-20 min. Purification of proteins. The 87, 60 and 19 kDa protein were purified from preparative SDS-PAGE gels by eluting proteins from gel slices in a Model 422 Electro- Eluter (Bio-Rad, Hercules, CA) . Fragmentation of proteins by proteases . Proteins were digested with 40 μg/ml of α-chymotrypsin or staphylo¬ coccal V8 protease (Sigma) for 1 h on ice. Coagulase test. Coagulase tests were performed in Difco Coagulase Plasma (Difco) with or without the presence of the protease inhibitors N-ethylmaleimide (NEM; 2 mM) , EDTA (6 mM), aprotinin (Sigma; 200 U/ml) (14), heparin (Sigma; 40 U/ml) (15), and PMSF (1 mM). Coagulate tests were also performed in the presence of 5 mM diisopropyl fluorophosphate (DFP; Janssen Chimica, Beerse, Belgium) (16). SUBSTITUTE SHEET Determination of NH2~terminal seguences . Samples were analysed in a 470 Protein Sequencer (Applied Bio- systems, Foster City, CA) . Bacterial strains and cloning vectors . Escherichia coli strain Y1090 (Clontech, Palo Alto, CA) was used to for the screening of the λgt-11 library (Clontech) containing genomic DNA from Staphylo¬ coccus aureus strain FDA 486. For subcloning E. coli strains XL-1 and JM103 were used with the cloning vectors Bluescript SK+ (Stratagene, La Jolla, CA), pGEM7Zf(+) (Promega) and M13mpl8 or M13mpl9 (Promega). Media and chemicals . E. coli were grown in Luria Bertani medium at 37°C. Ampicillin (50 μg/ml) and tetracyclin (5 g/ml) were added when appropriate. Restriction enzymes were purchased from Promega. IPTG and X-gal were from Boehringer-Mannheim. All other chemicals were purchased from Sigma (Sigma Che i- cal Co, St. Louis, MO) or Merck (E. Merck AG, Darmstadt, Germany) . Affinity chromatography Staphylococcal proteins were affinity purified as described previously (Bodέn and Flock, 1989). Briefly, fibrinogen-Sepharose was prepared by coupling human fibri¬ nogen (IMCO, Stockholm, Sweden) to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden), by the procedure recom¬ mended by the manufacturer. The Sepharose was equilibrated with phosphate-buffered saline (PBS; 145 mM NaCl, 10 mM phosphate, pH 7.4) containing 0.05% Nonidet P-40. Staphylococcal culture supernatants supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM EDTA and 0.05% Nonidet P-40 were applied. The absorbed material was eluted SUBSTITUTE SHEET with 0.7% acetic acid containing 0.05% Nonidet P-40. The eluted material was concentrated by acetone precipitation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), affinity- and immuno-blotting. SDS-PAGE and subsequent diffusion blotting was performed using the Phast System (Pharmacia) as described previously (Bodέn and Flock, 1989). Nitrocellulose filters were incubated for 1 hour at room temperature with human fibrinogen at 10 μg/ml in PBS supplemented with 0.05% Tween 20. Primary antibodies (rabbit anti(human)fibrinogen [Dakopatts, Glostrup, Denmark] and rabbit anti-fib protein) were diluted 1:500 or 1:1000 and incubated with the filters for 2 hours. The rabbit anti-fib protein antibodies (anti- fib serum) were obtained by subcutaneous immunization of rabbits with a highly purified 19-kDa protein preparation emulsified in complete Freund's adjuvant. Alkaline phos- phatase (ALP) conjugated goat anti-rabbit im unoglobulin G antibodies (Sigma) were diluted 1:1000 and incubated with the filters for 1 hour. The ALP reaction was developed in 100 mM Tris hydrochloride (pH 8.0) containing 10 M MgCl2, 0.02 mg α-naphtylphosphate per ml (E. Merck AG, Darmstadt, Germany) and 0.02 mg Fast Blue (Merck) per ml for 10-20 min. Screening of the λgt-11 library. Plates were grown and induction with IPTG was performed according to the protocol recommended by the manufacturer (Clontech). Nitrocellulose filters (Schleicher and Schull) were incubated with anti-fib sera diluted 1:500, as described above. DNA seguencing and seguence analysis. The DNA sequence was determined by the