EP0771351A1 - Surface protein of staphylococcus aureus - Google Patents

Surface protein of staphylococcus aureus

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Publication number
EP0771351A1
EP0771351A1 EP95922211A EP95922211A EP0771351A1 EP 0771351 A1 EP0771351 A1 EP 0771351A1 EP 95922211 A EP95922211 A EP 95922211A EP 95922211 A EP95922211 A EP 95922211A EP 0771351 A1 EP0771351 A1 EP 0771351A1
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EP
European Patent Office
Prior art keywords
protein
fibrinogen
clone
sequence
aureus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP95922211A
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German (de)
French (fr)
Inventor
Ambrose Cheung
Vincent A. Fischetti
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Rockefeller University
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Rockefeller University
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Publication date
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Publication of EP0771351A1 publication Critical patent/EP0771351A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to methods and compositions for preventing infections by Staphylococcus aureus (S. aureus) , especially those triggered by the use of catheters prosthetic devices and heart valve replacements. More specifically, it relates to methods and compositions useful for inhibiting the ability of a surface protein of S. aureus to implement adhesion of the organism to endothelical cells or to catheters at the skin-catherter junction thereby initiating infection.
  • Staphylococcus aureus Staphylococcus aureus
  • S. aureus is one of the most frequently encountered pathogens in infections acquired in the hospital. It accounts for 25% of all hospital acquired infections resulting from the use of catheters or similar structures. Since a great majority of patients entering a hospital require some sort of intravenous device, there is a very high probability of infection. The same type of risk applies to the use of prosthetic devices such a as hip and other joint replacements because the ability of staphylococci to adhere to such devices.
  • a surface protein, and the gene which expresses it, have now been discovered. This protein enables the invading bacteria to adhere to the fibrinogen. Antibodies to fibrinogen or to the surface protein will prevent bacterial adhesion and thereby inhibit infection.
  • the protein and segments of the protein are useful as vaccines or for the production of antibodies useful for passive protection of patients prior to the use of a catheter or equivalent device.
  • This invention therefore, comprises the protein itself and segments thereof, the gene and segments thereof which produce such products, vectors for the gene and its useful segments, organisms transformed by such vectors, monoclonal and polyalonal antibodies to the protein and its useful segments, vaccines produced utilizing the protein and its segments and methods of preventing S. aureus infections utilizing such products.
  • the invention also includes diagnostic probes utilizing the gene products described herein. THE FIGURES
  • Fig. 1 Western blot of cellular fractions of clone number 14 probes with fibrinogen followed by anti-fibrinogen antibody conjugate.
  • the control contains the lysate of an E. coli clone with pBR322 insert in f Zap.
  • the arrows indicate a 34 kD reactive band together with an upper band which may be a dimer.
  • R Reessuullttss o. f a duplicate blot probed with 125I fibrinogen were similar.
  • FIG. 2 Western blot (A) and silver-stained gel (B) of the periplas ic extract of clone number 14 fractionated in a fibrinogen column.
  • the Western blot was probed with fibrinogen/anti-fibrinogen conjugate.
  • the short arrow indicates the fibrinogen-reactive band from crude lysate positive control.
  • the long arrow marks a 34 kD protein that reacts with fibrinogen [4th lane in (A)] and is eluded with 3 M potassium thiocyanate, but not with PBS with 0.5 M NaCl nor with acid elusion.
  • Fig. 3 Is the complete sequence of clone 36 (C36) encoding fibrinogen reactive protein.
  • Fig. 4 Sequence comparison of strain DB to that of coagulases from strains 8325-4, 213 and BB with the Pileup program under the GCG package.
  • the arrows indicate the 11 amino acid sequence that is unique to the protein. This sequence shares homology with a cell wall anchor motif found in other gram positive wall protein However, there is no complete identify to this motif in the Genbank.
  • the arrows at resiues 409 and 419 indicate a unique sequence segment of this protein.
  • the amino half (residues 59-325) of this protein is primarily helical as predicted by the Gamier analysis.
  • Two areas (residues 58-194 and 264-297) reveal a 7 residue periodicity in which residues in position a / and 'd' in a heptad motif 'abcdefg' are either hydrophobic or nonpolar. This pattern is consistent with a coiled-coil conformational structure.
  • the sequence is analyzed by the Matcher Program (22) .
  • Fig.5 Sequence comparison of our protein (strain DB) to that of coagulases from strains 8325-4, 213 and BB with the Pileup program under the GCC package.
  • the arrows indicate the 11 amino acid sequence that is unique to our protein. This sequence shares homology with a cell wall anchor motif found in other gram positive cell wall protein. However, there is no complete identify to this motif in the Genbank.
  • Strain DB a wild strain of S. aureus N2Y broth - a commercially available growth medium LB - a commercially available growth medium IPTG - Isopropyl-beta-D- thiogalactopyronoside BSA - bovine serum albumin O 95/34655
  • Strain DB has been deposited at the American Type Colture Collection under the accession number .
  • Strain DB which has been phenotypically characterized (5) was used in the construction of a genomic library.
  • E. coli strain Sure (Stratagene) was the host cell for the"fZap vector.
  • Clones 14 and 36 were phagemids derived from fibrinogen-reactive plaques containing the insert.
  • a pBluescript phagemid, pAC8, which contained a protein A clone was derived from a Zap genomic library of DB.
  • NZY broth and LB (23) were used for the growth of E. coli strains.
  • Goat anti-human fibrinogen antibody obtained commercially was affinity-purified on a fibrinogen column as previously described (7) .
  • the fibrinogen column was prepared with glutardialehyde activated beads (Boehringer Mannheim, Indianapolis, IN) as described in the manufacturer's insert (4) .
  • the monospecificity of the affinity purified goat anti-human fibrinogen IgG was verified by an immunoblot using purified fibrinogen and plasma as antigens (7) .
  • the protein concentration of the affinity purified antibody was determined by the BCA protein assay reagent (Pierce Chemicals, Rockeford, IL) .
  • Affinity purified goat anti-fibrinogen antibody was conjugated to bovine intestinal alkaline phosphates (Sigma, St. Lousis, MO) as described by Voller (32) .
  • a genomic library of strain DB was constructed with the Zap vector that has been digested with EcoRI and dephosphorylated (Stratagene cloning kit) .
  • DB chromosomal DNA was extracted from lysostaphin-lysed cells as previously described (5,29). Genomic DNA was sheared with a 26 gauge syringe and subject to gel filtration on Sepharose CL2B to remove fragments smaller than lkb.
  • recombinant phage was incubated with the E. coli host on NZY agar (23) at 42 C for gene expression. Following transfer to duplicate nitrocellulose filters (82 mm in diameter, Schleicher & Schuell, Keene, NH) , the filters were blocked with 10ml of TNT buffer (10 mM Tris with 0.15 M NaCl and 0.05% Tween 20) containing 1% BSA for 1 hour at RT and then incubated at 37°C for 3h with 25 ug of fibrinogen (Sigma #4883) .
  • TNT buffer 10 mM Tris with 0.15 M NaCl and 0.05% Tween 20
  • This fibrinogen preparation had been further purified over a protein A sepharose column to remove contaminated IgG and was found to be essentially free of contaminants as determined by a silver-stained SDS-gel containing this protein.
  • the filters were then washed once with TNT containing 0.1% BSA and 0.1% NP40, and twice with TNT with 0.1% BSA for 5 min. each.
