CA2247072A1 - Bacterial elastin binding protein, nucleic acid sequence encoding same and diagnostic and therapeutic methods of use thereof - Google Patents

Abstract

A microbial surface component recognizing adhesive matrix molecules (MSCRAMM) is disclosed which is characterized by its ability to bind to elastin, its inhibited activity in the presence of SDS and its enhanc ed activity in the presence of thiol reductants. More particularly, the MSCRAMM is bacterial in origin and is a member of the family of elastin bind ing proteins. In one embodiment, the MSCRAMM comprises a polypeptide having an amino acid sequence defined herein as SEQ ID N O:2, and extends to active fragments thereof. The role of MSCRAMMs in bacterial infection and its sequelae and related conditions is no ted and the MSCRAMM may be prepared and used in diagnostic procedures and tests, including drug discovery assays, as well as in pharmaceutical compositions applicable for corresponding therapeutic methods. Both agonists and antagonists of the MSCRAMM are proposed a nd illustrated.

Description

W O 98/38312 PCT~US97/03106 BACTERL~L ELASTIN BINDING PROTE~IN, NUCLEIC
ACID SEQUENCE ENCODING SAME AND DIAGNOSTIC
AND THERAPEUTIC METHODS OF USE THEREOF

S GOVERNMENTAI S~JPPORT

The research leading to the present invention was supported in part by NIH grants HL-26499 and HL-41926. The government may have certain rights in the invention.

The present invention relates generally to a microbial surface component recognizing adhesive matrix molecules (MSCRAMM) and to active polypeptide fragments thereof, and to nucleotide sequences encoding the protein and active polypeptide15 fragments thereof. More specifically, the invention relates to a protein on the surface of bacteria which binds a component of the extracellular matrix (ECM), which component is terrned elastin, and to diagnostic and thcrapeutic methods which relate to this protein-protein interaction.

20BAC~GROIrND OI~ THE INVENTIOI~

The extracellular matrix (ECM) is a ubiquitous structure that contributes to architecture, elasticity, and regidity of virtually all vertebrate tissues and organs.
Within the last several decades. additional biologic activities of the ~CM have been 25 identified. Distinct components of the ECM have been found to mediate one or several cellular events such as adhesion, proliferation. and regulation of gene expression (1-4). These cellular ECM interactions in turn dircct many physiologic and pathologic processes including developmcnt. wound hcaling, and tumor cell metastasis (5-7). lt is now known that cell surface ECM receptors are the key 30 mediators of these biologic events. Most of these ECM receptors belong to a family of dimeric receptor complexes called integrins (8,9), although non-integrin ECM
receptors have been identified (10). In addition to eukaryotic cells, various RECTiFlE~ SHEET (RULE 9t~
ISA/EP

2 PCTrUS97/03106 pathogenic bacteria also interact specifically with the host ECM tnrough cell surface - ECM binding molecules. Cell surface ECM binding molecules of pathogenic bacteria belong to a group of proteins known collectively as adhesins or microbial sur~ace components recognizing adhesive matrix molecules (MSCRAMMS). and are widely 5 believed to play important roles in key steps of disease pathogenesis ( I 1,12).

Among many important pathogenic bacteria, few are as efficient in developing multiple resistance to antibiotics and causing a wide spectrum of diseases as Stap~2ylococcus aureus (S. Aureus). S. aureus has been identified as one of the 10 causative agents of diseases such as infective endocarditis, osteomyelitis, aortitis, pneumonia, and scalded skin syndrome (13-15). Furthermore, several strains of S.aureus have a propensity to extravasate into the circulation to cause bacteremia and subsequent formation of metastatic abscesses. These properties imply that S. aureus is capable of interacting with various components of the host expressed in respective 15 target tissues. In agJreement with this hypothesis, S aureus has been found to associate with many host determinants including major ECM components such as collagen (16), fibronectin (17), l~minin (18), proteoglycans (19), and elastin ('7~)).

Most clinical isolates of S aure21s bind specifically to fibronectin. and mutant strains 20 defective in fibronectin binding have decreased ability to colonize damaged heart valves in animal models of endocarditis (21). The S. aureus-collagen binding interaction has been implicated in osteomyelitis and septic arthritis by Swita}ski et al ( 2), in which expression of the collagen adhesin has been found to be both necessary and sufficient for attachrnent of S. aureus to the type II collagen-rich cartilage. They 25 also demonstrated in a murine experimental septic arthritis model that greater than 70% of ~nim~ injected with collagen adhesin-positive strains developed septic arthritis whereas less than '77% of ~nim:~ls challenged with isogenic mutant strains lacking the collagen adhesin developed clinical symptoms of the disease (23). As one of the main component of the basement membrane ECM, l~minin has been proposed 30 to play an important role in tumor cell metastasis. It has been shown that invasive S.

W O 98/38312 PCT~US97/03106 aureus interacts with the basement membrane component l~minin, but, non-invasive- S. epidermidis shows no binding (18). Talcen together~ these observations indicate that S. aureus-EC~I interactions are playing critical roles in targeting host tissues for o attachment, colonization, and invasion.
s The primary physiologic role of elastin is to confer the property of reversible elasticity to tissues and organs (24). Elastin expression is highest in the lung? skin, and blood vessels, but the protein is widely expressed in m~mm~ n hosts for S. aureus. Since elastin binding may be a mechanism for S. aureus to target elastin-rich tissues of the 10 host, the cellular and biochemical properties of this interaction (20) were investigated in a previous study. S. aureus binding to elastin was found to be rapid, reversible, saturable, of high affinity (low nM), and ligand specific. Furthermore, a 25 kDa cell surface elastin binding protein named EbpS (for_lastin binding ~rotein of Staphylococcus aureus) was isolated. and EbpS has been proposed to mediate S.
15 au~eus binding to elastin-rich host ECM. EbpS is structurally distinct from the m~mm~ n cell surface elastin binding protein. and the two elastin binding proteins recognize different regions in elastin. EbpS binds to a region in the N-terminal 30 l~Da fragment of elastin, whereas the m~mm~lian elastin receptor recognizes the hexapeptide sequence VGVAPG in the C-terrninal half of elastin (25).
From the foregoing it is apparent that a greater understanding of EbpS would contribute to the effort to develop new diagnostic and therapeutic strategies against bacterial infection and the sequelae thereof. and that a concomitant need exists in the art for compositions and methods for such diagnostic and therapeutic intervention.
25 Accordingly, it is toward the fulfillment of that need that the present invention responds.

W O 9~/38312 PCTnUS97/03106 SUMMARY OF THE INVENTIO~

In accordance with the present invention, a nucleotide sequence is provided thatencodes a bacterial MSCRAMM, which is implicated in the attachment, colonization Q
Sand invasion of host cells by the bacteria.

In a further embodiment, the invention provides the MSCRAMM protein, biologically active fragments thereof, and antibodies and binding partners thereto, which are useful in the diagnosis and treatment of bacterial infections. These reagents are particularily 10useful in treating infections of elastin-cont~inin~ host tissues, for example lung, skin and blood vessels. Corresponding pharmaceutical compositions and therapeutic methods involving the MSCRAMMs of the present invention are contemplated, that may be directed to a broad range of diseases and other conditions. including by way of non-limiting illustration, such conditions as tumor cell metastasis, wound he~l;n~, 15infective endocarditis, osteomyelitis, aortitis, pneumonia and scalded slcin syndrome.

One aspect of the present invention includes an MSCRAMM having the following characteristics:
a) it binds to elastin;
b) its activity is inhibited in the presence of SDS; and c) it has enhanced activity in the presence of thiol reductants.
In a further aspect~ the MSCRAMM has a predicted molecular weight of about 25 kDa, and a predicted PI of about 4.9. More particularly~ the present MSCRAMM is isolated from Staphylococcus aureus.
~nother aspect of the present invention extends to active polypeptide fragments of an MSCRAMM and related polypeptides which retain the property of binding elastin. In one embodiment, such a polypeptide comprises an amino acid sequence that corresponds to the elastin binding site of a MSCRAMM and consists of bet~veen 8 and 80 amino acids. In one such embodiment the polypeptide has the amino acid W O 98/38312 PCT~US97/03106 sequence consisting of the N-terrninal 59 amino acids of the MSCRAMM. In another- embodiment the polypeptide consists of between 10 and 46 amino acids. In still another embodiment, the polypeptide consists of between 12 and 21 amino acids. In yet another embodiment, the polypeptide contains about 10 amino acids. In preferred S embodiments the MSCRAMM has the amino acid sequence of SEQ ID NO:2 or SEQ
ID NO:2 comprising a conservative substitution thereof.

In another such embodiment the polypeptide corresponds to an elastin binding site of a MSCRAMM that comprises the arnino acid sequence of SEQ ID NO: 16 or SEQ ID
10 NO: 16 comprising a conservative substitution thereof (amino acids (AAs) 18-23 of SEQ ID NO:2: TNS~QD). In one particular embodiment the polypeptide has the amino acid sequence of SEQ ID NO: 10 (AAs I -78 of SEQ ID NO:2) or SEQ ID
NO: 10 comprisin~ a conservative substitution thereof. ln another particular embodiment the polypeptide has the amino acid sequence of SEQ ID NO:12 (AAs 1-15 34 of SEQ ID NO:2) or SEQ ID NO:12 comprising a conservativc substitution thereof. In yet another particular embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO:13 (AAs 14-34 of SEQ ID NO:2) or SEQ ID NO:13 comprising a conser~ative substitution thereof. In still another particular embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO:I~ (AAs 14-23 of 20 SEQ ID NO:2) or SEQ ID NO:14 comprising a conservative substitution thereof. In still another particular embodiment the polypeptide consists of the amino acid sequence of SEQ ID NO: 15 (AAs 14-59 of SEQ ID NO:2) or SEQ ID NO: 15 comprising a conservative substitution thereof. In yet another particular embodiment the polypeptide of comprises the amino acid sequence of SEQ ID NO: 18 (AAs 18-3425 of SEQ ID NO: ) or SEQ ID NO: 18 comprising a conservative substitution thereof.
In a preferred embodiment the polypeptide further inhibits the binding of S. aureus to elastin.

The present invention also relates to a recombinant DNA molecule or cloned gene, or 30 a degenerate variant thereof, which encodes a MSCRAMM or an active polypeptide WO 98/38312 PCT~US97/03106 fragment thereof; preferably a nucleic acid molecule~ in particular a recombinant - DNA molecule or cloned gene. In one particular embodiment the nucleic acid encodes a MSC~IM having the DNA sequence shown in FIGURE 3 (SEQ ID
NO~
In another such embodiment a nucleic acid encodes a polypeptide that binds elastin;
consists of between 8 and 80 amino acids; and comprises an amino acid sequence that corresponds to the elastin binding site of a microbial surface component recognizing adhesive matrix molecules (MSCRAMM). In one particular embodiment the nucleic 10 acid has the nucleic acid sequence of SEQ ID NO:9. In another particular embodiment, the nucleic acid has the nucleic acid sequence of SEQ ID NO: I 1.