dideoxy- chain termination method (Sanger et al 1977), using SUBSTITUTE SHEET [α-35s]dATP (Amersham Corp.) and Sequenase 2.0 (United States Biochemical Corporation (USB), Cleveland, OH). Recombinant M13mpl8 or M13ir.pl9 phage was used as template. Ml3 Universal primer (USB) as well as custom made primers from the Unit for. Nucleotide Synthesis, CBT, Novum (Huddinge, Sweden) were used as sequencing primers. The sequencing reaction products were resolved on 8% poly- acrylamide-urea gels. Gels were run at 40W for 1-3 h on a Sequencing Unit from Cambridge Electrophoresis Ltd (Cam- bridge, England), fixed in 10% methanol, 10% acetic acid for 15 min and dried on Whatman 3MM papers under vacuum. DNA bands were visualized by autoradiography. DNA fragments containing the fib gene from strain FDA 486 and strain Newman were produced by the polymerase chain reaction (PCR) using a Perkin Elmer Cetus DNA Thermal Cycler (Perkin Elmer, Norwalk, CT) and Taq polymerase (Boehringer- Mannheim) . The PCR generated DNA fragments were sequenced in an Applied Biosystems 373A DNA Sequencer (Applied Biosystems Inc., Foster City, CA) using fluorescent nucleotide terminators ( ) . Computer assisted analysis of DNA sequences was performed with GCG software package (Genetics Computer Group 1991) and with Seq Ed software (ref). Competition in fibrinogen binding The three different fibrinogen binding proteins from S. aureus 19, 60 and 87 kD are identified. We show that each of the FgBPs, separately or together with an additive effect can block S. aureus binding to immobilized fibrinogen in vitro. In short, the experimental procedure was as follows: Microtiter plates were coated with fibrino¬ gen free of contaminating plasma components (IMCO). After- coating to block non specific adherence was done with BSA. Radiolabelled bactera were added, 2-5 x 106 per well. Simultaneously, various amounts of 19, 60 and/or 87 kD FgBP SUBSTITUTESHEET were added. Bacterial adherence was measured after two hours incubation. Figure 12: Binding of radiolabelled S. aureus to fibrinogen in the presence of various amounts of 19 kD FgBP. Figure 13: Binding of radiolabelled S. aureus to fibrinogen in the presence of various amounts of 60 kD FgBP. Figure 14: Binding of radiolabelled S. aureus to fibrinogen in the presence of various amounts of 87 kD FgBP. Figure 15: Binding of radiolabelled S. aureus to fibrinogen in the presence of various combinations of FgBPs. Incidence of FgBPs The incidence of the 19 and the 87 kD FgBPs were measured. Thirty nine S. aureus isolates of human origin and thirty seven bovine mastitis isolates, taken from a wide variety of sources, were tested by PCR for the gene and in affinity blotting for the proteins. All (100%) of the human isolates were positive in both PCR and affinity blotting for the 19 kD protein and 95% were positive for the 87 kD (only tested by affinity blotting). Of the bovine isolates, 45% were positive in affinity blot for the 19 kD but 95% in PCR (some variation in the genome size was found). Fifty five were positive for the 87 kD FgBP. Vaccination The 19 and 87 kD proteins in combination were used to immunize mice which were subsequently subjected to experimental mastitis caused by S. aureus . A control group was given only the adjuvant (Freund's). Histopathological SUBSTITUTESHEET examination and bacterial count was performed after 24 hours. A significant (p<0.05) difference in the number of colonizing bacteria was found between the two groups. The present fibrinogen binding proteins can be used in immunization, whereby the proteins, preferably in combination with a fusion protein in order to form a larger antigen to react upon, are injected in doses creating an immunological reaction in the host mammal. Thus the fibri¬ nogen binding proteins can be used in vaccination of mam- mals to protect against infections caused by staphylococcal infections. Further, the fibrinogen