  • Affinity purified goat anti-fibrinogen antibody alkaline phosphatase conjugate diluted 1:1000 in TNT buffer with 1% BSA was then incubated with the filter for 1 hour at RT.
  • fibrinogen-reactive plaques were visualized with 5-bromo- 4-cloro-3-indolyl phosphate as a substrate as described by Blake et al (l).
  • AfZap vector with a pBR322 insert plated on E.coli or E. coli cells alone served as negative controls. Positive clones detected by fibronogen/anti-fibrinogen conjugate were confirmed by allowing plaques on duplicate filters to react with
  • 125 I fibrinogen This screening procedure was similar to the method described above except that 125 fibrinogen (100,000 cpm) was used in place of the cold unlabeled fibrinogen. Following reaction, the nitrocellulose filters were washed twice with TNT with 0.1% BSA and 0.1% NP40 and three times with TNT with 0.1%
  • Purified plaques were isolated by rescreening each positive clone at least 4 times.
  • a single strand DNA containing the pBluescript phagemid with the insert can be packaged for recirculation, thereby generating subclones in E. coli cells when plated in LB/a picillin agar (Statagene cloning kit instructions) .
  • Plasmids for sequencing were purified from E. coli by equilibrium centrifugation in Cesium chloride-ethidiu bromide gradients (23) . The purity of the plasmid was confirmed by digestion with restriction enzymes (New England BioLab, Beverly, MA) .
  • DNA probes were labeled with 32P ( -32P deoxycyti.dm. e trl.phosphate, Amersham) using the random primed DNA labeling kits (Boehringer Mannheim) . The membrane was then hybri .di.zed wi.th the 32P labeled DNA probe at
  • the membrane was then subject to autoradiography with an intensifying screen at -70°C.
  • E. coli cells containing this clone was grown in 10ml of LB with 50 ug/ml of ampicillin at 37°C until the OD 6Q0 reached 1.0. Cells were collected by cetrifugation at 7,000 g for 10 min and resuspended in 1.25 ml of ice cold TSE buffer (100 mM Tris, pH 8.0, containing 20% sucrose and 5mM EDTA) . Lysozyme was added to a final concentration of 0.5 mg/ml and the sample was iced for 20 min.
  • cytoplasmic fraction 0.25 ml of DNase solution was added to the pellet along with 0.75 of water.
  • the spheroplasts were aspirated vigorously several times with a pasteur pipette, frozen and thawed twice as described above.
  • the lysate generated by this treatment was centrifuged for 49,000 g for 1 hr.
  • the supernatant filtered through a 0.4 um membrane was designated cytoplasmic fraction.
  • the transformed E. coli has been deposited at the American Type Culture Collection under the accession number .
  • Cellular extracts (10 ul each) were separated on 9% SDS- polyacrylamide gel slabs by the method of Laemmli (21) . Prestained molecular standards (BRL, Gaithersburg, MD) were run concurrently in adjacent wells. After electrophoresis, the gel was either stained with silver (Pierce Chemicals) or transferred onto nitrocellulose (30) . After transfer, the nitrocellulose filters were allowed to react with fibrinogen followed by affinity purified goat anti-fibrinogen alkaline phosphatase conjugate and reactive substrate as described for the screening of the genomic library. In some experiments, the filters were incubated with 125I fibrinogen (250,000 cpm), washed and autoradiographed as described above.
  • the specific antibody diluted in blocking buffer (Tris 10mN with 0.5 M NaCl and 0.05% Tween 20, pH 8.2) was incubated with the blot for 2 hours at RT. This was followed by incubation with an appropriate alkaline phosphate conjugate for 1 hour and then processed for band visualization as previously described (3) .
  • periplasmic extracts from 4 L of culture were prepared as previously described. Briefly, cells were harvested by centrifugation (7,000 g for 20 min) and resuspended in 75 ml of TSE buffer containing 37.5 mg of lysozyme. After incubation on ice for 20 min, MgCl_ was added to a final concentration of 50 mM and spheroplasts segmented at 7,000 g for 30 min. The periplasmic fraction was aspirated from the supernatant, filtered through a 0.45 um membrane and immediately applied to a fibrinogen column (2.5 x 20 cm) followed by rotation at 4°C overnight.
  • the fibrinogen column was prepared by mixing 25 mg of fibrinogen and 5 gm of glutardialdehyde beads as described (4) . After collecting the fall through, the column was washed with 150 ml. of PBS followed by 150 ml of PBS with 0.5 NaCl. The fibrinogen-binding protein was then eluded by rotating the column with 10 ml of 3 M potassium thiocyanate at RT for 20 Min followed by collection. In preliminary studies, a similar elusion procedure with 0.1M glycine pH 3.0 was not successful. Fractions from the column were concentrated in a Centricon 10 (Amicon, Danvers, MA) and analyzed by SDS-PAGE and immunoblots with fibrinogen as described. Computer analysis of sequence data
  • clones 14 and 30 were similar. Although clone 36 contained the complete gene as determined by sequence analysis, expression of the fibrinogen reactive protein with this clone was found to be difficult. Notably, a culture of clone 14 when grown to late stationary phase (i.e. °D 600 1.5) also resulted in a significantly decreased yield in fibrinogen-reactive protein. This result can be explained either by toxicity of this protein on E. coli or by increased proteolytic breakdown during stationary phase. For these reasons, the expression of the partial protein was evaluated in clone 14 that has been grown to early stationary phase (OD 600 ⁇ mn -l.O).
  • these bands did not react with polyclonal and monoclonal anti- beta-galactosidase antibody (1:1000 dilution) (Boehringer Mannheim) on immunoblots.
  • the fibrinogen reactive band also did not react with affinity purified chicken anti-protein A antibody (Accurate Chemicals, Westbury, NY) , thus providing additional evidence that protein A was not cloned which, by binding to IgG, could lead to false positive results.
  • periplasmic extracts which contained fewer contaminating bands were harvested from 4 L of E. coli cells expressing the protein of clone 14 and applied to an affinity column with fibrinogen linked beads.
  • the cloned proteins of interest as analyzed by silver stain and Western blots with 125I fibrinogen and fibrinogen/anti-fibrinogen conjugate, was found in precolumn fractions and the 3M potassium thiocyanate eluant.
  • the complete sequence of the fibrinogen protein expressed by clone 30 is shown in Fig. 3.
  • the sequence revealed an open reading frame of 1,935 nucleotides.
  • the sequence has a guanosine-cytosine (GC) content of 34.7%, in contrast to the 30% GC content in the staphylococcal genome (10) .
  • the higher GC content is attributable to the carboxy terminal half of the molecule (39.7%).
  • Putative transcription and translation signals and ribosomal binding sites are indicated in Fig. 3.
  • the first 26 amino acids have features characteristic of a bacterial signal peptide (16) . Based on the predicted cleavage site, the mature protein has a predicated size of 69,991 Da with a deduced pi of 6.5.
  • the second domain between residues 326 and 505 denotes a proline and glycine rich region (20%) .
  • residues 326 and 505 denote a proline and glycine rich region (20%) .
  • residues 326 and 505 there are 17 proline and 19 glycine residues.
  • the carboxyl-terminal domain (residues 506-645) consists of 5 tandem, direct repeats of 27 amino acids each followed by 5 terminal amino acids (PRVTK) . Divergence is observed mainly in the outside repeats. Conformational analysis indicated that this repeat region is nonhelical and contains mostly beta-sheets.
  • residues 423 and 645 comprising the five repeated units to homologous regions in the coagulase sequences increased markedly to 93.9%. 95.1% and 96.9% for strains 8325-4, BB and 213, respectively (Fig. 5).
  • the C-termini of coagulase sequences of strains 8325-4, BB and 213 are composed of repeating units of 27 homologous, but no identical, amino acids followed by the terminal sequence PRVTK (Fig. 6) .
  • the number of repeating units differ among strains.
  • fibrinogen reactive protein sequence displayed features that are common to the coagulase sequence, a careful comparison revealed a unique stretch of 11 amino acids between residues 409 and 419 (SVTLPSITGES) in the middle of the proline/glycine rich region (Fig. 5) .