The bacterial DNA sequences of the MSCRAMM of the present invention or portions thereof. may be prepared as probes to screen for complementary sequences and 15 genomic clones in the same or alternate species. The present invention extends to probes so prepared that may be provided for screening cDNA and a'enomic libraries for the MSCRAMM. In particular, such probes may be degenerate probes based on SEQ ID NO:3. In addition, primers directed to the 5' and 3' sequences of the MSCRAMM, such as SEQ ID NOS: 4 and 5, may be used to amplifv related 20 sequences by PCR. For example, the probes may; be prepared with a variety of known vectors, such as the phage ~ vector. The present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors e~pressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences set forth in FIGURE 3 (SEQ ID NO: 1, 25 respectively). Correspondingly, the preparation of antisense RNA and ribozymes are included herein.

The present invention also includes MSCRAMM proteins having the activities notedherein, and that display the amino acid sequences set forth and described above and 30 selected from SEQ ID NO:2.

CA 02247072 l998-08-27 W O 98/38312 PCTrUS97/031~6 In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or 5 recombinant DNA molecule comprising a DNA sequence encoding the present MSCRAM(s) active polypeptide fragments thereof and the polypeptides of the present invention. More particularly, a unicellular host is transformed with the complete DNA sequence determined from the sequences set forth above or a fragment of the DNA sequence, e.g. SEQ rD NO: 1, or SEQ ID NOs: 9 or 11.
According to other preferred features of certain preferred embodiments of the present invention, a recombinant expression system is provided to produce biologically active animal or human MSCRAMMs or the active polypeptide fragments thereof~ or the polypeptides of the present invention.
The concept of the MSCRAMM contemplates that specific bacterial cell surface proteins exist for correspondingly specific components in host tissue. such as elastin and thc lilce as described earlier. Accordingly. the exact structure of each MSCRAMM will understandably vary so as to achieve this binding and activity 20 specificity. It is this specificity and the direct involvement of the MSCRAMM in the chain of events leading to bacterial infection. that offers the promise of a broad spectrum of dia~nostic and therapeutic utilities.

The present invention naturally contemplates several means for preparation and 25 isolation of the MSCRAMM, including isolation from a natural source and/or asillustrated herein. through known recombinant techniques. The invention is accordingly intended to cover such preparations within its scope. Subsequent isolation of the MSCRAMM~ active polypeptide fragments thereof and polypeptides of the present invention as illustrated herein, is also included as part of the invention.
30 The isolation of the genomic DNA and amino acid sequences disclosed herein W O98/38312 PCTrUS97/03106 facilitates the reproduction of the MSCRAMM by such recombinant techniques, and accordingly. the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.

l[ he invention includes an assay system for screening of potential drugs effective to modulate binding activity to target m~mm~ n cells by interrupting or potentiating the binding of ehe MSCRAMM to host tissue. In one instance, the test drug could be ~ mini~tered to a cellular sarnple with the MSCRAMM that binds the host tissue, or 10 an extract containing the MSCRAMM, to deterrnine its effect upon the binding activity of the MSCRAMM to any chemical sample (including DNA), or to the test drug, by comparison with a control.

The assay system could more importantly be adapted to identify drugs or other entities 15 that are capable of binding to the MSCRAMM, thereby inhibiting or potentiating infectivity. Such an assay would be useful in the development of drugs that would be specific against particula} infections For example. such drugs might be used to prevent infection. or to treat infection. as for example, in association with anantibiotic .
In yet a further embodiment, the invention contemplates antagonists of the activity of a MSCI~AMM, in particular, an agent or molecule that inhibits MSCRAMM binding to elastin. In a specific embodiment. the antagonist can be a peptide having thesequence of a portion of an elastin-binding domain of a MSCRAMM.
One of the characteristics of the present MSCRAMM is that it binds to a 30 kDa N-terminal fragment of elastin. which is present in tissues which require elasticity, such as lungs. skin and blood vessels.

=

The diagnostic utility of the present invention extends to the use of the present MSCRAMM in assays to screen for bacterial infection.

The present invention likewise extends to the development of antibodies against the 5 MSCRAMM(s) and active polypeptide fragments thereof and polypeptides of the invention, including naturally raised and recombinantly prepared antibodies. For example. the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the MSCRAMM(s). Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well 10 as bi-specific (chimeric) antibodies, and antibodies including other functionalities thereby suiting them for additional diagnostic use conjunctive with their capability of mo~ ting bacterial infectivity.

Thus, the MSCE~AMMs active polypeptide fragements thereof, polypeptides of the 15 invention, their analogs, cognates and/or mimics, and any antagonists or antibodies that may be raised thereto. are capable of use in connection with various diagnostic techniques~ including immunoassays, such as a radioimmunoassay~ using for example, an antibody, to the MSCRAMM that has been labeled by either radioactive addition~
or radioiodination.

In an immunoassay a control quantity of the antagonists or antibodies thereto. or the like may be prepared and labeled with an enzyme~ a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites 25 within the sample. the resulting mass may be examined by known techniques~ which may vary with the nature of the label attached.

In the instance where a radioactive label, such as the isotopes iE~ C, ;'P, 3'S. 36Cl, '- 5ICr, 57Co, 58Co, 59Fe, 90Y, ~251, ~3~I, and ~36Re are used, known currently available 30 counting procedures may be ~ltili7~d In the instance where the label is an enzyme, WO 98/38312 PCTrUS97/03106 detection may be accomplished by any of the presently utilized colorimetric, - spectrophotometric fluorospectrophotometric, amperometric or gasometric techniques known in the art.

5 The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the MSCRAMM?
or to identify drugs or other agents that may mimic or block their activity. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the MSCRAMMs, 10 their agonists and/or antagonists, and one or more additional imrnunochemicalreagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).

15 In a further embodirnent. the present invention relates to certain therapeutic methods which would be based upon the activity of the MSCRAMM(s). its (or their) subuni~s, or active pol~/peptide fragments thereof, or polypeptides of the present invention or upon agents or other drugs determined to possess the same activity A first therapeutic method is associated with the prevention of the manifestations of 20 conditions causallv related to or following from the binding activity of the MSCR~MM or its subunits, and comprises ~Amini~tering an agent capable of mo~ ting the production and!or activity of the MSCRAMM or subunits thereof, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host. For example, drugs or other binding25 partners to the MSCRAMM or proteins may be ~lmini~tered to inhibit bacterial infection. More particularly in the treatment of S aureus infection.

More specifically~ the therapeutic method generally referred to herein could include the method for thc treatment of various pathologies or other cellular dysfunctions and 30 derangements by the z~lmini~tration of ph~rm~reutical compositions that may W O 98/38312 PCT~US97/03106 comprise effective inhibitors or enhancers of the MSCRAMM or its subunits, or other - equally effective drugs developed for instance by a drug screening assay prepared and used in accordance ~vith a further aspect of the present invention. For exarnple, drugs or other binding partners to the MSCRAMM or proteins~ as represented by SEQ ID
5 NO:2, may be ~lmini.stered to inhibit bacterial infectivity.
-In particular. the MSCRAMS active polypeptide fragments thereof and peptides of thepresent invention, their antibodies, agonists, antagonists, could be prepared in pharmaceutical formulations for ~mini~tration in instances wherein antibiotic therapy 10 is appropriate.

Accordingly, it is a principal object of the present invention to provide a MSCRAMM
and its subunits in purified form that exhibits certain characteristics and activities associated with bacterial infectivity.
It is a further object of the present invention to provide antibodies to the MSCRAMM
and its subunits, and methods for their preparation, including recombinant means.

It is a further object of the present invention to provide a method for detecting the 20 presence of the MSCRAMM and its subunits in mammals in which invasive.
spontaneous. or idiopathic pathological states are suspected to be present.

It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially 2~ effective in either mimicking the activitv or combating the adverse effects of the MSCRAMM and/or its subunits in mzlmm~lc.

It is a still further object of the present invention to provide a method for the treatment of m~mm:~l.c to control the amount or activity of the MSCRAMM or subunits thereof, W O 98/38312 PCTnUS97/03106 so as to alter the adverse consequences of such presence or activity~ or where beneficial, to enharlce such activity.

It is a still further object of the present invention to provide a method for the treatment 5 of m~mm~l~ to control the arnount or activity of the MSCRAMM-cont:~inin~ bacteria or its subunits, so as to treat or avert the adverse consequences of invasive~ ~
spontaneous or idiopathic pathological states.

It is a still further object of the present invention to provide pharmaceutical 10 compositions for use in therapeutic methods which comprise or are based upon the MSCRAMM, its subunits, active polypeptide fragments thereof, polypeptides of thepresent invention binding partner(s) to MSCRAMMs, or upon agents or drugs that control the production~ or that mimic or antagonize the activities of the MSCRAMM.

15 Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the followin~illustrative drawings.

BRTFF DESCRIPTION OF T~IE DRAWINGS
Figure 1: Southern analysis of genomic DNA from S aurez~s. ~enomic DNA
isolated from S. aureus strain 12598 was digested with ~;coR I (lanes A & C) or EcoR
I/Hind III/Hinc II (lane D) and pEBPS-1 was digested with EcoR I (lane B). Samples were fractionated by 1% TAE-agarose electrophoresis and Southem blotted to 25 nitrocellulose. The membranes were hybridized to a degenerate oligonucleotide(lanes A & B) or to the 2.6 kb Hind III/Hinc II insert of pKS-2.6 (lanes C & D). Sizes of the hybridized fragments were deterrnined from the migration pattern of Hind III-digested ~ DNA markers.

CA 02247072 l998-08-27 W O 98/38312 PCTrUS97/03106 Figure 2: Physical map of the pKS-2.6 insert. Sites recognized by various restriction - endonucleases are indicated. Location and direction of the ~bpS open reading frame is shown by the hatched box and arrow, respectively~
~, 5 Figure 3: Primary sequence of ebpS. Nucleotide and predicted amino acid sequences are numbered starting at the first nucleotide of the open reading frame and translation initiation codon, respectively. The putative -35 and -10 hexamers, and ribosomalbinding site are indicated. The experimentally determined N-terminal sequence ofcell surface EbpS is shown in bold letters, and the experimentally determined arnino 10 acid sequences of EbpS are underlined. The N-terminal amino acids of the recombinant construct were sec~uenced from both full length rEbpS and N-terminalfragment of CNBr-cleaved rEbpS. The in-frame termination codon is indicted by anasterisk.

15 Figure 4: E~cpression of ~bpS gene in E. coli The ebpS open reading frame wasPCR-arnplified and expressed in E. coli as a fusion protein with polyhistidine residues attached to the N-terminus. rEbpS purified from three different positive clones by Ni++-NTA affinity chromatography was fractionated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (lanes B-D). Migration pattern of the sizc 20 standard is shown in lane A.