binding proteins can be used to block an infection in an open skin lesion. Wounds can be treated by using a suspension comprising the fibri- nogen binding protein. Thus the fibrinogen binding proteins can be used to treat wounds, e.g., for blocking bacterial binding sites in fibrinogen, or for immunization (vaccina¬ tion) . In the latter case the host produces specific anti¬ bodies which can protext against attachment by bacterial strains comprising such fibrinogen binding proteins. Hereby the antibodies block the adherence of the bacterial strains to damaged tissue. Examples of colonizing of tissue damage are: a) colonizing of wounds in skin and connective tissue, which wounds have been caused by a mechanical trauma, chemical damage, and/or thermical damage; b) colonizing of wounds on mucous membranes such as in the mouth cavity, or in the mammary glands, urethra or vagina; c) colonizing of connective tissue proteins, which have been exposed by minimal tissue damage (micro lesions ) in connection with epithelium and endothelium (mastitis, heart valve infection, hip exchange surgery). When using the present fibrinogen binding pro- teins, prepared by isolation from living cells, by means of SUBSTITUTESHEET hybrid-DNA technique, or synthesized, for immunization (vaccination) in mammals, including humans, the proteins, or polypeptides thereof, are dispersed in sterile isotonic saline solution, optionally while adding a pharmaceutically acceptable dispersing agent. Different types of adjuvants can further be used in order to sustain the release in the tissue, and thus expose the protein for a longer period of time to the immuno defence system of a body. A suitable dose to obtain immunization is 0.5 to 5 /μg of fibrinogen binding protein per kg body weight and injection at immunization. In order to obtain durable immu¬ nization, vaccinations should be carried out at consecutive occasions with an interval of 1 to 3 weeks, preferably at three occasions. Adjuvants are normally not added when re- peating the immunization treatment. When using the present fibrinogen binding pro¬ teins or polypeptides thereof for local topical administra¬ tion the protein is dispersed in an isotonic saline solu¬ tion to a concentration of 25 to 250 /μg per ml. The wounds are then treated with such an amount only to obtain a comp¬ lete wetting of the wound surface. For an average wound thus only a couple of millilitres of solution are used in this way. After treatment using the protein solution the wounds are suitably washed with isotonic saline solution or another suitable wound treatment solution. Further the fibrinogen binding protein, or synthetized polypeptide thereof can be used to diagnoze bacterial infections caused by Staphylococcus aureus strains, whereby a fibrinogen binding protein of the present invention is immobilized on a solid carrier, such as small latex or Sepharose R beads, whereupon sera con¬ taining antibodies are allowed to pass and react with the fibrinogen binding protein thus immobilized. The agglutina¬ tion is then measured by known methods. SUBSTITUTESHEET Further the fibrinogen binding protein or poly- peptide can be used in an ELISA test (Enzyme Linked Immuno Sorbent Assay; E Engvall, Med. Biol. 5_5, 193 (1977)). Here¬ by wells in a polystyrene microtitre plate are coated with the fibrinogen binding protein and incubated over night at 4°C. The plates are then thoroughly washed using PBS con¬ taining 0.05% Tween 20, and dried. Serial dilutions of the patient serum made in PBS-Tween, are added to the wells, and are incubated at 30°C for 1.5 hrs. After rinsing anti- human IgG conjugated with an enzyme, or a horseradish peroxidase, or an alkaline phosphatase is added to the wells and further incubated at 30°C for 1.5 hrs. During these incubations IgG from patient serum, and added anti- human IgG-enzyme conjugate, respectively, has been bound thereto. After