  • motif LPSITGES shares homology with a cell wall anchor motif (LPXTGX) found in other gram positive surface protein (12,28).
  • LPXTGX cell wall anchor motif
  • a novel fibrinogen reactive protein of S. aureus has been cloned.
  • This protein is both structurally and functionally different from other apparently similar proteins such as the coagulases.
  • the protein and the gene which expresses it are illustrated in Fig. 3.
  • the figure shows the complete clone 36 and the protein it expresses.
  • Clone 14 runs from nucleotide 684 to nucleotide 1935 in Fig. 3.
  • the protein expressed by clone 14 runs from amino acid residue 229 to 645.
  • Clone 30 is substantially the same as clone 14 and expresses substantially the same protein.
  • fibrinogen binding protein from staphylococcal whole cell lysates with conventional chromatographic methods has been reported in two studies (11,31). However, the molecular weight (420 vs 62 kD) and the amino acid composition differs widely between the two studies. In contrast to the 62 kD protein, methionine and tyrosine, but not cysteine residues, are present in the protein of this invention.
  • the fibrinogen reactive protein described here as expressed by clone 36 comprises predominately lysine (11.2%), threonine (9.3%) and glutamic acid (9%) while glycine (16.8%), glutamic acid (15%) and lysine (13.3%) were the abundant amino acids in the 62 kD protein (31) .
  • the deduced isoelectric point (pI-6.5) of the fibrinogen reactive protein also differs from the basic pl (about 10.2) of the 62kD protein.
  • fibrinogen binding component of S. aureus is a cell wall constituent because it is absent in staphylococcal L form (10) and because bacterial clumping in the presence of fibrinogen is abolished upon whole cell digestion with proteinases (4) .
  • a charged tail (4-7 amino acids) is usually preceded by a highly hydrophobic membrane anchor (about 16-20 amino acids) , the hexamer LPXTGX, a proline-glycine rich domain and a C- terminal repeat region (14) .
  • the C-terminal region of the fibrinogen reactive protein is different from the model described.
  • the region preceding the stop codon lacks a charged tail and a hydrophobic membrane anchor.
  • the five terminal amino acids (PRVTK) are preceded by five repeats of 27 amino acid each.
  • the region N- terminal to these repeats is a broad proline/glycine region (residues 326-505) in the middle of which is a unique sequence (LPSITGE) that shares homology with a cell wall anchor motif (LPXTGX) found in other gram positive surface proteins.
  • this molecular architecture at the C-terminus is similar to those described for pneumococal surface protein A (35) .
  • the amino terminal half of pneumococcal surface protein A is ⁇ -helical and is consistent with an ⁇ *>-helical coiled protein conformation.
  • the -helical region is followed by a proline-rich domain and a repeat domain consisting of ten 20-amino-acid repeats. In addition, it also lacks a classic membrane anchor and a charged tail.
  • novel proteins of this invention are surface proteins of S. aureus. No such proteins have previously been detected, isolated and characterized. They are principally characterized by their ability to bind fibrinogen.
  • the molecular weight of the protein expressed by clone 36 is about 69,991 Da and its isoelective point 6.5.
  • the gene which expresses this protein contains about 1935 nucleotides. Other characteristic features of the protein and of segments C14 and C30 are described above. Because of the difficulty in expressing protein from clone 36, the preferred clone of this invention is clone 36.
  • the preferred fibrinogen binding protein is the protein expressed by this clone.
  • the protein of this invention will be recognized by the skilled artisan as representative of a class of protein which may differ amongst the various strains of S. aureus, but will all be characterized as having substantially the same number of amino acid residues, the same tertiary structure and binding activity. They may differ slightly in the identity of the amino acid residues at specific positions in the protein chain. All such proteins are included within the scope of this invention.
  • genes which generate the proteins of this invention may also differ slightly amongst various strains, but they all have the common characteristic of producing a protein of this invention.
  • genes of this invention may be employed, as will be recognized by the skilled artisan, to produce plasmids or other vectors which, in turn are useful for transforming organisms such as E. coli to produce novel strains of this bacteria which will express the proteins of the invention.
  • Inhibition of the binding of S. aureus to endothelial cells is a major factor in preventing infection. Accordingly antibodies to proteins expressed by clone 36, its segments clone 14 and clone 30 and even smaller segments are important factors in controlling infection.
  • the proteins and protein segments of this invention are therefore useful to form vaccines to inhibit S. aureus infections of mammals, including humans, by administering an amount of the selected protein which will stimulate the production of protective quantities of antibodies to limit adhesion of S. aureus.
  • the proteins may also be used to produce antibodies in vitro which may be employed for passive immunization.
  • proteins and polypeptide or peptide segments of this invention may be obtained by any of a number of known process including the recombinant DNA techniques described above.
  • Polypeptide and peptides within the scope of the invention containing, for example from about 6 to 20 or more amino acid segments may be synthesized be standard solid phase procedures with appropriate amino acids using the protection, deprotection and cleavage techniques and reagents appropriate to each specific amino acid or peptide.
  • a combination of manual and automated (e.g., Applied Biosystem 430A) solid phase techniques can be used to synthesize the novel peptides of this invention.
  • classical methods of peptide synthesis can also be employed.
  • the products of the invention are amphoteric. They can exist and be utilized as free bases or as pharmaceutically acceptable metallic or acid addition salts.
  • Suitable metallic salts include alkali and alkaline earth metal salts, preferably sodium or potassium salts.
  • Acid addition salts may be prepared from a wide variety of organic and inorganic acids including mineral acids, for example citric, lactic, maleic, tartaric, phosphoric and hydrochloric acids. These salts can be prepared by procedures well known to those skilled in the art.
  • a vaccine For use as a vaccine, it is presently preferred to administer the selected product in a pharmaceutically acceptable carrier such as a buffer.
  • a pharmaceutically acceptable carrier such as a buffer.
  • Mice or other mammals, including humans, when so immunized are protected from colonization and subsequent infection by S . aureus.
  • the patient to be protected will be treated with product of the invention in an amount which is effective to elicit a protective immune response.
  • the selected agent may be administered alone or in a pharmaceutically acceptable liquid or solid carrier in which it may be dispersed, dissolved or suspended. If, for example, the patient is to be treated intravenously, the peptide may be suspended as a free base or dissolved as a metallic salt in isotonic aqueous buffer. Other methods of treatment and pharmaceutically acceptable carriers will be apparent to the skilled artisan.
  • proteins, polypeptides and peptides of this invention and the genes or oligonucleotides which are employed in their expression are useful as probes for genes and proteins. They are also useful to raise antibodies by which specific strains of streptococci can be identified.
  • sequence of nucleotides which elicit the unique segment from position 409 to position 419 and modification of this sequence are especially useful as diagnostic probes to identify S. aureus strains. The procedure O 95/34655
  • Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to culture human endothelial cells. J. Clin.Invest. 87:2236-2245.