Figure 5: rEbpS binds specifically to immobilized elastin peptides. Appro~cimately 1 o6 cpm of radiolabeled rEbpS was incubated with 1 ml of the elastin peptide affinity resin in the absence (lane C) or presence (lane D) of 2 mg of unlabeled elastin 25 peptides for 2 h at room temperature in 1.5 ml of binding buffer. After thorough washing7 bound proteins were eluted with 1% SDS buffer and analyzed by 10% SDS-PAGE and autoradiography. The starting material contained a 40 kDa degradation product in addition to the intact 45 kDa rEbps (lane B). Migration of ''IC-labeled size standards is shown in lane A.

WO 9X/38312 PCT~US97/03106 Figure 6: rEbpS specifically inhibits S. aureus binding to radiolabeled elastin.Radioiodinated elastin (20 ng) was incubated with 2 .Y 10 8 live S. aurclls cells in the absence or presence of 1.0, 2.0, 5.9, 9.8, or 19.6 IlM of unlabeled rEbpS or 26 ~lM of mouse DHFR for I h at room temperature in 200 ~LI of TSB. The cells were pelleted 5 by centrifugation, and the supernatant was discarded. Pellets were resuspended in 1 ml of TSB, transferred to new tubes, and washed two more time with TS13.
Radioactivity associated with cells was measured using a gamma counter. Results are presented as mean % binding + standard deviation of triplicate determinations.

10 Figure 7: Cell surface labeled EbpS binds to an antibody against rEbps. Cell surface labeled extracts (107 cpm) ~.cL,aLcd by IODOGEN radioiodination and subsequent lysostaphin digestion (lane A) were pre-absorbed to 3 ml of pig IgG-Affi-Gel 10 to remove protein from the starting material. Starting material devoid of surface labeled protein A (lane B) was incubated with I ml of the anti-rEbpS IgG affinity resin in the 15 absence (lane C) or presence (lane D) of 2 mg unlabeled rEbpS in ml of binding buffer for 2 h at room temperature. After washing with buffer until radioactivity of the flow through reached background levels, bound surface proteins were eluted with 1% SDS and analyzed by 15% SDS-PAGE and autoradiography. Tl1c band intensity of proteins (25 & 35 kDa) that associated with the affinity resin was scanned to20 quantify binding.

Figure 8: Fab fragments of anti-rEbpS IgG inhibit S. at~reus binding to elastin.Radiolabeled elastin was incubated with live S. aureus cells in the absence or presence of 6. 10, 20, 50, and 1 00~g of immune rEbpS IgG Fab or 2û and 100 ~g of pre-25 immune Fab fragments for 1 h at room temperature in 2 ml of binding buffer, andbinding was quantified as previously described. Data are presented as mean %
binding + standard deviation of triplicate measurements.

Figure 9: The elastin binding site in EbpS is contained within residues 14-59. Elastin 30 binding properties of various EbpS fragments and recombinant constructs (described CA 02247072 l998-08-27 W O 98/38312 PCTrUS97/03106 in Example 2) were assessed by their capacity to specifically bind to tropoelastin.
Inactivity of the amino terminal synthetic peptide was determined by an inability to inhibit binding. Residues 14-59 (shaded area) are common to all fragments with elastin binding activity.

Figure 10: Expression of recombinant EbpS proteins. Recombinant EbpS proteins were purified by Ni-NTA affinity chromatography, fractionated by 15% SDS-PAGE, and stained with Coomassie Brilliant Blue R-''50 (a) or transferred to nitrocellulose membranes and reacted with anti-rEbpS IgG (b) or anti-rEbpS IgG that had been pre-absorbed to trEbpS-2 (c). Lane A: ovalbumin, lane B: rEbpS, lane C: trEbpS-I, lane D: trEbpS-2~ and lane E: Iysozyme. Molecular masses of the recombinant proteins were appro~imated from the migration pattern of ovalbumin~ Iysozyme, and pre-stained size standards.

Figure 11: Recombinant trEbpS-I and trEbpS-2 bind to elastin. Tropolastin (3 llg) that was fractionated by 10% SDS-PAGE and Western blotted to nitrocellulose membranes was reacted with 5 tlM biotinylated trEbpS-I (lanes A and B) or trEbpS-2 (lanes C and D) in the absence (lanes A and C) or presence (lanes B and D) of 3 mg/ml elastin peptides. Binding of tr mcated EbpS proteins was visualized by subsequent incubation with avidin-horse radish peroxidase and 4-chloro-naphthol.
Figure 12: Truncated recombinant EbpS proteins inhibit binding of S. au-eus cells to radiolabeled elastin. Live S. aureus cells (2x 108) were incuhated with radioiodinated elastin ( 10 ng) in the absence or presence of increasing concentrations of rEbpS, trEbpS-1, or trEbpS-2 for 1 hour at room temperature in 200 ~LI of TSB. The assay was terrnin~3tecl by centrifugation, and cell pellets were washed twice with 1 ml of TSB. E~tent of binding was quantified by measuring radioactivity associated with the pellets. Results are presented as mean relative % binding+SD of triplicate determinations~ with measurements obtained in the absence of recombinant EbpS

3 0 proteins defined as 100%.

WO 98/38312 PCTrUS97/03106 Figure 13: The inhibitory effect of anti-rEbpS antibody is neutralized by pre-absorption with trEbpS-2. Fab fragments from the original (control) and anti-rEbpS IgGs ehat had been pre-absorbed with trEbpS-~ (trEbpS-2 negative) were 5 prepared by papain digestion. Increasing concentrations of Fab fragments were incubated with live S. aureus cells and radiolabeled elastin as previously described.
Data are shown as mean relative % binding+SD from triplicate deterrninations.

Figure 14: Elastin binding site defined by peptide inhibition studies. Seven 10 overlapping synthetic peptides spanr~ing residues 14-36 were tested for their ability to inhibit binding of S. aureus to tropoelastin. Inhibition activity, qualitatively scored as or -, is indicated next to the peptide number. Some arnino acids in peptides P2 and P7 were substituted as indicated. The shaded bo~ indicates the predicted active sequence required for elastin binding.
Figure 15: Peptide P1 spanning EbpS residues 18-34 specifically inhibits binding of S. aureus to radioiodinated elastin. Increasing concentrations of the P I and P2peptides were incubated with S. aureus cells and radiolabeled elastin for 1 hour at room temperature. Binding was quantified as previously described. Results are 20 presented as mean relative % binding~SD (n=3).

Figure 16: A synthetic peptide corresponding to EbpS residues 14-23 specificallyinhibits staphylococcal elastin binding. Overlapping synthetic 1 Omers corresponding to residues 14-23 (P3), 21-30 (P4), and 27-36 (P5) were generated as described. S.
~5 aurezls cells were incubated with labeled elastin in the absence or presence of 0.5, 1.0, or '~.0 mg/ml of P3-P5 peptides. Binding assays were processed as described previously. Error bars represent SD calculated from triplicate determinations.

DET~TT.Fn DESCRIPTION

W O 98/38312 PCT~US97/03106 Early studies on the binding of Staphylococcus aure2~s were described in Park et al, J.
Biol. Chem. 266: 3399-23406 (1991), which is hereby incorporated by reference in its entirety. MSCRAMM-mediated adherence of microorg~ni~mc to host tissues is reviewed in Patti et al. Annu. Kel~. Microbiol. 48:58~-617 (1994), which is also hereby 5 incorporated by reference in its entirety.

In accordance with the present invention there may be employed conventional molecular biology~ microbiology, and recombinant DNA techniques within the skillof the art. Such techniques are explained fully in the literature. ~ee, e.g., Sambrook 10 et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols inMolecular Biology" Volumes l-III ~Ausubel, R. M.~ ed. (1994)]; "Cell Biology: A
Laboratory Handbook" Volumes I-III [J. E. Celis. ed. (1994))]; "Current Protocols in Immunology" Volumes I-III [Coligan~ J. E., ed. (lg94)]; "Oligonucleotide Synthesis"
(M.J. Gait ed. 1984): "Nucleic Acid Hybridization" [B.D. Harnes & S~J. Higgins eds.
~ 15 (1985)]; "Transcription And Translation" [B.D. Hames & S.J. Higgins, eds. (1984)];
"Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes"
[IRL Press. (1986)]; B. Perbal~ "A Practical Guide To Molecular Cloning" (1984).
Therefore. if appearing herein~ the following terms shall have the definitions set out 20 below.

The terms "MSCR~MM," "bacterial cell surface protein~" "elastin binding protein (ebpS)," and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous2~ material including single or multiple proteins, and extends to those proteins having the amino acid sequence data descri'oed herein and presented in FIGURE 3 (SEQ IDNO:2), and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substanti:~lly equivalent or altered activity are likewise contemplated. These modifications mav be deliber,~te, for example, such as 30 modifications obtained through site-directed mutagenesis~ or may be accidental, such W O 98/38312 PCT~US97/03106 as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terrns "MSCRAMM," "bacterial cell surface protein" and"elastin binding protein (ebpS)" are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic 5 variations.

As used herein, an "active polypeptide fragment" of a MSCRAMM is a polypeptide ~ragment of an MSCRAMM that binds to elastin. A "polypeptide of the present invention" is a polypeptide that comprises an arnino acid sequence that corresponds to 10 the elastin binding site of an MSCRAMM. Such polypeptides consist of bet~,veen 8 and 80 arnino acids.

The arnino acid residues described herein are preferred to be in the "L" isomeric form.
However, residues in the "D" isomeric form can be substituted for any L-amino acid 15 residue, as long as the desired fuctional property of immunoglobulin-binding is retained by the polypeptide. NH, refers to the free amino group present at the amino terrninus of a polypeptide. COO~I refers to the free carboxy group present at the carbo~cy terminus of a polypeptide. In keeping with standard polypeptide nomenclature~ J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid 20 residues are shown in the following Table of Correspondence:

TABLE OF CORRESPO~DENCE
SY~IBOL AMINO ACID
l-T.etter~-Letter Y Tyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Ala alanine S Ser serine -W O 98/38312 PCT~US97/03106 Ile isoleucine L Leu leucine T Thr threonine V Val valine p Pro proline K Lys Iysine H His histidine Q Gln ghlt~mine E Glu glutarnic acid W Trp tryptophan R Arg arginine D Asp aspartic acid N Asn asparagine C Cys cysteine It should be noted that all amino-acid residue sequences are represented herein by forrnulae whose left and right orientation is in the conventional direction of amino-terminus to carbox~,-terrninus. Furtherrnore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptidc bond to a20 further sequence of one or more amino-acid residues. The above Table is prescnted to correlate the three-letter and one-letter notations which may appear alternately herein.