rinsing, an enzyme substrate is added, p- nitrophosphate in case of an alkaline phosphatase, or orthophenylene diamine substrate (OPD) in case a peroxidase has been used, respectively. The wells of the plates are then rinsed using a citrate buffer containing 0.055% OPD, and 0.005% H 2 0 2 , and incubated at 30°C for 10 min. The enzyme reaction is stopped by adding a 4N solution of H2SO4 to each well. The colour development is measured using a spectrophotometer. Depending on the type of enzyme substrate used a fluoroscence measurement can be used as well. Another method to diagnoze £>_;_ aureus infections is by using the DNA gene probe method based on the nucleo¬ tide sequence for the fibrinogen binding protein or part thereof. Thereby the natural or synthetic DNA sequence is attached to a solid carrier, such as a nitrocellulose filter, a nylon filter, or a polystyrene plate as mentioned above, by e.g., adding a body fluid, to the surface. The DNA gene probe, optionally labelled enzymatically, or by a radioactive isotope, is then added to the solid surface plate comprising the DNA sequence, whereby the DNA gene SUBSTITUTESHEET probe attaches to the membrane associated sequence where appearing. The enzyme or radioactive isotope can readily be determined by known methods. Above the term fibrinogen binding protein in- eludes any of polypeptide thereof as well, which constitute the minimal fibrinogen binding site of the complete pro¬ tein. The fibrinogen binding protein/s can be used for raising antibodies by administering the protein and then isolating said antibodies, whereupon these are administered for passive immunization purposes . SUBSTITUTE SHEET References 1. Duthie ES. Evidence for two forms of staphylococcal coagulase. J Gen Microbiol 1954;10:427-36. 2. Gorrill RH, Klyhn KM, McNeil EM. The initiation of infection in the mouse kidney after intravenous in¬ jection of bacteria. J Path Bacteriol 1966;91:157-72. 3. Johanovsky J. The significance of the clumping factor for pathogenicity of staphylococci. Folia Biol (Prague) 1957;3:338-42. 4. Kapral FA. Factors involved in experimental staphylo¬ coccal peritonitis. Ann NY Acad Sci 1965;128:259-73. 5. Proctor RA, Christman G, Mosher DF. Fibronectin- induced agglutination of Staphylococcus aureus corre¬ lates with invasiveness. J Lab Clin Med 1984; 104:455-69. 6. Espersen F, Clemmensen I. Clumping of Staphylpcoccus aureus by human fibronectin. Acta Pathol Microbiol Scand [B] 1981;89:317-21. 7. Espersen F, Schiotz PO. Normally-occurring precipi- tating antibodies against Staphylococcus aureus in human serum and colostrum. Acta Pathol Microbiol Stand [C] 1981;89:93-8. 8. Ljung A, Hjertέn S, Wadstrδm T. High surface hydro- phobicity of autoaggregating Staphylococcus aureus strains isolated from human infections studied with the salt aggregation test. Infect Immun 1985; 47:522-6. 9. Jeljaszewicz J, Switalski LM, Adlam C. Staphylo- coagulase and clumping factor. In: Easmon CSF, Adlam C, eds . Staphylococci and staphylococcal infections, vol. 2. London: Academic Press, 1983; 525-57. 10. Cheung AL, Krishnan M, Jaffe EA, Fischetti VA. Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial cells. J Clin Invest 1991;87:2236-45. SUBSTITUTE SHEET 11. Cheung AL, Fischetti VA. The role of fibrinogen in staphylococcal adherence to catheters in vitro. J Infect Dis 1990;161:1177-86. 12. Vaudaux P, Pittet D, Haeberli A et al . Host factors selectively increase staphylococcal adherence on in¬ serted catheters: a role for fibronectin and fibrino¬ gen or fibrin. J Infect Dis 1989;160:865-75. 13. Bodέn MK, Flock J-I. Fibrinogen-binding protein/ clumping factor from Staphylococcus aureus . Infect Immun 1989;57:2358-63. 14. Jacherts D. Experimentelle Untersuchungen ϋber die Identitat freier und gebundener coagulase. Hyg Infektionskr 1956;142:502-9. 