Abstract

Methods and compositions are provided for preventing Staphylococcus aureus infections associated with the use of catheters and similar devices. They are based on the neutralization of ability of surface proteins of staphylococcis to adhere to the surface of the catheters at the skin-catheter junction as the first step in the systemic invasion of the host by the organism.

Description

SURFACE PROTEIN OF STAPHYLOCOCCUS AUREUS
FIELD OF THE INVENTION
This invention relates to methods and compositions for preventing infections by Staphylococcus aureus (S. aureus) , especially those triggered by the use of catheters prosthetic devices and heart valve replacements. More specifically, it relates to methods and compositions useful for inhibiting the ability of a surface protein of S. aureus to implement adhesion of the organism to endothelical cells or to catheters at the skin-catherter junction thereby initiating infection.
BACKGROUND OF THE INVENTION
S. aureus is one of the most frequently encountered pathogens in infections acquired in the hospital. It accounts for 25% of all hospital acquired infections resulting from the use of catheters or similar structures. Since a great majority of patients entering a hospital require some sort of intravenous device, there is a very high probability of infection. The same type of risk applies to the use of prosthetic devices such a as hip and other joint replacements because the ability of staphylococci to adhere to such devices.
One of the initial reactions of the mammalian host to the presences of a catheter is to coat the object with fibrinogen and other matrix proteins as a prelude to the systemic reaction which is intended to expel the invasion. In a hospital setting, this provides an opportunity of infection by S. aureus which attaches itself to the fibrinogen at the skin-catheter injunction and thereafter works its way through the skin and into the blood. Since the strains prevalent in hospitals, nursing homes and other patient care facilities are often antibiotic resistant, these types of infections are extremely serious and very difficult to contain. Accordingly, the art has expended much effort to prevent such opportunistic infections.
BRIEF SUMMARY OF THE INVENTION
A surface protein, and the gene which expresses it, have now been discovered. This protein enables the invading bacteria to adhere to the fibrinogen. Antibodies to fibrinogen or to the surface protein will prevent bacterial adhesion and thereby inhibit infection. The protein and segments of the protein are useful as vaccines or for the production of antibodies useful for passive protection of patients prior to the use of a catheter or equivalent device.
This invention, therefore, comprises the protein itself and segments thereof, the gene and segments thereof which produce such products, vectors for the gene and its useful segments, organisms transformed by such vectors, monoclonal and polyalonal antibodies to the protein and its useful segments, vaccines produced utilizing the protein and its segments and methods of preventing S. aureus infections utilizing such products. The invention also includes diagnostic probes utilizing the gene products described herein. THE FIGURES
There follows a brief description of the figures.
Fig. 1 Western blot of cellular fractions of clone number 14 probes with fibrinogen followed by anti-fibrinogen antibody conjugate. The control contains the lysate of an E. coli clone with pBR322 insert in f Zap. The arrows indicate a 34 kD reactive band together with an upper band which may be a dimer. R Reessuullttss o. f a duplicate blot probed with 125I fibrinogen were similar.
Fig. 2 Western blot (A) and silver-stained gel (B) of the periplas ic extract of clone number 14 fractionated in a fibrinogen column. The Western blot was probed with fibrinogen/anti-fibrinogen conjugate. The short arrow indicates the fibrinogen-reactive band from crude lysate positive control. The long arrow marks a 34 kD protein that reacts with fibrinogen [4th lane in (A)] and is eluded with 3 M potassium thiocyanate, but not with PBS with 0.5 M NaCl nor with acid elusion.
Fig. 3 Is the complete sequence of clone 36 (C36) encoding fibrinogen reactive protein.
Fig. 4 Sequence comparison of strain DB to that of coagulases from strains 8325-4, 213 and BB with the Pileup program under the GCG package. The arrows indicate the 11 amino acid sequence that is unique to the protein. This sequence shares homology with a cell wall anchor motif found in other gram positive wall protein However, there is no complete identify to this motif in the Genbank. The arrows at resiues 409 and 419 indicate a unique sequence segment of this protein.
The amino half (residues 59-325) of this protein is primarily helical as predicted by the Gamier analysis. Two areas (residues 58-194 and 264-297) reveal a 7 residue periodicity in which residues in position a/ and 'd' in a heptad motif 'abcdefg' are either hydrophobic or nonpolar. This pattern is consistent with a coiled-coil conformational structure. The sequence is analyzed by the Matcher Program (22) .
Fig.5 Sequence comparison of our protein (strain DB) to that of coagulases from strains 8325-4, 213 and BB with the Pileup program under the GCC package. The arrows indicate the 11 amino acid sequence that is unique to our protein. This sequence shares homology with a cell wall anchor motif found in other gram positive cell wall protein. However, there is no complete identify to this motif in the Genbank.
The following abbreviations are employed in the description of this invention:
Strain DB - a wild strain of S. aureus N2Y broth - a commercially available growth medium LB - a commercially available growth medium IPTG - Isopropyl-beta-D- thiogalactopyronoside BSA - bovine serum albumin O 95/34655
X-gal - 5-bromσ-4-chlerσ-3-indolyl- beta-D-galactopyranoside
SDS - sodium dodecyl sulfate NP40 - a commercially available nonionic detergent pBR322 - a commercially available plasmid of known structure pBluescript - a commercially available phagemid of known structure
SSPE ' - O.lSNaCl, lOmM NaH 2_PO4„,
Im MEDTA pH7.4
TSE buffer - O.IM Tris, 20% sucrose, 5mM EDTA pH8
PBS - phosphate buffered saline
Strain DB has been deposited at the American Type Colture Collection under the accession number .
The following Materials and Methods section is provided for convience and ease of understanding of this invention.
MATERIALS AND METHODS
Bacteria, plasmids and phage
Strain DB which has been phenotypically characterized (5) was used in the construction of a genomic library. E. coli strain Sure (Stratagene) was the host cell for the"fZap vector. Clones 14 and 36 were phagemids derived from fibrinogen-reactive plaques containing the insert. A pBluescript phagemid, pAC8, which contained a protein A clone was derived from a Zap genomic library of DB.
Media and Antibiotics
Unless otherwise indicated, the following media wew used: NZY broth and LB (23) were used for the growth of E. coli strains.
Preparations of affinity purified goat anti-fibrinogen conjugate
Goat anti-human fibrinogen antibody obtained commercially (Cappel, West Chester PA) was affinity-purified on a fibrinogen column as previously described (7) . The fibrinogen column was prepared with glutardialehyde activated beads (Boehringer Mannheim, Indianapolis, IN) as described in the manufacturer's insert (4) . The monospecificity of the affinity purified goat anti-human fibrinogen IgG was verified by an immunoblot using purified fibrinogen and plasma as antigens (7) . The protein concentration of the affinity purified antibody was determined by the BCA protein assay reagent (Pierce Chemicals, Rockeford, IL) . Affinity purified goat anti-fibrinogen antibody was conjugated to bovine intestinal alkaline phosphates (Sigma, St. Lousis, MO) as described by Voller (32) .
Construction and screening of staphylococcal genomic library
A genomic library of strain DB was constructed with the Zap vector that has been digested with EcoRI and dephosphorylated (Stratagene cloning kit) . DB chromosomal DNA was extracted from lysostaphin-lysed cells as previously described (5,29). Genomic DNA was sheared with a 26 gauge syringe and subject to gel filtration on Sepharose CL2B to remove fragments smaller than lkb. Fractions containing 4-5 kb fragments were pooled, treated with T4 polymerase to produce blunt ends, methylate with EcoRI methylase (New England Biolabs, Boston, MA) , insert into the EcoRI site of the Zap vector with EcoRI linkers and packaged in vitro with Gigapack packing extracts (Statagene) . Over 90% of the recombinant phages were recognized as white plaques when plated on lac" host strain Sure in the presence of IPTG and X- gal.
For the screening of fibrinogen reactive plaques, recombinant phage was incubated with the E. coli host on NZY agar (23) at 42 C for gene expression. Following transfer to duplicate nitrocellulose filters (82 mm in diameter, Schleicher & Schuell, Keene, NH) , the filters were blocked with 10ml of TNT buffer (10 mM Tris with 0.15 M NaCl and 0.05% Tween 20) containing 1% BSA for 1 hour at RT and then incubated at 37°C for 3h with 25 ug of fibrinogen (Sigma #4883) . This fibrinogen preparation had been further purified over a protein A sepharose column to remove contaminated IgG and was found to be essentially free of contaminants as determined by a silver-stained SDS-gel containing this protein. The filters were then washed once with TNT containing 0.1% BSA and 0.1% NP40, and twice with TNT with 0.1% BSA for 5 min. each. Affinity purified goat anti-fibrinogen antibody alkaline phosphatase conjugate diluted 1:1000 in TNT buffer with 1% BSA was then incubated with the filter for 1 hour at RT. After washing the filters twice with TNT with 0.1% BSA and 0.1% NP40 and three times with TNT with 0.1% BSA for 5 min each, fibrinogen-reactive plaques were visualized with 5-bromo- 4-cloro-3-indolyl phosphate as a substrate as described by Blake et al (l). AfZap vector with a pBR322 insert plated on E.coli or E. coli cells alone served as negative controls. Positive clones detected by fibronogen/anti-fibrinogen conjugate were confirmed by allowing plaques on duplicate filters to react with