The tern~ "polypeptide" is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs~ or peptidomimetics. The subunits may 25 be linked by peptide bonds. In another embodiment, the subunit may be linked by other the bonds, e.g.. ester, ether, etc.
-A "replicon" is any genetic element (e.g., plasmid. chromosome, virus) that functionsas an autonomous unit of DNA replication in vivo; i.e., capable of replication under its 30 own control.

W O 9~/38312 PCT~US97/03106 A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNAsegment may be attached so as to bring about the replication of the attached segment.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, S guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This terrn refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this terrn includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-10 skanded DNA molecules, sequences may be described herein according to the normalconvention of giving only the sequence in the S' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homolo~ous to the mRNA).

15 An "origin of replication" refers to those DNA sequences that participate in DNA
synthesis.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate 20 regulatory sequences. The boundaries of the codin(~ sequence are determined by a start codon at the ~' (amino) terrninus and a translation stop codon at thc 3' (carboxyl) terminus. A codin_ sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mz~mm7~ n) DNA, and even synthetic DNA sequences. A polyadenylation 25 signal and transcription terrnination sequence will usually be located 3' to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters. enhancers, polyadenylation signals, terrninators, and the like, that 30 provide for the expression of a coding sequence in a host cell.

W O 98/38312 PCTrUS97/03106 A "promoter sequence" is a DNA regulatory region capable of binding RNA
- polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is - bounded at its 3' terminus by the transcription initiation site and extends upstream (5' 5 direction) to include the minimurn nurnber of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping ~,vith nuclease S I ), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, 10 contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -3~ consensus sequences.

An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under 15 the control" of transcriptional and translational control sequences in a cell when RNA
polymerase transcribes the coding sequence into mRNA. which is then translated into the protein encoded by the coding sequence.

A "signal sequence" can be included before the coding sequence. ~his sequence 20 encodcs a sianal peptide~ N-terminal to ~he polypeptide. that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
The term "oligonucleotide~" as used herein in referring to thc probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferabiy more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

W O 98/38312 PCT~US97/03106 The term "primer" as used herein refers to an oligonucleotide~ whether occurring- naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid 5 strand, is induced. i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature,10 source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides. although it may contain fewer nucleotides.

15 The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must besufficientlv complementary to hybridize with their respective strands. Therefore. the primer sequence need not reflect the e,Yact sequence of the template. For example, a non-complementary nucleotide fragmcnt may be attached to the S' end of the primer.
20 with the remainder of the primer sequence bein~ complementary to thc strand.
Alternatively, non-complementary bases or longcr sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template ~or the synthesis of the extension product.
As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded ~NA at or near a specific nucleotide sequence.

A cell has been "transforrned" by exogenous or heterologous DNA when such DNA
has been introduced inside the cell. The transforming DNA may or may not be integrated (covalentlv linked) into chromosomal DNA making up the genome of the cell. In prokaryotes~ yeast, and m~mm~ n cells for example, the transforrning DNA
- 5 may be m~int~ined on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforrning DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a prima;y cell that is capable of stable growth in vitro for many generations.

Two DNA sequences are "substantially homologous" when at least about 75%
(preferably at least about 80%. and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantiallv homologous can be identified by comparing the sequences using standard software available in sequence data banks~ or in a Southern hybridization experiment under~ for example. stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.
See, e.g., Maniatis et al., supra; DNA Clonin~" Vols. I & II, supra: Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the present invention are DNA
se~Lences enco~din~MSCRAMM which code for an MSCRAMM having the same amino acid sequence as SEQ ID NO: but which are degenerate to SEQ ID NO: .
By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:

CA 02247072 l998-08-27 W O 98/38312 PCTnUS97/03106 Phenylalanine (Phe or F) UUU or UUC
- Leucine (Leu or L) WA or UUG or CUU or CUC or CUA or CUG
Isoleucine (Ile or I~ AUU or AUC or AUA
Methionine (Met or M) AUG ..
Valine (Val or V) GUU or GUC of GUA or GUG
Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC
Proline (Pro or P) CCU or CCC or CCA or CCG
Threonine (Thr or T) ACU or ACC or ACA or ACG
Alanine (Ala or A) GCU or GCG or GCA or GCG
Tyrosine (Tyr or Y) UAU or UAC
Histidine (His or H) CAU or CAC
Glutamine (Gln or Q) CAA or CAG
Asparagine (Asn or N) AAU or AAC
Lysine (~ys or K) AAA or AAG
Aspartic Acid (Asp or D) GAU or GAC
Glutamic Acid (Glu or E) GAA or GAG
Cysteine (Cys or C) UGU or UGC
Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG
Glycine (Gly or G) GGU or GGC or GGA or GG~
Tryptophan (Trp or W) UGG
Termination codon UAA (ochre~ or UAG (amber) or UGA (opal) It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U.
Mutations can be made in S~Q ID NO:1 such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by mzlkinp the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative 30 manner (i.e., by ch~np:ing the codon from an amino acid belonging to a grouping of W O 98/38312 PCTAJS97/~3106 amino acids having a particular size or characteristic to an amino acid belonging to - another grouping) or in a conservative manner (i.e., by ch~nging the codon from an amino acid belonging to a grouping of arnino acids having a particular size or characteristic to an arnino acid belonging to the same grouping). Such a conservative 5 change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered toinclude sequences cont~ininE~ conservative changes which do not significantly alter the activity or binding characteristics of the resulting polypeptide. Accordingly, such 10 conservative changes are defined herein as a "conservative substitution".

The following is one example of various groupings of amino acids:

,Q mino acids with nonpolar R ~roups Alanine Valine Leucine Isoleucine 20 Proline Phenylalanine Tryptophan Methionine 25 Amino acids with uncharged polar R ~roups Glycine Serine Threonine 30 Cysteine W O98/38312 PCT~US97/03106 Tyrosine - Asparagine Glutamine S Amino acids with char~ed polar R yroups (negatively charged at ph 6.0) Aspartic acid Glutamic acid 10 Basic amino acids (positively charged at pH 6.0) Lysine Arginine Histidine (at pH 6.0) Another grouping may be those amino acids with phenyl groups:

Phenylalanine Tryptophan 20 Tyrosine Another grouping may be according to molecular weight (i.e., size of R groups):

Glycine 75 25 Alanine 89 Serine 1 05 Proline 1 15 Valine 1 17 Threonine 1 19 30 Cysteine 121 W O 98138312 PCTrUS97/03106 Leucine 13 1 Isoleucine 13 1 Asparagine 1 32 ., Aspartic acid 133 Ghlt~Tnine 146 Lysine 1 46 Glutarnic acid 147 Methionine 1 49 Histidine (at pH 6.0) 155 Phenylalanine 165 Arginine l 74 Tyrosine 18 1 Tryptophan 204 15 Particularly preferred substitutions are:
- Lys for Arg and vice versa such that a positive charge may be maintained;
- Glu for Asp and vice versa such that a ne~ative charge may be maintained;
- Ser for Thr such that a free -OH can be maintained; and - Gln for Asn such that a free NH. can be m~int~ined.
Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced into a potential site for disulfide bridges with another Cvs. A His may be introduced as a particularly "catalytic" site (i.e.. His can act as an acid or base and is the most 25 common amino acid in biochemical catalysis). Pro may be introduced bccause of its particularly planar structure, which induces ,~-tu~ns in the protein's structure.

Two amino acid sequences are "substantially homologous" when at least about 70%
,, of the amino acid residues (preferably at least about 80%, and most preferably at least 30 about 90 or 95%) are identical, or represent conservative substitutions.

W O 9$/38312 PCT~US97/03106 A "heterologous" region of the DNA construct is an identifiable segment of DNA
within a larger DN.~ molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a m~mm~ gene, the gene will usually be flanked by DNA that does not flank the m~mrn~ n genomicS DNA in the genome of the source org~ni.~rn Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of 10 DNA as defined herein.

An "antibody" is any immunoglobulin, including antibodies and fragments thereof,that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies the last mentioned described in further detail in U.S. Patent Nos.
15 4,816,397 and 4,~16~567.

An "antibody combining s;te" is that structural portion of an antibody molecule comprised of hea~r - and light chain variable and hypervariable rePions that specifically binds antigen.
~he phrase "antibody molecule" in its various grammatical ~orms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

25 Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin moleculethat contains the paratope, including those portions known in the art as Fab. Fab', F(ab')~ and F(v), which portions are preferred for use in the therapeutic methods described herein.

W O 98/38312 PCT~US97/03106 Fab and F(ab')7 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin. respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4~342.566 to Theofilopolous et al. Fab' antibody molecule portions are also well-known and are S produced from F(ab'), portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol~ and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred herein.

10 The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. Amonoclonal antibody may therefore contain an antibody molecule having a plurality 15 of antibody combining sites, each immunospecific for a different antigen; e.g., a bispccific (chimeric) monoclonal antibody.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an 20 allergic or similar untoward reaction, such as gastric upset. dizziness and the like, when ~rlmini~tered to a human.

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent~ and preferably reduce by at least about 30 percent. more 25 preferably by at least 50 percent~ most preferably by at least 90 percent. a clinically significant change in the S phase activity of a target cellular mass~ or other feature of pathology such as for example~ elevated blood pressure, fever or white cell count as may attend its presence and activity.

W O 9~/38312 PCTAUS97/03106 A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and m~int~ining S the correct reading frarne to perrnit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA
molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
The terrn "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65~C for both hybridization and wash.
However, one skilled in the art will appreciate that such "standard hybridization conditions" are dependent on particular conditions including the concentration of 15 sodium and magnesium in the buffer. nucleotide sequence length and concentration.
percent mi~m~tch, percent formamide, and the like. Also important in the deterrnination of "standard hybridization conditions" is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily, determined by one skilled in the art according to well known 20 forrnulae, wherein hybridization is typically l 0-20~C below tlle predicted or determined Tm with ~vashes of higher stringency, i~ desired.

In its primary aspect the present invention concerns the identification of a bacterial MSCRAMM which binds a component of the extracellular matrix. mediating the 25 attachment, colonization and/or invasion of the bacterial into a host tissue.
In a particular embodiment. the present invention relatcs to all members of the herein disclosed elastin binding proteins.

W O 98/38312 PCTrUS97/03106 As stated above, the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a MSCRAMM, or a fragment thereof, that possesses a predicted molecular weight of about 25 kD and an amino acid sequence set forth in FIGURE 3 (SEQ ID NO:2); preferably a nucleic acid 5 molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 25 - kD has a nucleotide sequence or is complementary to a DNA sequence shown in FIGURE 2 (SEQ ID NO:l).