15. Ushioda H, Terayama T, Sakai S, Zen-Yoji H, Nishiwaki M, Hidano A. Coagulase typing of Staphylococcus aureus and its application in routine work. In: Jeljaszewicz J, ed. Staphylococci and staphylococcal infections, Suppl. 10. Gustav Fischer Verlag, Stuttgart, Federal Republic of Germany, 1981;77-83. 16. Reeves MW, Drummond MC, Tager M. Partial purification and characterization of the multiple molecular forms of staphylococcal clotting activity (coagulase). J Bacteriol 1981;148:861-8. 17. Kawabata S, Morita T, Iwanaga S, Igarashi H. Enzymatic properties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin. J Biochem 1985;98:1603-14. 18. Hendrix H, Lindhout T, Mertens K, Engels W, Hemker HC. Activation of human prothrombin by stoichiometric levels of staphylocoagulase. J Biol Che 1983;258: 3637-44. 19. Okano K, Saito Y, Matsushima A, Inada Y. Protamine interacts with the D-domains of fibrinogen. Biochim Biophys Acta 1981;671:164-7. SUBSTITUTE SHEET 20. Peng H-L, Novick RP, Kreiswirth B, Kornblum J, Schlievert P. Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus . J Bacteriol 1988;170:4365-72. 21. Devereux, Haeberli, Smithies. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 1984;12:387-95. 22. Heczko PB, Wegrzynowicz A, Bulanda M, Jeljaszewicz J, Pulverer G. Taxonomic implications of the pseudo- coagulase activity of staphylococci. In: Jeljaszewicz J, ed. Staphylococci and staphylococcal infections, Suppl. 10. Gustav Fischer Verlag, Stuttgart, Federal Republic of Germany, 1981;43-7. 23. Wegrzynowicz Z, Heczko PB, Jeljaszewicz J, Neugebauer M, Pulverer G. Pseudocoagulase activity of staphylo¬ cocci. J Clin Microbiol 1979;9:15-19. 24. Drummond MC, Tager M. Enzymatic activities associated with clotting of fibrinogen by staphylocoagulase and coagulase-reacting factor and their inhibition by diisopropylfluorophosphate. J Bacteriol 1962;83:975- 80. 25. Kawabata S, Miyata T, Morita T, Miyata T, Iwanaga S, Igarashi H. The amino acid sequence of the procoa- gulant- and prothrombin-binding domain isolated from staphylocoagulase. J Biol Chem 1986;261:527-31. 26. Kawabata S, Morita T, Miyata T, Iwanaga S, Igarashi H. Isolation and characterization of staphylocoagulase chymotryptic fragment. J Biol Chem 1986;261:1427-33. 27. Kaida S, Miyata T, Yoshizawa Y et al. Nucleotide sequence of the staphylocoagulase gene: its unique COOH-terminal 8 tandem repeats. J Biochem 1987; 102: 1177-86. SUBSTITUTESHEET 28. Kaida S, Miyata T, Yoshizawa Y, Igarashi H, Iwanaga S. Nucleotide and deduced amino acid sequences of staphylocoagulase gene from Staphylococcus aureus strain 213. Nucleic Acids Res 1989;17:8871. 29. Phonimdaeng P, O'Reilly M, O'Toole PW, Foster TJ. Molecular cloning and expression of the coagulase gene of Staphylococcus aureus 8325-4. J Gen Microbiol 1988;134:75-83. 30. Phonimdaeng P, O'Reilly M, Nowlan P, Bramley AJ, Foster TJ. The coagulase of Staphylococcus aureus 8325-4. Sequence analysis and virulence of site- specific coagulase-deficient mutants. Mol Microbiol 1990;4:393-404. 31. Carr ME, Gabriel DA, Herion JC, Roberts HR. Granulo- cyte lysosomal cationic proteins alters fibrin assembly: a possible mechanism for granulocyte control of clot structure. J Lab Clin Med 1986;107:199-203. 32. Boden MK, Flock J-I. (1992). Evidence for three different fibrinogen-binding protein with unique pro- perties from Staphylococcus aureus strain Newman. Microbial Pathogen. 12(4 ) :289-298. 33. Duthie ES. (1954). Evidence for two forms of staphylococcal coagulase. J Gen Microbiol. 10:427-436. 34. Espersen F. (1987). Interactions between human plasma proteins and cell wall components of Staphylococcus aureus. Dan Med Bull. 34(2):59-69. 35. Hawiger J, Kloczewiak M, Timmons S. (1983). Inter¬ action of fibrinogen with staphylococcal clumping factor and with platelets. Ann N Y Acad Sci . 408:521- 535. SUBSTITUTE SHEET 36. Hermann M, Jaconi ME, Dahlgren C, Waldvogel FA, Stendahl 0, Lew DP. (1990). Neutrophil bacterici¬ dal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms. J Clin Invest. 