125 I fibrinogen. This screening procedure was similar to the method described above except that 125 fibrinogen (100,000 cpm) was used in place of the cold unlabeled fibrinogen. Following reaction, the nitrocellulose filters were washed twice with TNT with 0.1% BSA and 0.1% NP40 and three times with TNT with 0.1%
BSA for 5 min each, and finally subjected to autoradiography.
Purified plaques were isolated by rescreening each positive clone at least 4 times.
DNA sequencing of the fibrinogen reactive clones
By infecting the E. coli host strain simultaneously with a fl helper phage (R408) and the"-*- Zap vector containing the insert, a single strand DNA containing the pBluescript phagemid with the insert can be packaged for recirculation, thereby generating subclones in E. coli cells when plated in LB/a picillin agar (Statagene cloning kit instructions) . Plasmids for sequencing were purified from E. coli by equilibrium centrifugation in Cesium chloride-ethidiu bromide gradients (23) . The purity of the plasmid was confirmed by digestion with restriction enzymes (New England BioLab, Beverly, MA) . By using both T3 and T7 primers flanking the insert, plasmid sequencing of clones 14 and 36 were performed with the Sequenase kit (U.S. Biochemicals, Cleveland, OH) following the manufacture's instruction (27) . Additional primers were obtained for sequencing from within the insert. Southern blot hybridization
Southern blot hybridization was performed with random primed samples of gel-purified DNA fragments as probes (12,23). Briefly, chromosomal DNA digested with restriction enzymes was resolved on 0.7% TBE gel and transferred onto Hybond-N membrane
Amersham, Arlington Heights, IL) (14) . DNA probes were labeled with 32P ( -32P deoxycyti.dm. e trl.phosphate, Amersham) using the random primed DNA labeling kits (Boehringer Mannheim) . The membrane was then hybri .di.zed wi.th the 32P labeled DNA probe at
65 C overnight, washed twice with 2X SSPE with 0.1% SDS at RT for
10 min each followed by IX SSPE with 0.1% SDS at 65°C for 15 min.
The membrane was then subject to autoradiography with an intensifying screen at -70°C.
Expression of fibrinogen-reactive protein of S. aureus in E. coli
One of the fibinogen-binding clones, clone 14 was evaluated for the expression of the fibrinogen binding protein in E. coli. E. coli cells containing this clone was grown in 10ml of LB with 50 ug/ml of ampicillin at 37°C until the OD 6Q0 reached 1.0. Cells were collected by cetrifugation at 7,000 g for 10 min and resuspended in 1.25 ml of ice cold TSE buffer (100 mM Tris, pH 8.0, containing 20% sucrose and 5mM EDTA) . Lysozyme was added to a final concentration of 0.5 mg/ml and the sample was iced for 20 min. For whole cell lysate, 0.25 ml of this suspension was removed and 7.5 ul of Triton X-100 and 50 ul of DNase solution (10 mM MgCl_ with 100 ug/ml of DNase) were added. The sample was frozen (-70 C) and thawed twice. Magnesium chloride was added to the remaining 1 ml. cell suspension (50 mM final concentration) to stabilize the spheroplasts which were then pelted at 7,000 g for 15 min. The supernatant was filtered through a 0.45 um Millipore membrane to obtain the periplasmic fraction.
To lyse the spheroplasts, 0.25 ml of DNase solution was added to the pellet along with 0.75 of water. The spheroplasts were aspirated vigorously several times with a pasteur pipette, frozen and thawed twice as described above. The lysate generated by this treatment was centrifuged for 49,000 g for 1 hr. The supernatant filtered through a 0.4 um membrane was designated cytoplasmic fraction.
The transformed E. coli has been deposited at the American Type Culture Collection under the accession number .
SDS-PAGE and immunoblot analysis
Cellular extracts (10 ul each) were separated on 9% SDS- polyacrylamide gel slabs by the method of Laemmli (21) . Prestained molecular standards (BRL, Gaithersburg, MD) were run concurrently in adjacent wells. After electrophoresis, the gel was either stained with silver (Pierce Chemicals) or transferred onto nitrocellulose (30) . After transfer, the nitrocellulose filters were allowed to react with fibrinogen followed by affinity purified goat anti-fibrinogen alkaline phosphatase conjugate and reactive substrate as described for the screening of the genomic library. In some experiments, the filters were incubated with 125I fibrinogen (250,000 cpm), washed and autoradiographed as described above. To detect other proteins in the cell extracts, the specific antibody diluted in blocking buffer (Tris 10mN with 0.5 M NaCl and 0.05% Tween 20, pH 8.2) was incubated with the blot for 2 hours at RT. This was followed by incubation with an appropriate alkaline phosphate conjugate for 1 hour and then processed for band visualization as previously described (3) .
Partial purification of the fibrinogen-reactive protein of S. aureus
In an attempt to purify the fibrinogen-reactive protein from clone 14, periplasmic extracts from 4 L of culture were prepared as previously described. Briefly, cells were harvested by centrifugation (7,000 g for 20 min) and resuspended in 75 ml of TSE buffer containing 37.5 mg of lysozyme. After incubation on ice for 20 min, MgCl_ was added to a final concentration of 50 mM and spheroplasts segmented at 7,000 g for 30 min. The periplasmic fraction was aspirated from the supernatant, filtered through a 0.45 um membrane and immediately applied to a fibrinogen column (2.5 x 20 cm) followed by rotation at 4°C overnight. The fibrinogen column was prepared by mixing 25 mg of fibrinogen and 5 gm of glutardialdehyde beads as described (4) . After collecting the fall through, the column was washed with 150 ml. of PBS followed by 150 ml of PBS with 0.5 NaCl. The fibrinogen-binding protein was then eluded by rotating the column with 10 ml of 3 M potassium thiocyanate at RT for 20 Min followed by collection. In preliminary studies, a similar elusion procedure with 0.1M glycine pH 3.0 was not successful. Fractions from the column were concentrated in a Centricon 10 (Amicon, Danvers, MA) and analyzed by SDS-PAGE and immunoblots with fibrinogen as described. Computer analysis of sequence data
DNA protein sequence analysis, and sequence comparison with database were conducted with the Sequence Analysis Software Package from the Genetics Computer Group (University of Wisconsin, Madison, WI) (9) . The deduced amino acid sequence of the putative protein was compared to a sequence database by the algorithm of Pearson and Lipman (TFASTA implementation of GenBank) (25) . The fibrinogen reactive protein sequence shown in the figures has been assigned to clone 36.
RESULTS
Isolation of fibrinogen reactive clones
Using the foregoing procedures, aT Zap library of strain DB, was screened for clones that were reactive with fibrinogen. Of
100,000 plaques screened, three novel clones, designated 14, 30 and 36, were found to be highly reactive with both 125I fibrinogen and fibrinogen/antifibrinogen conjugate on immunoblots. Subclones containing the pBluescript phagemid together with the insert were subsequently generated in E. coli strain Sure. Plasmid DNA from alkaline lysis minipreps of clones
14, 30 and 36, upon digestion with EcoRI which released the inserts, revealed DNA fragments of 4.6, 3.6 and 3.2kb, respectively. Using the 4.6, 3.6 and 3.2 kb fragments as separate probes, Southern blot analysis of Eco RI digests of these clones established that they hybridized each other. These clones did not hybridize with the EcoRI fragments of pAC8, a protein A probe of DB, thus eliminating the possibility of a false positive reaction between expressed protein A gene product and goat anti-fibrinogen antibody conjugate during the screening procedure. Further analysis indicated that clone 14 comprises about 2/3 of the mature molecule, C 36, extending into the C- ter inus.
Expression studies of the fibrinogen reactive protein of S. aureus
Based on restriction analysis, clones 14 and 30 were similar. Although clone 36 contained the complete gene as determined by sequence analysis, expression of the fibrinogen reactive protein with this clone was found to be difficult. Notably, a culture of clone 14 when grown to late stationary phase (i.e. °D600 1.5) also resulted in a significantly decreased yield in fibrinogen-reactive protein. This result can be explained either by toxicity of this protein on E. coli or by increased proteolytic breakdown during stationary phase. For these reasons, the expression of the partial protein was evaluated in clone 14 that has been grown to early stationary phase (OD600πmn-l.O).
Expression studies of different fractions from clone 14 with Western Blots probed with either 125I fibrinogen or fibrinogen/anti-fibrinogen conjugate established that the protein, which has a molecular size of 34 kD was found in the whole cell, periplasmic and membrane fraction (Fig 1) . In contrast, a crude lysate of an E. coli clone which contained a pBluescript phagemid with a pBR322 insert did not react with fibrinogen (Fig.l). With some fractions (e.g. membrane), there was also a higher molecular weight band, possibly a dimer, which reacted with fibrinogen. Neither of these proteins were fusions proteins as they were not inducible with IPTG. Additionally, these bands did not react with polyclonal and monoclonal anti- beta-galactosidase antibody (1:1000 dilution) (Boehringer Mannheim) on immunoblots. The fibrinogen reactive band also did not react with affinity purified chicken anti-protein A antibody (Accurate Chemicals, Westbury, NY) , thus providing additional evidence that protein A was not cloned which, by binding to IgG, could lead to false positive results.
To confirm the binding specificity of this protein to fibrinogen, periplasmic extracts which contained fewer contaminating bands were harvested from 4 L of E. coli cells expressing the protein of clone 14 and applied to an affinity column with fibrinogen linked beads. The cloned proteins of interest, as analyzed by silver stain and Western blots with 125I fibrinogen and fibrinogen/anti-fibrinogen conjugate, was found in precolumn fractions and the 3M potassium thiocyanate eluant.