Initial steps for purifying an MSCRAMM or active polypeptide fragment thereof of10 the present invention include salting in or salting out, such as in amrnonium sulfate fractionations; solvent exclusion fractionations, e.g., an ethanol precipitation;
detergent extractions to free membrane bound proteins using such detergents as TRITON X-100, TWEEN-20 etc.; or high salt extractions. Solubilization of proteins or polypeptides may also be achieved using aprotic solvents such as dimethyl 15 sulfoxide and hexarnethylphosphoramide. In addition, high speed ultracentrifugation may be used either alone or in conjunction with other extraction techniques.

Generally good secondary isolation or purification steps include solid phase absorption using calcium phosphate gel or hydroxyapatite; or solid phase binding.
20 Solid phase binding may be performed through ionic bonding, with either an anion exchanger, such as diethylaminoethyl (DEAE), or diethyl [ -hydroxypropyl]
aminoethyl (QAE) SEPHADEX or cellulose; or with a cation exchanger such as carboxymethyl (CM) or sulfopropyl (SP) SEPHADEX or cellulose. Alternative means of solid phase binding includes the exploitation of hydrophobic interactions 25 e.g, the using of a solid support such as phenylSepharose and a high salt buffer;
affinity-binding, using, e.g., elastin on an activated support; imrnuno-binding~ using e.g., an antibody to an MSCRAMM or active polypeptide fragment thereof bound to an activated support; as well as other solid phase supports including those that contain specific dyes or lectins etc. A further solid phase support technique that is often used 30 at the end of the purification procedure relies on size exclusion, such as SEPHADEX

and SEPHA~OSE gels, or pressurized or centrifugal membrane techniques, using size exclusion membrane filters.

Solid phase support separations are generally perforrned batch-wise ~-ith low-speed S centrifugations or bv colurnn chromatography. High performance liquid chromatography (HPLC), including such related techniques as FPLC. is presently the most cornmon means of performing liquid chromatography. Size e~cclusion techniques may also be accomplished with the aid of low speed centrifugation.

10 In addition size permeation techniques such as gel electrophoretic techniques may be employed. These techniques are generally perforrned in tubes, slabs or by capillary electrophoresis .

Almost all steps in~,-olving protein purification employ a'oufr'ered solution. Unless otherwise specified generally 25-100 ~LM salt concentrations of buffer salts are used.
Low concentration buffers generally imply 5-25 ~M concentrations. High concentration buffers generally imply concentrations of the buffering agent of between 0.1-2M concentrations. Typical buffers can be purchased from most biochemical cataloc7ues and include the classical buffers such as Tris. p~r rophosphate.
20 monophosphate and diphosphate and the Good buffers [Good~ N.E.~ et al.
Biochemislry, ~:467 (1966); Good, N.E. and Izawa, S., Meth. En~mol.. 24B:53 (1972); and Fergunson, W.J. and Good, N. E., Anal. Biochen,., 10'~:300 (1980] such as Mes, Hepes, Mops, tricine and Ches.

25 Materials to perforrn all of these techniques are available from ~ varietv of sources such as Sigma Chemical Company in St. Louis, Missouri.

The possibilities both diagnostic and therapeutic that are raised by the existence of the MSC~AMM, derive from the fact that the factors appear to participate in direct and 30 causal protein-protein interaction between the MSCRAMM, and those factors that are W O 98/38312 PCT~US97/03106 in the extracellular matrix to which the MSCRAMM binds. As suggested earlier andelaborated further on herein. the present invention contemplates pharmaceutical intervention in the cascade of reactions in which the MSCRAMM is implicated, to modulate the activitv initiated by the MSCRAMM.

r Thus, in instances where it is desired to reduce or inhibit the deleterious effects resulting from interaction of the MSCRAMM with a particular stimulus or factor. an appropriate inhibitor of the MSCRAMM could be introduced to block the interaction of the MSCRAMM with those factors causally connected with bacterial adhesion 1 0 thereby.

As discussed earlier, the MSCRAMM or their binding partners or other ligands or agents exhibiting either mimicry or antagonism to the MSCRAMM or control over their production, may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for ~tlmini~tration by various means to a patient experiencing an adverse medical condition associated with specific bacterial infection for the treatment thereof. A variety of administrative techniques may be utilized.
among them parenteral techniques such as subcutaneous. intravenous and intraperitoneal injections~ catheterizations and the like. Average quantitics of the MSCRAMM or their subunits may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.

Also, antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the MSCRAMM and/or their subunits may possess certain diagnostic applications and may for example~ be utilized for thepurpose of detecting and/or measuring conditions such as bacterial infection or the like. For example, the MSCRAMM or its subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media~ by known techniques such as the hybridoma technique ~ltili7in~, for example, fused mouse spleen Iy~nphocytes and myeloma cells. Likewise~ small molecules that mimic or antagoni~e the activity(ies) of the MSCRAMM of the invention may be discovered or synthesized. and may be used in diagnostic and/or therapeutic protocols.

The general methodology for making monoclonal antibodies by hybridomas is well S known. Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenicDNA, or transfection with Epstein-Barr virus. See~ e.g., M. Schreier et al..
"Hybridoma Techni~ues" (1980); Harnmerling et al., "Monoclonal Antibodies And T-cell Hybridomas" ( 1981 ); Kennett et al., "Monoclonal Antibodies" ( 1980); see also 10 U.S. Patent Nos. 4,341,761, 4,399,121, 4,427,783; 4,444,887; 4,451,570; 4,466.917;

4~472,500; 4,491,63 '; 4,493,890.

Panels of monoclonal antibodies produced against MSCRAMM peptides can be screened for various properties; i.e., isotype~ epitope. affinity, etc. Of particular 15 interest are monoclonal antibodies that neutralize the binding activity of the MSC~AMM or its subunits, in particular the binding activity of the first 59 amino acids of the molecule to~the amino terminal portion of elastin. Such monoclonals can be readily identified in binding activity assays such as ELISA or WESTERN BLOT.
High affinity antibodies are also useful when immunoaffinity purification of native or 20 recombinant MSCRAMM is possible.

Preferably, the anti-MSCRAMM antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb). In addition~ it is preferable for the anti-MSCRAMM
25 antibody molecules used herein be in the form of Fab, Fab', F(ab')~ or F(v) portions of whole antibody molecules.

As suggested earlier, the diagnostic method of the prescnt invention comprises ex~mining a cellular sample or medium by means of an assay including an effective 30 amount of an antagonist to a MSCRAMM/protein, such as an anti-MSCRAMM

antibody, preferably an affinity-purified polyclonal antibody~ and more preferably a mAb. In addition. it is preferable for the anti-MSCRAMM antibody molecules used herein be in the form of Fab, Fab', F(ab')7 or F(v) portions of whole antibody molecules. As pre~iously discussed~ patients capable of benefiting from this method 5 include those suffering from bacterial infections associated with cancer, a pre-cancerous lesion, a viral infection or other like pathological derangement. Methods for isolating the MSCRAMM and inducing anti-MSCRAMM antibodies and for determining and optimi~in~ the ability of anti-MSCRAMM antibodies to assist in the e~C~min~tion of the target cel}s are all well-known in the art.
Methods for producing polyclonal anti-polypeptide antibodies are well-known in the art. See U.S. Patent No. 4,493,795 to Nestor et al. A monoclonal antibody, typically cont~ining Fab and/or F(ab'), portions of useful antibody molecules, can be prepared using the hybridoma technology described in Antibodi~s - A Laboratory Manual, 15 Harlow and Lane, eds.. Cold Spring Harbor Laboratory, New York (1988). which is incorporated herein by reference. Briefl~ to form the hybridoma from which the monoclonal antibodv composition is produced. a myeloma or other self-perpetuating cell line is fused ~-ith Iymphocytes obtained from the spleen of a mammal hyperimmunized ~~ith an elastin-binding portion thereof.
Splenocytes are typically fused with myeloma cells using polyethylene glvcol (PEG) 6000. Fused hybrids are selected by their sensitivity to HAT. Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present MSCRAMM and their ability to inhibit specified 25 MSCRAMM activitv in target cells.

A monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
30 The culture is mzlint~ined under conditions and for a time period sufficient for the W O 98/38312 PCT~US97/03106 hybridoma to secrete the antibody molecules into the medium. The antibody-con~ining medium is then collected. The antibody molecules can then be further isolated by well-kno~,vn techniclues.

5 Media useful for the l,iepaldLion of these compositions are both well-known in the art and commercially available and include synthetic culture media, inbred mice and the lil;e. An exemplarv synthetic medium is Dulbecco's rninim~l essential medium (DMEM; Dulbecco et al., ~irol. 8:396 (1959)) supplemented with 4.5 gm/l glucose,20 mm gl-1t~mine, and 20% fetal calf serum. An exemplary inbred mouse strain is the 1 0 Balb/c.

Methods for producing monoclonal anti-MSCRAMM antibodies are also well-known in the art. See Niman et al., Proc. Natl. ~lcad. Sci. US~l, 80:4949-4953 (1983).Typically, the present MSCRAMM or a peptide analog is used either alone or 15 conjugated to an immunogenic carrier. as the immunogen in the before described procedure for producing anti-MSCRAMM monoclonal antibodies. The hybridomas are screened for the ability to produce an antibody that immunoreacts with the MSCRAMM peptide analog and the present MSCRAMM.

20 The present invention further contemplates therapeutic compositions useful inpracticing the therapeutic methods of this invention. A subject therapeutic composition includes, in admixture, a pharrnaceutically acceptable excipient (carrier) and one or more of a MSCRAMM, polypeptide analog thereof or fragment thereof. asdescribed herein as an active ingredient. In a preferred embodiment, the composition 25 comprises an antigen capable of modulating the specific binding of the present MSCRAMM to a target cell.

The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, such ., 30 compositions are prepared as injectables, either as liquid solutions or suspensions.

CA 02247072 l998-08-27 WO 98/38312 PCTrUS97/03106 however, solid forms suitable for solution in, or suspension in, liquid prior to injection - can also be prepared. The ple,odldLion can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and ~ compatible with the active ingredient. Suitable excipients are, for example~ water, 5 saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

10 A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts tformed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example hydrochloric or phosphoric acids. or such organic acids as 15 acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example. sodium.
potassium, ammonium, calcium, or ferric hydroxides. and such organic bases as isopropylamine trimethylamine, 7-ethylamino ethanol histidine, procaine and the like.
The therapeutic polypeptide-~ analog- or active fragment-containing compositions are conventionally ~tlrnini~tered intravenously, as by injection of a unit dose, for example.
The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for 25 humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

,, The compositions are ~fiminictered in a manner compatible with the dosage 30 formulation, and in a therapeutically effective amount. The quantity to be _ ~lmini~tered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition or neutralization of MSCRAMM binding capacity desired. Precise amounts of active ingredient required to be ~lminictered depend on the judgment of the practitioner and are peculiar to each 5 individual. Howev, er, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilo~ram body weight of individual per day and depend on the route of ~1ministration. Suitable regimes for initial ~lminictration and booster shots are also variable, but are typified by an initial ~ ministration followed by repeated doses at 10 one or more hour intervals by a subsequent injection or other ~-lmini~tration.
Alternatively, continuous intravenous infusion sufficient to m~intzlin concentrations of ten nanomolar to ten micromolar in the blood are contemplated.