86(3) :942-51. 37. Herrmann M, Vaudaux PE, Pittet D, Auckenthaler R, Lew PD, Schumacher PF, Peters G, Waldvogel FA. (1988). Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material. J Infect Dis. 158(4): 693-701. 38. Hook M, McGavin MJ, Switalski LM, Raja R, Raucci G, Lindgren PE, Lindberg M, Signas C. (1990). Inter¬ actions of bacteria with extracellular matrix proteins. Cell Differ Dev. 32(3):433-8. 39. Kuusela P. (1978). Fibronectin binds to Staphylo¬ coccus aureus . Nature (London). 276:718-720. 40. Kuusela P, Vartio T, Vuento M, Myhre EB. (1985). Attachment of staphylococci and streptococci on fibronectin, fibronectin fragments, and fibrinogen bound to a solid phase. Infect Immun. 50(1):77-81. 41. McDevitt D, Vaudaux P, Foster TJ. (1992). Genetic evidence that bound coagulase of Staphylococcus aureus is not clumping factor. Infect Immun. 60(4):1514-23. 42. Mohammad SF, Topham NS, Burns GL, 01sen DB. (1988). Enhanced bacterial adhesion on surfaces pre- treated with fibrinogen and fibronectin. Asaio Trans. 34(3):573-7. 43. Muller E, Takeda S, Goldmann DA, Pier GB. (1991). Blood proteins do not promote adherence of coagulase- negative staphylococci to biomaterials. Infect Immun, 59(9):3323-6. SUBSTITUTESHEET 44. Phonimdaeng P, O'Reilly M, Nowlan P, Bra ley AJ, Foster TJ. (1990). The coagulase of Staphylococcus aureus 8325-4. Sequence analysis and virulence of site-specific coagulase-deficient mutants. Mol Micro- biol. 4(3):393-404. 45. Pugsley AP. (1989). Early stages in the secretory pathway. In Protein targeting, (ed.). San Diego: Academic Press, Inc., pp.46-48. 46. Raus J, Love DN. (1991). Comparison of the affinities to bovine and human prothrombin of the staphylocoagulases from Staphylococcus intermedius and Staphylococcus aureus of animal origin. J Clin Microbiol. 29(3):570-2. 47. Strong DD, Laudano AL, Hawiger J, Doolittle RF. (1982). Isolation, characterization, and synthesis of peptides from human fibrinogen that block the staphylococcal clumping reaction and construction of a synthetic clumping particle. Biochemistry. 21:1414-1420. 48. Vaudaux P, Pittet D, Haeberli A, Huggler E, Nydegger UE, Lew DP, Waldvogel FA. (1989). Host factors selectively increase staphylococcal adherence on inserted catheters: a role for fibronectin and fibrinogen or fibrin. J Infect Dis. 160(5 ):865-75. SUBSTITUTESHEET Figure legends Fig. 1. Analysis of affinity purified material from fibrinogen-Sepharose. Arrows indicate molecular masses (in kDa). Immunoblot probed with anti-19 serum. Lanes: 1, fibrinogen-proteins from S. aureus strain Newman; 2, fibri¬ nogen-proteins from S■ aureus strain FDA 486; 3, fibrino¬ gen-proteins from E. coli XL-1 harbouring plasmid pBfiblll; 4, fibrinogen-proteins from E. coli XL-1 harbouring plasmid pBfibT. Fig. 2. Restriction map and sequencing strategy of the insert containing the fib gene. Subcloning of the fib gene from the original λ clone on a Hindlll - Hindlll fragment resulted in the pBfiblll vector. This was further subcloned into the pBfibT and pBfib J vectors . Boxes show the regions for which the sequence was deduced. SS denotes the signal sequence and fib the structural gene for the mature fib protein. Arrows indicate the primers used for sequencing. Fig. 3. Nucleotide and amino acid sequence for the fib protein gene. The box denotes a possible Shine- Dalgarno sequence. Putative promoter sequences are under¬ lined. The vertical arrow indicates the cleavage site of the signal sequence. Fig. 4. Comparison of the nucleotide sequences for the fib gene from strain FDA 486 (top sequence) and strain Newman. Similarity is shown by blank spaces, dif¬ ferences in sequence is indicated by the diverging nucleo¬ tide of the Newman fib gene. Fig. 5. Comparison of the amino acid sequences for the fib protein from strain FDA 486 (top sequence) and strain Newman. Similarity is shown by blank spaces, dif¬ ferences