However, they were not found in the fall-through, PBS with 0.5 M
NaCl eluant, nor in the acid eluant (glycine pH 3.0) Fig. 2).
Although the protein was not purified to homogeneity in this one step procedure (Fig. 2) , these results clearly indicate the binding specificity of this protein to fibrinogen.
Sequence analysis of the fibrinogen reactive protein
The complete sequence of the fibrinogen protein expressed by clone 30 is shown in Fig. 3. The sequence revealed an open reading frame of 1,935 nucleotides. The sequence has a guanosine-cytosine (GC) content of 34.7%, in contrast to the 30% GC content in the staphylococcal genome (10) . The higher GC content is attributable to the carboxy terminal half of the molecule (39.7%). Putative transcription and translation signals and ribosomal binding sites are indicated in Fig. 3. The first 26 amino acids have features characteristic of a bacterial signal peptide (16) . Based on the predicted cleavage site, the mature protein has a predicated size of 69,991 Da with a deduced pi of 6.5.
Analysis of the deduced amino acid sequence revealed three distinct domains in this protein. With the exception of residues 27-58, the N-terminal half (residues 58-325) of the protein is primarily helical as predicted by the Gamier analysis (15) . Two areas (residues 58-194 and 264-294) within the helical portion of the molecule reveal a 7 residue periodicity in which residue in positions 'a' and d' in a heptad motif 'abcdefg' are either hydrophobic or nonpolar (Fig. 4) . This finding is suggestive of a stable coiled-coil conformational structure in these areas (14.22). The second domain between residues 326 and 505 denotes a proline and glycine rich region (20%) . Of the 180 residues present, there are 17 proline and 19 glycine residues. This contrasts with the N-terminal portion of the molecule in which only 3% of the residues are either proline or glycine while the remaining carboxyl portion reveals a composition of 14% proline/glycine residues. The carboxyl-terminal domain (residues 506-645) consists of 5 tandem, direct repeats of 27 amino acids each followed by 5 terminal amino acids (PRVTK) . Divergence is observed mainly in the outside repeats. Conformational analysis indicated that this repeat region is nonhelical and contains mostly beta-sheets. Comparison of the protein sequence with others in the Genbank database revealed significant homology to three published S. aureus coagulases from S. aureus strains 8325-4, BB and 213 (18,19,26). With the exception of residue 7 in the primary translation product of the fibrinogen reactive protein, the N-terminal 33 amino acid residue which include the leader peptides among all four sequences were identical and therefore are likely to possess identical sequence cleavage sites (see Fig. 3) . Comparing residues 1 to 422 in the fibrinogen reactive protein to coagulases in strains 8325-4, BB and 213, there are 56.2%, 73.2% and 56.2% identity, respectively. The identity between residues 423 and 645 comprising the five repeated units to homologous regions in the coagulase sequences increased markedly to 93.9%. 95.1% and 96.9% for strains 8325-4, BB and 213, respectively (Fig. 5). Like that of fibrinogen- reactive protein, the C-termini of coagulase sequences of strains 8325-4, BB and 213 are composed of repeating units of 27 homologous, but no identical, amino acids followed by the terminal sequence PRVTK (Fig. 6) . However, the number of repeating units differ among strains. Although the fibrinogen reactive protein sequence displayed features that are common to the coagulase sequence,a careful comparison revealed a unique stretch of 11 amino acids between residues 409 and 419 (SVTLPSITGES) in the middle of the proline/glycine rich region (Fig. 5) . Of interest is the fact that the motif LPSITGES shares homology with a cell wall anchor motif (LPXTGX) found in other gram positive surface protein (12,28). However, there is no complete identity to this heptad motif among sequences in the Genbank database.
Based on all of the foregoing, it is clear that a novel fibrinogen reactive protein of S. aureus has been cloned. This protein is both structurally and functionally different from other apparently similar proteins such as the coagulases. The protein and the gene which expresses it are illustrated in Fig. 3. The figure shows the complete clone 36 and the protein it expresses. Clone 14 runs from nucleotide 684 to nucleotide 1935 in Fig. 3. The protein expressed by clone 14 runs from amino acid residue 229 to 645. Clone 30 is substantially the same as clone 14 and expresses substantially the same protein.
Sequence analysis clearly indicates that the fibrinogen- reactive proteins of this invention shares significant homology with staphylococcal coagulases. Recent evidence by Boden and Flock also suggested that the fibrinogen binding protein of S. aureus may possess cross-reactivity with anti-coagulase antibody (2) . However, several lines of evidence indicate that clone 36 expresses a unique fibrinogen-binding protein as do clones 14 and 30. First, expression studies of clone 14 which expresses amino acid residues 229 to 645 show that the expressed protein is necessary for fibrinogen binding. In contrast, classical coagulase has been found to complex with prothrombin to from staphylothrombin which subsequently converts fibrinogen to fibrin (10,17). Secondly, there is a unique stretch of 11 amino acids in the sequence (residues 409-419) that are not found in any of coagulase sequence described. Third, this unique amino acid sequence shares homology with a cell wall anchor motif (LPXTGX) that is found to be necessary for anchoring in a variety of gram positive surface proteins (13,28). Based on these findings, it would appear that the fibrinogen reactive protein may belong to a family of coagulase-like proteins, yet it is both structurally and functionally distinct from any of the coagulases previously described.
The isolation of fibrinogen binding protein from staphylococcal whole cell lysates with conventional chromatographic methods has been reported in two studies (11,31). However, the molecular weight (420 vs 62 kD) and the amino acid composition differs widely between the two studies. In contrast to the 62 kD protein, methionine and tyrosine, but not cysteine residues, are present in the protein of this invention. The fibrinogen reactive protein described here as expressed by clone 36 comprises predominately lysine (11.2%), threonine (9.3%) and glutamic acid (9%) while glycine (16.8%), glutamic acid (15%) and lysine (13.3%) were the abundant amino acids in the 62 kD protein (31) . In addition, the deduced isoelectric point (pI-6.5) of the fibrinogen reactive protein also differs from the basic pl (about 10.2) of the 62kD protein.
Previous studies have revealed that the fibrinogen binding component of S. aureus is a cell wall constituent because it is absent in staphylococcal L form (10) and because bacterial clumping in the presence of fibrinogen is abolished upon whole cell digestion with proteinases (4) . In reviewing the molecular architecture of the C-terminal region of other gram positive surface-anchored proteins, it is evident that they contain several conserved features (13,14). In the C-terminal of these proteins, a charged tail (4-7 amino acids) is usually preceded by a highly hydrophobic membrane anchor (about 16-20 amino acids) , the hexamer LPXTGX, a proline-glycine rich domain and a C- terminal repeat region (14) . Clearly, the C-terminal region of the fibrinogen reactive protein is different from the model described. In particular, the region preceding the stop codon lacks a charged tail and a hydrophobic membrane anchor. Instead, the five terminal amino acids (PRVTK) are preceded by five repeats of 27 amino acid each. In addition, the region N- terminal to these repeats is a broad proline/glycine region (residues 326-505) in the middle of which is a unique sequence (LPSITGE) that shares homology with a cell wall anchor motif (LPXTGX) found in other gram positive surface proteins. Notably, this molecular architecture at the C-terminus is similar to those described for pneumococal surface protein A (35) . The amino terminal half of pneumococcal surface protein A, like that of fibrinogen reactive protein, is< -helical and is consistent with an<*>-helical coiled protein conformation. The -helical region is followed by a proline-rich domain and a repeat domain consisting of ten 20-amino-acid repeats. In addition, it also lacks a classic membrane anchor and a charged tail. In contrast to the fibrinogen reactive protein, however, there is no LPXTGE motif in the C-terminal region of pneumococcal surface protein A.
The novel proteins of this invention are surface proteins of S. aureus. No such proteins have previously been detected, isolated and characterized. They are principally characterized by their ability to bind fibrinogen. The molecular weight of the protein expressed by clone 36 is about 69,991 Da and its isoelective point 6.5. The gene which expresses this protein contains about 1935 nucleotides. Other characteristic features of the protein and of segments C14 and C30 are described above. Because of the difficulty in expressing protein from clone 36, the preferred clone of this invention is clone 36. The preferred fibrinogen binding protein is the protein expressed by this clone. The protein of this invention, as specifically described herein, will be recognized by the skilled artisan as representative of a class of protein which may differ amongst the various strains of S. aureus, but will all be characterized as having substantially the same number of amino acid residues, the same tertiary structure and binding activity. They may differ slightly in the identity of the amino acid residues at specific positions in the protein chain. All such proteins are included within the scope of this invention.