The therapeutic compositions may further include an effective amount of the 15 MSCRAMM/MSCRAMM antagonist or analog thereof, and one or more of the following active ingredients: an antibiotic, a steroid. E~cemplary forrnulations are given below:

Formulations lntravenous Formulation I
Ingredient mg/ml cefotaxime 250.0 MSCRAMM 1 0.0 dextrose USP 45.0 sodium bisulfite USP 3 .
edetate disodium USP 0.1 water for injection q.s.a.d. 1.0 ml 30 Tntravenous Formulation II

In~redient mg/ml - ampicillin 250.0 MSCRAMM I 0.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml Intravenous Formulation III
In~redient m~/ml gentamicin ~charged as sulfate) 40.0 MSCRAMM I 0.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q s.a.d. 1.0 ml Intravenous Formulation IV
Ingredient m~/ml MSCRAMM I 0.0 dextrose USP 45 ~
sodium bisulfite USP 3.
edetate disodium USP 0.1 water for injection q.s.a.d 1.0 ml Intravenous Formulation V
Ingredient mg/ml MSCE~AMM antagonist 5.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml W O 98/38312 PCT~US97/03106 As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "~Lg" mean micrograrn, "mg" means milligram, "ul" or "~LI" mean microliter, "ml" means milliliter, "I" means liter.

5 The polypeptides of the present invention can be can be chemically synthesized. The synthetic polypeptides are prepared using the well known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc ~ amino protected Na-t-butyloxycarbonyl) amino acid resin 10 with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield [.~. Am. Chem. Soc., 85:2149-2154 (1963)], or the base-labile N~-amino protected 9-fluorenylmethoxycarbonyl (Fmoc)amino acids first described by Carpino and ~an [J. (~rg. Chenl., 37:3403-3409 (1972)]. Both Fmoc and Boc N~-amino protected amino acids can be obtained from 15 Fluka. E~achem. Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem. or Peninsula Labs or other chemical companies familiar to those who practice this art. In addition, the method of the invention can be used ~vith other Na-protecting groups that are f~miliAr to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and 20 provided. for example. in Stewart and Young, 1984? Solid Phase Synthesis. Second Edition, Pierce Chemical Co., Rockford, IL; Fields and Noble,1990, Int. J. Pept.Protein Res.35: 161 -214, or using automated synthesizers, such as sold by ABS.
Thus, polypeptides of the invention may comprise D-amino acids. a combination ofD- and L-amino acids, and various "designer" amino acids (e.g., ~-methyl amino 25 acids, Ca-methyl amino acids, and Na-methyl amino acids, etc.) to convey special properties. Synthetic amino acids include ornithine for Iysine, fluorophenyl~l~nine for phenylalanine, and norleucine for leucine or isoleucine. Additionally, by assigning specific amino acids at specific coupling steps, o~-helices. ,13 turns. ,B sheets, ~-turns, and cyclic peptides can be generated.

W O 98/38312 PCT~US97/03106 In a further embodiment, subunits of peptides that confer usefill chemical and - structural properties will be chosen. For example, peptides comprising D-amino acids will be resistant to L-arnino acid-specific proteases in vivo. In addition~ the present invention envisions preparing peptides that have more well defined structural S properties, and the use of peptidomimetics, and peptidomimetic bonds such as ester - bonds, to prepare peptides with novel properties. In another embodiment, a peptide may be generated that incorporates a reduced peptide bond. i.e., R,-CH2-NH-~2, where R, and R. are arnino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Such a molecule would be resistant to peptide 10 bond hydrolysis, e.g, protease activity. Such peptides would provide ligands with unique function and activity, such as extended half-lives in vivo due to resistance to metabolic breakdo~n, or protease activity. Furtherrnore, it is well known that in certain systems constrained peptides show enhanced filnctional activity [Hruby, Life Sciences, 31:189-199 (198 ~)]; [Hruby et al., Biochem J.. 268:249-26~ (1990)]; the present invention provides a method to produce a constrained peptide that incorporates random sequences at all other positions.

Non-classical amino acids that induce conformnIional constraints:
The following non-classical amino acids may be incorporated in the peptide in order to introduce particular conformational motifs: 1~3~itetrahydroisoquinoline-3-carboxylate [k;azrnierski et aZ., J. A~71. Chem. Soc., 113:~ '75-2283 (1991)~; (2S.3S)-methyl-phenyl~ nine, ( S,3R)-methyl-phenylalanine, ( 'R,3S)-methyl-phenylalanineand (2R,3R)-methyl-phenylalanine [Kazrnierski and Hruby, Tetrahedron Lett., (1991)]; 2-arninotetrahydronaphthalene-2-carboxylic acid [Landis~ Ph.D. Thesis, UniversityofAri-ona~ (1989)]; hydroxy-1,~.3.4-tetrahydroisoquinoline-3-carboxylate [Miyake et al.. J. Takeda Res. Labs., 43:53-76 (1989)]; ~-carboline (D and L) ~Kazrnierski~ Ph. D. Thesis, Universih~ of Ari_ona. (1988)]; HIC (histidine isoquinoline carbo~ylic acid) [Zechel et al., Int. J. Pep. Protein Kes., 43 (1991)]; and HIC (histidine cyclic urea) (Dharanipragada).

W O 98/38312 PCTrUS97/03106 The follo~ing amino acid analogs and peptidomimetics may be incorporated into a peptide to induce or favor specific secondary structures: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a ~-turn inducing dipeptide analog (Kemp et al., .1.
Org Chem., 50:583~-5838 (1985)]; ~B-sheet inducing analogs ~Kemp et al., S Te~ahedron Lett., 29:5081-5082 (1988)~; ~-turn inducing analogs [Kemp ef al.,Tetrahedron Lett., 29:5057-5060 (1988)]; ~-helix inducing analogs ~Kemp et al., Tetrahedron Lett., 29:4935-4938 (1988)]; y-turn inducing analogs [Kemp et al., J.
Org Chem., 54: 109: 115 (1989)]; and analogs provided by the following references:
Nagai and Sato, Tetrahedron Lett., 26:647-650 (1985); DiMaio et al., J. Chem. Soc.
10 Perkin Trans., p. 1687 (1989); also a Gly-Ala turn analog [Kahn et al., Tetrahedron Lett., 30:''3 } 7 (1989)]; arnide bond isostere ~Jones et al., Tetrahedron Lctt., 29:3853-3856 (1988)]; tretrazol ~Zabrocki et al., J. An~l. Chem. Soc., 110:5875-5880 (1988)];
DTC [Samanen et al.. Int. J. Protein Pep. Res., 3~:501:509 (1990)]; and analogs taught in Olson et al., J. ~m. Chem. Sci., 112:323-333 (1990) and Garvey et al., J.
15 Org Chem., ~6:436 (1990). Conformationally restricted mimetics of beta turns and beta bulges. and peptides containing them. are described in U.S. Patent No. 5,440,013.
issued August 8, 1995 to Kahn.

Another feature of this invention is the expression of the DNA sequences disclosed 20 herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and emploving that expression vector to transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to an expression control 25 sequence, of course, includes, if not already part of the DNA sequence. the provision of an initiation codon, ATG, in the correct reading frarne upstream of the DNA
sequence.

A wide variety of host/expression vector combinations may be employed in 30 expressing the DNA sequences of this invention. Useful expression vectors, for CA 02247072 l998-08-27 W O 98/38312 PCT~US97/03106 example, may consist of segments of chromosomal, non-chromosomal and synthetic - DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pB~32'', pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage ~, e.g., 5 NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2~ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mzlmm~ n cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to emplov phage DNA or other expression control sequences; and the like.
Any of a wide variety of expression control sequences -- sequences that control the expression of a DNA sequence operatively linked to it -- may be used in these vectors to express the DNA sequences of this invention. Such useful e:cpression control sequences include. for exarnple, the early or late promoters of SV40~ CMV, vaccinia, 15 polyoma or adenovirus, the lac system, the trp system, the TAC system~ the TRC
system, the LTR svstem, the major operator and promoter regions of phage ~. the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes. the promoters of acid phosphatase (e.g.. Pho5), the promoters of the yeast a-mating factors. and other sequences kno~,vn to control the expression of 20 genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressing the DNA
sequences of this invention. These hosts may include well known eukaryotic and 25 prokaryotic hosts. such as strains of E. coli, Pseudomonas~ Bacillus~ Streptomyces.
fungi such as yeasts. and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9~, and human cells and plant cells in tissue culture.

CA 02247072 l998-08-27 WO 98/38312 PCT~US97/03106 It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However. one skilled in the art will be able to select the proper vectors. expression control sequences, and 5 hosts ~,vithout undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number~ and the expression of any otherproteins encoded by the vector, such as antibiotic markers, will also be considered.
In selecting an expression control sequence, a variety of factors will normally be considered. These include, for exarnple, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly with regard to potential secondary structures. Suitable 15 unicellularhosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly~
and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed. and the ease of purification of the expression products.
Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on ferrnentation or in large scale animal culture.

25 It is further intended that MSCRAMM analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the presentinvention. Analogs, such as fragments, may be produced, for example. by pepsin digestion of bacterial material. Other analogs. such as muteins, can be produced by standard site-directed mutagenesis of MSCRAMM coding sequences. Analogs CA 02247072 l998-08-27 W O 98/38312 PCT~US97/031~6 exhibiting "elastin binding activity" such as small molecules, whether functioning as - promoters or inhibitors, may be identified by known in ~ivo and/or in vi~ro assays.

As mentioned abo~ e. a DNA sequence encoding MSC~AMM can be prepared synthetically rather than cloned. The '~NA sequence can be designed with the appropriate codons for the MSC~AMM amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete coding sequence See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al.,J Biol. Chem., 7~9:6311 (1984~.

Synthetic DNA sequences allow convenient construction of genes which will express MSCRAMM analogs or "muteins". Alternatively. DNA encoding muteins can be made by site-directed mutagenesis of native MSCRAMM genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.

A general method for site-specific incorpo}ation of unnatural amino acids into proteins is described in Christopher J Noren, Spencer J. Anthony-Cahill. Michael C
Griffith.PeterG Schultz, Scienc~ 21~:182-188(April 1989). Thismcthodmaybe used to create analogs with unnatural amino acids.

The present invention extends to the preparation of antisense oligonucleotides and ribozymes that may be used to interfere with the expression of the MSCRAMM at the translational level This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by m~king that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (See Weintraub, 1990; Marcus-Sekura, W O 98/38312 PCTrUS97/03106 1988.) In the cell, they hybridize to that mRNA. forming a double stranded molecule.
The cell does not translate an mRNA in this double-stranded form. Therefore, ~ntic~n~e nucleic acids interfere with the expression of mRNA into protein.
Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG
5 initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into MSCRAMM-producing cells. Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1988; Hambor et al., 1988).