in sequence is indicated by the diverging amino acid of the Newman protein. Fig. 6. Sequence homologies between the fib pro- tein and the coagulase from S. aureus. Bold letter show SUBSTITUTE SHEET homologies between the two repeats in the fib protein. Shaded letters show homologies between the fib protein and coagulase. Fig. 7. Coomassie blue-stained SDS-PAGE of fibri- nogen-binding material, affinity purified from S. aureus culture supernatants. Cells were grown in LB under low aeration conditions and samples were taken every hour. Lanes 1-6 represent samples taken after 1, 2, 3, 5, 7 and 9 h. Fig. 8. Analysis of affinity purified material from fibrinogen- and prothrombin-Sepharose. (a) Coomassie blue stained, undiluted eluate; (b) Immunoblot of eluate (diluted 1/100), probed with fibrinogen (10 μg/ml) and pre- absorbed antifibrinogen antibody; (c) immunoblot of eluate (diluted 1/100), probed with prothrombin (10 μg/ml) and pre-absorbed antiprothrombin antibody. Lanes: 1, eluate from fibrinogen-Sepharose purified from culture superna¬ tants of staphylococci grown in BHI for 3-4 h; 2, eluate from prothrombin-Sepharose purified from culture superna- tants of staphylococci grown in LB for 6-8 h and initially passed through fibrinogen-Sepharose. Fig. 9. Immunoblot analysis of eluate from fibri¬ nogen-Sepharose. Lanes: 1, eluate (undiluted) incubated with fibrinogen (20 ng/ml) and antifibrinogen antibody; 2, eluate (undiluted) incubated with anti-19 serum. Fig. 10. Immunoblot analysis of eluate (diluted 1/100) from fibrinogen- and prothrombin-Sepharose prepared as indicated in Fig. 2. (a) Anti-19 serum pre-absorbed with the 60-kDa protein; (b) Anti-19 serum pre-absorbed with the 19-kDa protein. Lanes: 1, eluate from fibrinogen-Sepharose; 2, eluate from prothrombin-Sepharose. Fig. 11. Analysis of purified proteins eluted from preparative SDS-PAGE gels, (a) Silver stain of un¬ digested sample; (b-d) immunoblots probed with fibrinogen and antifibrinogen antibodies; (b) undigested sample; (c) SUBSTITUTE SHEET samples digested with α-chymotrypsin; (d) samples digested with staphylococcal V8 protease. Lanes: 1, 19 kDa protein; 2, 87 kDa protein; 3, 60 kDa protein. SUBSTITUTE SHEET Applicant's or agent's file reference number 02 - 2 WO TtlTM / n B 7 S « INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) For receiving Office use only For International Bureau use only This sheet was received with the international application I I This sheet was received by the international Bureau on: Authorized officer Authorized officer Christina Senften Form PCT/RO/134 (July 1992) 36/2 GACTAGTGTA TAAGTGCTGA TGAGTCACAA GATAGATAAC TATATTTTGT CTATATTATA AAGTGTTTAT AGTTAATTAA TAATTAGTTA ATTTCAAAAG TTGTATAAAT AGGATAACTT AATAAATGTA AGATAATAAT TTGGAGGATA ATTAACATGA AAAATAAATT GATAGCAAAA TCTTTATTAA CAATAGCGGC AATTGGTATT ACTACAACTA CAATTGCGTC AACAGCAGAT GCGAGCGAAG GATACGGTCC AAGAGAAAAG AAACCAGTGA GTATTAATCA CAATATCGTA GAGTACAATG ATGGTACTTT TAAATATCAA TCTAGACCAA AATTTAACTC AACACCTAAA TATATTAAAT TCAAACATGA CTATAATATT TTAGAATTTA ACGATGGTAC ATTCGAATAT GGTGCACGTC CACAATTTAA TAAACCAGCA GCGAAAACTG ATGCAACTAT TAAAAAAGAA CAAAAATTGA TTCAAGCTCA AAATCTTGTG AGAGAATTTG AAAAAACACA TACTGTCAGT GCACACAGAA AAGCACAAAA GGCAGTCAAC TTAGTTTCGT TTGAATACAA AGTGAAGAAA ATGGTCTTAC AAGAGCGAAT TGATAATGTA TTAAAACAAG GATTAGTGAG ATAATACTTC TGTCATTATT TTAAGTTCAA AATAATTTAA TATTATATTA TTTTTTATTA ATAAAACGAC TATGCTATTT AATGCCAGGT TAATGTAACT TTCCTAAAAT TGACTATATA ATCGTTAAGT ATCAATTTTA AGGAGAGTTT ACAATGAAAT TTAAAAAATA TATATTAACA GGAACATTAG CATTACTTTT ATCATCAACT GGGATAGCAA CTATAGAAGG GAATAAAGCA GATGCAAGTA GTCTGGACAA ATATTTAACT GAAAGTCAGT TTCATGATAA ACGCATAGCA GAAGAATTAA GAACTTTACT TAACAAATCG AATGTATATG CATTAGCTGC AGGAAGCTT 36/3 ATAGATAACT ATATTTTGTC TATATTATAA AGTGTTTATA GTTAATTAAT AATTAGTTAA TTTCAAAAGT TGTATAAATA GGATAACTTA ATAAATGTAA GATAATAATT TGGAGGATAA TTAACATGAA AAATAAATTG ATAGCAAAAT CTTTATTAAC AATAGCGGCA ATTGGTATTA CTACAACTAC