The genes which generate the proteins of this invention may also differ slightly amongst various strains, but they all have the common characteristic of producing a protein of this invention.
The genes of this invention may be employed, as will be recognized by the skilled artisan, to produce plasmids or other vectors which, in turn are useful for transforming organisms such as E. coli to produce novel strains of this bacteria which will express the proteins of the invention.
Inhibition of the binding of S. aureus to endothelial cells is a major factor in preventing infection. Accordingly antibodies to proteins expressed by clone 36, its segments clone 14 and clone 30 and even smaller segments are important factors in controlling infection. The proteins and protein segments of this invention are therefore useful to form vaccines to inhibit S. aureus infections of mammals, including humans, by administering an amount of the selected protein which will stimulate the production of protective quantities of antibodies to limit adhesion of S. aureus. The proteins may also be used to produce antibodies in vitro which may be employed for passive immunization.
The proteins and polypeptide or peptide segments of this invention may be obtained by any of a number of known process including the recombinant DNA techniques described above.
Polypeptide and peptides within the scope of the invention containing, for example from about 6 to 20 or more amino acid segments, may be synthesized be standard solid phase procedures with appropriate amino acids using the protection, deprotection and cleavage techniques and reagents appropriate to each specific amino acid or peptide. A combination of manual and automated (e.g., Applied Biosystem 430A) solid phase techniques can be used to synthesize the novel peptides of this invention. Although less convenient, classical methods of peptide synthesis can also be employed. For background on solid phase techniques, reference is made to Andreu, D., Merrifield, R.B., Steiner, H. and Boman, H.G., (1983) Proc. Natl. Acad. Sci USA 80, 6475-6479; Andreu, D. Merrifield, R.B., Steiner, H. and Boman, H.G., (1985) Biochemistry 24, 1683-1688; Fink, J. , Boman, A., Boman, H.G., and Merrifield, R.B., (June 1989) Int. J. Peptide Protein Res. 33, 412-421; Fink, J. Merrifield, R.B., Boman, A. and Boman, H.G., (1989) J. Biol. Chem. 264-6260-6267; each of which being hereby incorporated herein by reference.
The products of the invention are amphoteric. They can exist and be utilized as free bases or as pharmaceutically acceptable metallic or acid addition salts. Suitable metallic salts include alkali and alkaline earth metal salts, preferably sodium or potassium salts. Acid addition salts may be prepared from a wide variety of organic and inorganic acids including mineral acids, for example citric, lactic, maleic, tartaric, phosphoric and hydrochloric acids. These salts can be prepared by procedures well known to those skilled in the art.
For use as a vaccine, it is presently preferred to administer the selected product in a pharmaceutically acceptable carrier such as a buffer. Mice or other mammals, including humans, when so immunized are protected from colonization and subsequent infection by S . aureus.
Typically, the patient to be protected will be treated with product of the invention in an amount which is effective to elicit a protective immune response. The selected agent may be administered alone or in a pharmaceutically acceptable liquid or solid carrier in which it may be dispersed, dissolved or suspended. If, for example, the patient is to be treated intravenously, the peptide may be suspended as a free base or dissolved as a metallic salt in isotonic aqueous buffer. Other methods of treatment and pharmaceutically acceptable carriers will be apparent to the skilled artisan.
The proteins, polypeptides and peptides of this invention and the genes or oligonucleotides which are employed in their expression are useful as probes for genes and proteins. They are also useful to raise antibodies by which specific strains of streptococci can be identified. The sequence of nucleotides which elicit the unique segment from position 409 to position 419 and modification of this sequence are especially useful as diagnostic probes to identify S. aureus strains. The procedure O 95/34655
is well known to the skilled artisan for identifying other infectious organisms. It involves the preparation of labeled oligonucleotides which aRe used to probe the DNA released from the suspected gram positive bacteria by all lysis, either mechanically or enzymatically.
The following citations are mentioned in the application. They are incorporated by reference.
CITATIONS
1. Blake, M.S., K.H.Johnston, G,J.Russell-Jones, and E.C. Gotschlich. 1984. A rapid sensitive method for detection of alkaline phosphatase-conjugated anti-body on Western blots. Anal.Biochem 136:175-179.
2. Boden, M.K. and J.I. Flock. 1989. Fibrinogen-binding protein/clumping factor from Staphylococcus aureus. Infect.Immun. 57:2358-2363.
3. Cheung, A.L. and V.A. Fischetti. 1989. The role of surface proteins in staphylococcal adherence to fibers in vitro. J.Clin.Invest. 83:2041-2049.
4. Cheung, A.L. and V.A. Fischetti. 1990. The role of fibrinogen in staphylococcal adherence to catheters in vitro. J.Infect.Dis. 161:1177-1186.
5. Cheung, A.L., J.M. Koomey, CA. Butler, S.J. Projan, and V.A. Fischetti. 1992. Regulation of exprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr.
Proc.Natl.Acad.Sci.USA.89:6462-6466. 6. Cheung, A.L., J.M. Koomey, S.Lee, E.A. Jafe, and V.A. Fischetti. 1991. Recombinant human tumor necrosis factor alpha promotes adherence of Staphylococcus aureus to endothelial cells. Infect.Immun.59:3827-3831.
7. Cheung, A.L., M.Krishman, E.A. Jaffe, and V.A. Fischetti. 1991. Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to culture human endothelial cells. J. Clin.Invest. 87:2236-2245.
8. Cohen, M.L. 1992. Epidemiology of drug resistance: implications for a post-antibiotic era. Science 257:1050-1055.
9. Devereux, J.,P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequences of analysis programs for the VAX. Nucl.Acids Res. 12:387-395.
10. Easom, C.S.F. and C. Adlam. 1983. Staphylσcocci and staphylococcal infections, Academic Press, New York.
11. Espersen,F. ,1. Clemmensen, and V. Barkholt. 1985. Isolation of Staphylococcus aureus clumping factor. Infect.Immun. 49:700- 708.
12. Feinberg, A.P. and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal.Biochem. 132:6-13.
13. Fischetti, V.A. , V. Pancholi, and 0. Schneewind. 1990. Conservation of a hexapeptide sequence in the anchor region of surface proteins from gram-bacteria. Mol.Microbiol. 4:1603-1605. O 95/34
14. Fischetti,V.A., V.Pancholi, P.Sellers, J.Schmidt, G.Landau, X.Xu, and 0. Schneewind. 1992.Streptococcal M protein: A common structural motif used by gram positive bacteria for biologically active surface molucules, p.31-38. In T.K. Korhonen, T.Hovi, and P.H. Malela (ed.), Molecular recognition in host-parasite interactions. Plenum Press, New York.
15. Gamier, J.,D.J. Osguthorpe, and B.Robson. 1978. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins.J.Mol.Biol.120:97- 120.
16. Halvorson,H.O. and D. Perlman. 1983. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides.J.Mol.Biol. 167:391-409.
17. Hemker, H.C., M.Bas, and A.D.Muller. 1975. Biochim Biophys.Acta 379:180-188.
18. Kaida, S.T.Miyata, Y.Yoshizawa, H.Igarashi, and S.Iwanaga. 1989. Nucleotide and deduced amino acid sequences of staphylocoagulase gene from Staphylococcus aureus strain 213. Nucl.Acids Res.17:8871.
19. Kaida,S., T.Miyata, Y.Yoshizawa, S.Kawabata, T.Morita, H.Igarashi, and S. Iwanaga. 1987. Nucleotide sequence of the staphylocoagulase gene: its unique COOH-terminal 8 tandem repeats. J.Biochem.102:1177-1186. O 95/34655
20. Kyte, J. and R.F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein.
J. ol.Biol.157:105-132.
21. Laemmli, U.K. 1970. Cleavage of structure proteins during th assembly of the head of bacteriophage T4, Nature 227:680-685.
22. Lupas, A.,M. Van Dyke, and J. Stock. 1991. Predicting coiled coils from protein sequences. Science 252:1162-1164.
23. Manaitis, T.,E.F. Fritsch, and J. Sa brook. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Col Spring Harbor, NY.
24. Neu,H.C. 1992. The crisis in antibiotic resistance. Science 257:1064-1072.
25. Pearson, W.R. and D.L. Lipman. 1988. improved tolls for biological sequence comparison. Proc.Natl.Acad.Sci.USA.88:4781- 4785.
26. Phonimdaeng,P.,M. O'Reilly, P. Nowlan, A.J. Bramley, and T. Foster. 1990. The coagualse of Staphylococcus aureus 8325-4. Sequence analysis and virulence of site-specific coagualse- deficient mutants.Mol.Microbiol. 4:393-404.
27. Sanger, F.,S. Nicklen, and A.R. Coulson. 1977. DNA sequencing with chain terminating inhibitors.
Proc.Natl.Acad.Sci.USA.74:5463. O 95/34655
28. Schneewind, 0.,P.Model, and V.A. Fischetti. 1992. Sorting of protein A to the staphylococcal cell wall. Cell 70:267-281.
29. So, M. , J.H. Crosa, and S. Falkow. 1975. Polynucleotide sequence relationships among Ent plasmids and the relationship between Ent and other plasmids. J. Bacteriol. 121:234-238.
30. Towbin, H., T. Staehelin, and J. Gordon. 1970. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:Procedure and some applications.
Proc.Natl.Acad.Sci.USA. 76:4350-4354.
31. Usui, Y. 1986. Biochemical properties of fibrinogen binding (clumping.factor) of the staphylococcal cell surface. Zentralbl.Bakteriol. ikrobiol.Hyg.[AJ262:287-297.
32. Voller, A.,D.E. Bidwell, and A. Barlett. 1976. Enzyme immunoassays in diagnostic medicine. Bull.World Health Organ. 53:55-65.
33. Waldvogel, F.A. 1985. Staphylococcus aureus, p.1097-1116. In G.L. Mandell, R.G.Jr. Douglas, and J.E. Bennett (ed.). Principles and Practice of Infectious Diseases, John Wiley & Sons, New York.
34. Watanakunakorn, C. and I.M. Baird. 1977. Staphylococcus aureus bacter ia and endocarditis associated with a removable infected intravenous device. Am.J.Med. 63:253-256. r'lll "* D-E- BrilβS- 1992- Strurtual " e«ies and evolutionary relationship of PspA, a surface protein of
J.Bacteriol. 174:601-609.