10 Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAshave the ability to e~;cise their o~vn introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific 15 nucleotide sequences in an RNA molecule and clea-e it [Cech, J. ,4~7t. Med Assoc., 260:3030 (1988)1. Because they are sequence-specific, only mRNAs with particularsequences are inactivated.

Investigators have identified two types of ribozymes. Tetrahl~mena-type and 20 "hammerhead"-type.(HasselhoffandGerlach. 198~) Tetrahyme}2a-type ribozymes recognize four-base sequences, while "hammerhead"-type recognize eleven- to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target mRNA species. Therefore. hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific 25 mRNA species. and eighteen base recognition sequences are preferable to shorter recognition sequences.

The DNA sequences described herein may thus be used to prepare antisense molecules zlg~in~t and ribozymes that cleave mRNAs for MSCRAMM and their 30 ligands.

CA 02247072 l998-08-27 W O 98/38312 PCT~US97/03106 The present invention also relates to a variety of diagnostic applications. including - methods for detecting the presence of stimuli such as the earlier referenced polypeptide ligands. by reference to their ability to elicit the activities which are mediated by the present MSCRAMM. As mentioned earlier, the MSCRAMM can be 5 used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular MSCRAMM activity in suspect target tissues.

As described in detail above, antibody(ies) to the MSCRAMM can be produced and 10 isolated by standard methods including the well known hybridoma techniques. For convenience~ the antibody(ies~ to the MSCRAMM will be referred to herein as Ab, and antibody(ies) raised in another species as Ab~.

The presence of MSCRAMM in cells can be ascertained by the usual immunological 15 procedures applicable to such determinations. A number of useful procedures are known. T'nree such procedures which are especiallv useful utilize cither the MSCRAMM labcled with a detectable label. antibody Ab, labeled with a detectable label. or antibody Ab, labeled with a detectable label. The procedures may be surnmarized by the following equations wherein the asterisk indicates that the particle 20 is labeled, and "M" stands for the MSCRAMM:
A. M* + Abl = M*Ab, B. M + Ab* = MAb,*
C. M + Ab, + Ab,* = MAblAb2*

25 The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. The "competitive" procedure, Procedure A~ is described in U.S. Patent Nos. 3.654,090 and 3,850,75 . Procedure C. the "sandwich" procedure. is described in U.S. Patent Nos.
R~ 31,006 and 4,016,043. Still other procedures are known such as the "double 30 antibody," or"DASP" procedure.

W O 98/383~2 PCTAJS97/03106 In each instance, the MSCRAMM forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label.
The fact that a complex has formed and, if desired, the amount thereof. can be determined by kno~,vn methods applicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab, is that it will react ' with Ab,. This is because Abl raised in one m~mmz~ n species has been used in another species as an antigen to raise the antibody Ab,. For example~ Ab, may beraised in goats using rabbit antibodies as antigens. Ab~ therefore would be anti-rabbit antibody raised in goats. For purposes of this description and claims. Ab~ will be referred to as a primary or anti-MSC~AMM antibody, and Ab, will be referred to as a secondary or anti-Ab, antibody.

The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when e~posed to ultraviolet light. and others.

A number of fluorescent materials are known and can be utilized as labels. Theseinclude, for example, fluorescein, rhodamine, auraminc, Te~;as Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.

The MSCRAMM or its binding partner(s) can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 3~ 14C 3-P 35S 36Cl 5~Cr, 57Co, 53Co, 59Fe, 90Y, 1'5I, ~3'I, and '36Re.

Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric~ amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by rcaction r 30 with bridging molecules such as carbodiimides. diisocyanates, glutaraldehyde and the W O 98/38312 PCT~US97/03106 like. Many enzymes which can be used in these procedures are known and can be - utilized. The preferred are peroxidase, 13-glucuronidase, 13-D-glucosidase, 13-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
U.S. Patent Nos. 3~654,090; 3,850,752; and 4.016,043 are referred to by way of 5 example for their disclosure of alternate labeling material and methods.
-A particular assay system developed and utilized in accordance with the presentinvention, is kno~n as a receptor assay. In a receptor assay. the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a 10 quantity of both the labeled and unlabeled material after which binding studies are conducted to deterrnine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.

Accordingly. a purified quantity of the MSCRAMM may be radiolabeled and 15 combined, for example, with antibodies or other inhibitors thereto, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombined MSCRAMM, and cell samples would then be inoculated and thereaf~er incubated. The resulting cell monolavers are then washed. solubilized and then counted in a gamma counter for a 20 length of time sufficient to yield a standard error of <5%. These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a 25 distinguishing characteristic.

An assay useful and contemplated in accordance with the present invention is known as a "cis/trans" assay. Briefly. this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest 30 when transfected into an appropriate cell line. and the second of which is a plasmid W O 98/38312 PCT~US97/03106 that expresses a reporter such as luciferase~ under the control of a receptor/ligand complex. Thus. for example, if it is desired to evaluate a compound as a ligand for a particular receptor, one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a 5 promoter linked to the luciferase gene in which the response element to the particular receptor is inserted. If the compound under test is an agonist for the receptor, the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene. The resulting chemiluminescence is then measured photometrically, and dose response curves are10 obtained and compared to those of kno~,vn ligands. The foregoing protocol is described in detail in U.S. Patent No. 4,981,7g~ and PCT International Publication No. WO 88/03168, for which purpose the artisan is referred.

In a further embodiment of this invention, commercial test kits suitable for use by a 15 medical specialist may be prepared to determine thc presence or absence of predetermined MSCRAMM activity or predetermined elastin binding activity capability in suspected target cells. In accordance with the testing techniques discussed above. one class of such kits will contain at least the labeled MSCRAMM
or its binding partner~ for instance an antibod~ specific thereto, and directions, of 20 course, depending upon the method selected, e.g"competitive~" "sandwich~"
"DASP" and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of the presence orcapability of cells for predetermined elastin binding activity, comprising:
(a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the present MSCRAMM
factor or a specific binding partner thereto, to a detectable label;
(b) other reagents; and (c) directions for use of said kit.

W O 98/38312 PCTrUS97/031~6 More speeifically. the diagnostie test kit may eomprise:
(a) a known amount of the MSCRAMM as described above (or a binding partner) generally bound to a solid phase to form an immunosorbent, or in the alternative, bound to a suitable tag, or plural sueh end produets, etc. (or their binding partners) one of eaeh;
(b) if neeessary, other reagents; and (e) direetions for use of said test kit.

In a further variation. the test kit may be prepared and used for the purposes stated above, whieh operates aecording to a predetermined protocol (e.g. "competitive,""sandwich," "double antibody," etc.), and comprises:
(a) a labeled component whieh has been obtained by eoupling the MSCRAMM to a detectable label;
(b) one or more additional immunoehemieal reagents of whieh at least one reagentis a ligand or an immobilized ligand, whieh ligand is seleeted from the group consisting of:
(i) a ligand capable of binding with the labeled component (a);
(ii) a ligand capable of binding with a binding partner of the labeled eomponent (a);
(iii) a ligand eapable of binding with at least one of the component(s) to be determined; and (iv) a ligand capable of binding with at least one of the binding partners of at least one of the eomponent(s) to be determined; and (e) directions for the performanee of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between the MSCRAMM and a specifie binding partner thereto.

In aceordance with the above, an assay system for screening potential drugs effective - to modulate the activity of the MSCRAMM may be prepared. The MSCRAMM may be introduced into a test system, and the prospective drug may also be introdueed into W O 98/38312 PCT~US97/03106 the resulting cell culture, and the culture thereafter e~camined to observe any changes in the MSCRAMM activity of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known MSCRAMM.

PRF.T TMINARY CONSIDERATIONS

The interaction between staphylococci and components of the extracellular matrixmediates attachment to host tissues and organs, and is an important mechanism leading to colonization, invasion, and formation of metastatic abscesses. A specific 10 binding interaction has been demonstrated between Staphylococcus aureus and elastin, one of the major protein components of the extracellular matri~c. Available evidence suggests that this association is mediated by a cell surface 25 kDa staphylococcal elastin binding protein (EbpS). To study the molecular structure and function of EbpS, the gene encoding EbpS was cloned. sequenced. and expressed in15 E. coli. DNA sequence data indicate that the ebpS open reading frame consists of 606 bp, and encodes a novel polypeptide of 202 amino acids. EbpS protein has a predicted molecular mass of 23,345 daltons and pI of 4.9. EbpS was e~pressed in coli as a fusion protein with polyhistidine residues attached to the N-terminus. A
polyclonal antibody raised against recombinant EbpS interacted specifically with the 20 25 kDa cell surface EbpS and inhibited staphylococcal elastin binding. ~urthermore.
recombinant EbpS bound specifically to immobilized elastin and inhibited binding of Staphylococcus aureus to elastin. A degradation product of recombinant EbpS
lacking the first 59 amino acids of the molecule and a C-teFminal fragment of CNBr-cleaved recombinant EbpS, however~ did not interact with elastin. These results 25 strongly suggest that EbpS is the cell surface molecule me~ ting binding of Staphylococcus auretls to elastin. The finding that some constructs of recombinant EbpS do not interact with elastin suggests that the elastin binding site in EbpS is contained in the first 59 amino acids of the molecule.

W O 98138312 PCT~US97/03106 The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed. however, as limiting the broad scope of the invention.

F.XAMPLE I
-Materials and Methods Materials 10 Restriction endonucleases, calf intestinal phosphatase? T4 DNA ligase, T4 polynucleotide kinase~ isopropyl-13-D-galactoside (X-gal), Wizard Miniprep plasmid purification kit~ and Hind III-digested 1 DNA markers were purchased from Promega (Madison, WI). DNase-free RNase was obtained from Boehringer Mannheim (Tn~ n~rolis IN). Luria-~ertani ~LB) medium and LB agar-medium capsules were 15 from BIO 101 (La Jolla, CA). Tryptic soy broth (TSB) was obtained from Remel (Lenexa, KS). High melting point agarose was purchased from Fisher (St. Louis.
MO) and SeaPlaque GTG Agarose (low melting) was obtained from FMC
BioProducts (Rocl;land. ME). Na'-5I,y-3'P-ATP~ and a-3'P-CTP were from ICN
(Costa Mesa. CA~. Papain and protein A immobilized to cross linked agarose. and 20 IODOGEN were purchased from Pierce (Rockford. IL). Rapid-hyb buffer and rediprime DNA labeling system were obtained from Amersham (Arlington Heights.
IL). Chroma Spin-1() columns were purchased from Clontech (Palo Alto, CA).
QIAexpress vector kit type IV and the midi-prep plasmid purification kit were obtained from Qiagen (Chatsworth~ CA). Nitrocellulose membrane and blotting paper 25 were from Schleicher & Schuell (E~eene, NH). Affi-Gel-10 affinity support was from Bio-Rad (Melville. NY). All other materials were purchased from Si<7ma Chemical (St. Louis, MO).