AATTGCGTCA ACAGCAGATG CGAGCGAAGG ATACGGTCCA AGAGAAAAGA AACCAGTGAG TATTAATCAC AATATCGTAG AGTACAATGA TGGTACTTTT AAATATCAAT CTAGACCAAA ATTTAACTCA ACACCTAAAT ATATTAAATT CAAACATGAC TATAATATTT TAGAATTTAA CGATGGTACA TTCGAATATG GTGCACGTCC ACAATTTAAT AAACCAGCAG CGAAAACTGA TGCAACTATT AAAAAAGAAC AAAAATTGAT TCAAGCTCAA AATCTTGTGA GAGAATTTGA AAAAACACAT ACTGTCAGTG CACACAGAAA AGCACAAAAG GCAGTCAACT TAGTTTCGTT TGAATACAAA GTGAAGAAAA TGGTCTTACA AGAGCGAATT GATAATGTAT TAAAACAAGG ATTAGTGAGA TAATACTTCT GTCATTATTT TAAGTTCAAA A....TAATT TAATATTATA TTATTTTTTA TTAATAAAAC GACTATGCTA TTTAATGCCA GGTTAATGTA ACTTTCCTAA AATTGACTAT ATAATCGTTA AGTATCAATT TTAAGGAGAG TTTACAATGA AATTT 36/4 MKNKLIAKSL LTIAAIGITT TTIASTADAS EGYGPREKKP VΞINHNIVEY NDGTFKYQSR PKFNSTPKYI KFKHDYNILE FNDGTFEYGA RPQFNKPAAK TDATIKKEQK LIQAQNLVRE FEKTHTVSAH RKAQKAVNLV SFEYKVKKMV LQERIDNVLK QGLVR

Claims: - 37 - CLAIMS 1. A new fibrinogen binding protein derived from Staphylococci having a molecular weight of 60 kDa. 2. A new fibrinogen binding protein derived from Staphylococci having a molecular weight of 19 kDa. 3. A fibrinogen binding protein according to claim 2, having the NH2~terminal sequence SEGYG PREKK PVSIN HNIVE. 4. A fibrinogen binding protein of claim 1 having coagulase activity. 5. Hybrid-DNA-molecule comprising a nucleotide sequence from S. aureus coding for a protein or polypeptide having fibrinogen binding activity. 6. Plasmid or phage comprising a nucleotide sequence from S. aureus coding for a protein or polypeptide having fibrinogen binding activity. 7. A plasmid pBfiblll as contained in E. coli XL having the deposit number DSM 8. An E. coli strain expressing said fibrinogen binding protein. 9. A microorganism transformed by recombinant DNA molecule of claims 5-7. 10. Hybrid-DNA-molecule according to claim 5, characterized in that it comprises the following nucleotide sequence: GAGCGAAGGA TACGGTCCAA GAGAAAAGAA ACCAGTGAGT ATTAATCACA ATATCGTAGA GTACAATGAT GGTACTTTTA AATATCAATC TAGACCAAAA TTTAACTCAA CACCTAAATA TATTAAATTC AAACATGACT ATAATATTTT AGAATTTAAC GATGGTACAT TCGAATATGG TGCACGTCCA CAATTTAATA AACCAGCAGC GAAAACTGAT GCAACTATTA AAAAAGAACA AAAATTGATT CAAGCTCAAA ATCTTGTGAG AGAATTTGAA AAAACACATA CTGTCAGTGC ACACAGAAAA GCACAAAAGG CAGTCAACTT AGTTTCGTTT GAATACAAAG TGAAGAAAAT GGTCTTACAA GAGCGAATTG ATAATGTATT AAAACAAGGA TTAGTGAG SUBSTITUTESHEET - 38 - 11. A method for producing a fibrinogen binding pro¬ tein or polypeptide wherein a) at least one hybrid-DNA- molecule according to claim 5, is introduced into a micro¬ organism, b) said microorganism is cultivated in a growth promoting medium, and c) the protein thus formed is iso¬ lated. 12. A fibrinogen binding protein or polypeptide com¬ prising at least one amino acid sequence SEGYGPREKK PVSINHNIVE YNDGTFKYQS RPKFNSTPKY IKFKHDYNIL EFNDGTFEYG ARPQFNKPAA KTDATIKKEQ KLIQAQNLVR EFEKTHTVSA HRKAQKAVNL VSFEYKVKKM VLQERIDNVL KQGLVR 13. Pharmaceutical composition for the inhibition of Staphylococci binding to fibrinogen comprising a fibrinogen binding protein of a preceding claim in combination with a pharmaceutically inert carrier. 14. Method for inhibition of Staphylococci binding to fibrinogen in mammals including humans, by administering a therapeutically and/or prophylactically effective amount of a fibrinogen binding protein of any preceding claim together with an inert carrier. 15. Method for immunization, characterized in that the protein is administered in an amount sufficient to raise antibodies . 16. Method for immunization, whereby an antibody raised in accordance with claim 15 is administered to a mammal body in amount sufficient to provide a passive immu¬ nization. SUBSTITUTE SHEET

Inventors: BODEN MARIA K, FLOCK JAN-INGMAR, BODEN, MARIA, K., FLOCK, JAN-INGMAR
Applicants: ALFA LAVAL AGRI INT, BODEN MARIA K, FLOCK JAN INGMAR, ALFA-LAVAL AGRI INTERNATIONAL AKTIEBOLAG
Application year: 1993
Main classification: C07K15
IPC codes: A61K38, A61K39, C07K14, C12N9