Claims

WHAT IS CLAIMED IS:
1. A gene of the group clone 14, clone 30 and clone 36 which expresses a fibrinogen binding surface protein on S. aureus and segments and analogs of said gene capable of expressing surface proteins on S. aureus having substantially the same activity.
2. A surface protein expressed by a gene of claim 1.
3. A plasmid vector carrying a gene of claim 1.
4. A microorganism transformed by a gene of claim 1 and capable of expressing a fibrinogen binding protein.
5. An antibody to a surface protein of claim 2.
6. A vaccine effective to inhibit the adhesion of S. aureusto fibrinogen because it contains a protein of claim 2.
EP95922211A 1994-06-07 1995-06-05 Surface protein of staphylococcus aureus Withdrawn EP0771351A1 (en)

Applications Claiming Priority (3)

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US25496894A 1994-06-07 1994-06-07
US254968 1994-06-07
PCT/US1995/007100 WO1995034655A2 (en) 1994-06-07 1995-06-05 Surface protein of staphylococcus aureus

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EP0771351A1 true EP0771351A1 (en) 1997-05-07

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Publication number Priority date Publication date Assignee Title
AUPN350795A0 (en) * 1995-06-13 1995-07-06 Australian National University, The Nucleic acid molecule and its uses in determining pathogenicity of Staphylococcus
WO1999027109A2 (en) * 1997-11-26 1999-06-03 Inhibitex, Inc. EXTRACELLULAR MATRIX-BINDING PROTEINS FROM $i(STAPHYLOCOCCUS AUREUS)
US6680195B1 (en) 1997-11-26 2004-01-20 Inhibitex, Inc. Extracellular matrix-binding proteins from staphylococcus aureus
US6692739B1 (en) * 1998-08-31 2004-02-17 Inhibitex, Inc. Staphylococcal immunotherapeutics via donor selection and donor stimulation
WO2011008092A2 (en) 2009-07-15 2011-01-20 Aimm Therapeutics B.V. Gram-positive bacteria specific binding compounds

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Publication number Priority date Publication date Assignee Title
CA2124210A1 (en) * 1992-09-21 1994-03-31 Maria K. Boden Fibrinogen binding protein

Non-Patent Citations (1)

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Title
See references of WO9534655A3 *

Also Published As

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JPH10501418A (en) 1998-02-10
WO1995034655A2 (en) 1995-12-21
CA2192175A1 (en) 1995-12-21
WO1995034655A3 (en) 1996-02-01

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