W O 98/38312 PCT~US97~03106 E3acterial. plasmids and culture conditions S. aureus strain 1''598 (Cowan) was purchased from the American Type Culture Collection (Roclcville, MD). E. coli strains DH5c~ competent cells (MAX Efficiency3 S and M 15 (pREP4) were from Gibco BRL (C~aithersburg~ MD) and Qiagen, respectively. M 15 cells contain the plasmid pREP4 which constitutively expresses the lac repressor from the lacl gene. S. aureus cells were grown in TSB, and E. coli strains in LB media supplemented with appropriate antibiotics as described below.

10 The low copy number cloning plasmid, pHSG575 (26), was kindly provided by Dr.Michael Caparon (Department of Molecular Microbiology, Washington University School of Medicine). The plasmid pl31uescript KS+ was purchased from Stratagene (La ~olla, CA) and used for subcloning and sequencing purposes. The expression plasmid pQE-30 was obtained from Qiagen. All of these plasmids were propagated in 15 DH5a cells and purified using the Qiagen Plasmid Midi-Prep Kit for further applications.

Isolation of IS. aurez~s genomic DNA. preparation of p}obes and Southern blottin~

20 High molecular weight genomic DNA was isolated from 400 ml of an overnight culture of S. aureus strain 12598 cells by Iysostaphin Iysis, followed by treatment with DNase-free RNase, and subsequent purification by phenol/chloroform and chloroform extractions. After the final chloroforrn extraction, DNA in the aqueous layer was precipitated with ethanol and Iyophilized.
A degenerate 30mer oligonucleotide probe corresponding to the amino acid sequence NNFKDDFEKN was generated by chemical synthesis. The oligonucleotide was end-labeled with T4 polynucleotide kinase and y-3~P-ATP, and the radiolabeled oligonucleotide was separated from unincorprated 3'P by Chroma Spin- 10 spin 30 chromatography. The specific activity was approximately 5 x 108 cpm/~g of WO 98/38312 PCT~US97/03106 ~;~
oligonucleotide. The 2.6 kb ~Iind III/Hinc II probe was generated as described below and radiolabeled with a-3~P-CTP using the rediprime DNA labeling system.

Genomic and plasmid DNAs were digested to completion with restriction 5 endonucleases. Restriction endonuclease-cleaved DNAs were separated by TAE-agarose gel electrophoresis, and Southern blotted to nitrocellulose membranes. The membranes were baked at 80~C for 2h under vacuum, and pre-hybridization, hybridization, and washing of the membranes were performed according to instructions supplied with the Rapid-hyb buffer. Washed blots were air-dried andexposed to Kodak XAR-5 films at -70~C with intensifying screens for 0.5-2 days.

Cloning and sequencing of ebp~

Based on the observation that the 30mer oligonucleotide probe hybridized to a 4.2 kb 15 EcoR l-genomic DNA fragment (Fig. 1: lane A)~ a size selected genomic library in the 4.2 kb region was _enerated. Genomic DNA from ~S. aureus strain 12598 was digested with EcoR I and fractionated with 1% low melting agarose electrophoresis.
The 4.2 kb region was excised from the gel and melted at 68~C for 15 min. DNA inthe melted agarose was ligated in situ with pHSG575 trcated with EcoR I and alkaline 20 phosphatase according to instructions provided by FMC Products. Competent DH5cells were transformed with the ligated material. and different dilutions were plated out on LB agar-medium plates supplemented with chloramphenicol (20 llg/ml), IPTG(0.5 nM), and X-Gal (40 ~lg/ml) for antibiotic and blue/white selections. White colonies were collected, propagated overnight. and the Wizard plasmid mini-prep was 25 used to isolate plasmid DNA from cells. Purified plasmids were digested with EcoR I
and screened by Southern blotting using the radiolabeled oligonucleotide probe.

The cloned 4.2 kb fragment was digested with Hind III and Hinc Il. yielding a 2.6 kb fragment. which ~has subcloned into pBluescript KS+ and pUC19. The 2.6 kb 30 fragment was also used as a probe in Southern analyses with S. au~eus genomic DNA.

The insert was digested using the Exo III/mung bean nuclease system (Stratagene, La Jolla, CA) to generate two sets of nested deletions. Multiple clones covering both strands in their entirety were se~uenced by the Sanger dideoxynucleotide chain termination method as modified for TAQ polymerase cycle sequencing using an ABI373A automated DNA sequencer. Sequence data were assembled and discrepancies resolved using the Wisconsin Package (Genetics Computer Group, Madison, Wl). The primary sequence of ebpS as shown in Figure 3 has been assigned the GenBank accession number.

F.xpression of ebpS in ~. Coli and CNBr cleavage of recombinant F.bpS

A 2.6 kb Hind III/Hinc II fragment in pBluescript KS+(30ng) served as the template, and PCR reactions were perforrned with a Perkin Elmer thermocycler using standard reagents. The open reading frame of ebpS was PCR,amplified using the sense oligonucleotide, 5'-TGTGGATCCATAGAAAGGAAGGTGGCTGTG-3', and the z~nticçn.se oligonucleotide, 5'GCAAAGCTTGCTGTACCAGGACCAATT-3'. The sense oligonucleotide contained a BamH I site (underlined), and A of the two ATGcodons were changed to G (in bold letters) to avoid internal initiation of translation as recommended by Qiagen. The antisense oliconucieotide contained a Hind III
cleavage site ~underlined). The exact conditions for amplification were 90 ~C for I
min. followed by 30 cycles of 94~C for 30 s. 53 ~C for 30 s and 7~ ~C for 60 s. The PCR product was digested with Bam~l I and Hind III, and gel purified. This material was ligated to pQE-30 that had been digested with BamH I and Hind III and treated with calf intestinal alkaline phosphatase. Competent M 15 cells were transformedwith the ligation product, selected by arnpicillin (100 ~g/ml) and kanamycin (20~g/ml), and antibiotic-resistant cells were screened for recombinant protein expression.

Upon obtaining several positve clones, ideal conditions for maximum expression were examined. Based on results from these studies, the following protocol was used W O 98/38312 PCT~US97/03106 routinely for mediurn-scale purification of recombinant EbpS (rEbpS). A stock culture of the clone was grown overnight in 10 ml of LB media supplemented with ampicillin and kanamycin. On the following day. this culture was added to 100 ml of fresh LB media with antibiotics. Cells were allowed to re-grow until the OD600nm5 value reached 0.8 (~3 h). Then expression was induced with 1 mM IPTG for 4 h at 37~C. The cells were pelleted by centrifugation (5000 x g), resuspended in 15 ml of buffer A (8 M urea 100 mM NaH.PO" 10 mM Tris-HCI, pH 8)~ and vortexed gently for 15 min. The Iysed cells were centrifuged at 15.000 x g for 20 min at 4~C, and the supernatant was transferred to a tube containing 4 ml of nickel nitriloacetic acid (Ni~-10 NTA) resin pre-equilibrated with buffer A. The mixture was incubated for 30 min at room temperature with gentle agitation. The mixture was then transferred to a disposable polypropvlene column and washed consecutively with 100 ml of buffer Aand 100 ml of buffer B (same as buffer A except pH=6). The tightly bound recombinant protein was eluted with 10 ml of buffer c (same as buffer A. but pH=4).
The eluted material was dialyzed twice against 4L of 10 mM Tris-HCI. pH 7.5 and the concentration of the dialysate was measured by UV spectrophotometry based on thenumber of Tyr and Trp residues in rEbpS ( 1 A(280nm) =2.68 mg/ml). The yield of purified rEbpS under these conditions was approximately S mg per 100 ml of culture.

20 To generate CNBr-cleaved fragments, 500 ~g of rEbpS was incubated in the dark for 24 h at room temperature with I mg of CNBr in 200 ~11 of 70% formic acid. ~t theend of incubation, the sample was diluted with 14 ml of de-ionized H,O and speed-vac dried. The dried material was resuspended in 10 ml of de-ionized H.O and re-dried in 100 ~g alic~uots.
Generation of rabbit anti-rFbpS F~olyclonal antibodies Pre-immune sera were collected, and New England White rabbits were injected withhighly purified rEbpS (20~g) mixed 1:1 with complete Freunds adjuvant. Booster W O 98/38312 PCT~US97/03106 58 injections (20 ~Lg) mixed 1:1 with incomplete Freunds adjuvant were given at 57 7,10.
1~, and 19 weeks. Sera were tested by Western irnmunoblotting using rEbpS.

IgG fractions were purified from immune and pre-irnmune sera by either caprylic acid 5 precipitation (27) or protein A affinity chromatography. For generation of an antibody affinity resin, approximately 100 mg of anti-rEbpS IgG were covalently coupled to 5 ml of Affi-Gel-10 according to manufacturer's instructions. To generate anti-rEbpS Fab fragments, 50 mg of Iyophilized IgGs were reacted overnight at 37~C
with 2 ml of immobilized papain in 5 ml of papain digestion buffer (20 mM NaH~PO4, 10 20 mM cysteine-HC1, 10 mM EDTA, pH 6.5). Fab fragments were separated from undigested IgGs and Free Fc fragments by protein A affinity chromatography.

Bindin~ of radiolabeled rEbpS constructs to immobilized elastin peptides Preparation and coupling of elastin peptides to Affi-Gel- 10 were as described previously (20). Both rEbpS (20 ,ug) and CNBr-cleaved rEbpS (80 ilg) were iodinated with 300 IlCi of Na'~sI by the IODOGEN method. The specific activitieswere approximately 2.3 ~; 104 and 1.~ x 10~ cprn/ng protein for rSEBP and CNBr-cleaved rEbpS fragments, respectively. Radiolabeled rEbpS (45 ng) in 1.5 ml of binding buffer (50 mM Tris, 500 mM NaCI, 2 nM CaCI.~0. 1 mg/ml BSA, pE-I 7.5) was incubated with 1 ml of the elastin peptide affinity resin for 2 h at room temperature in the absence or presence of 2 mg unlabeled elastin peptides. The mixture was transferred to disposable polypropylene columns and washed with binding buffer by gravity flow until radioactivity of the flow through reached background. Bound rEbpS was eluted with 3 ml of 1% SDS buffer, spin concentrated. and analyzed by 10% SDS-PAGE and autoradiography. Binding of radiolabeled CNBr-cleaved rEbpS
to immobilized elastin was assessed similarly, except 80 ng of the starting material was used and bound material was visualized by 12% SDS-PAGE and autoradiography.

W O 98/38312 PCT~US97/03106 Detection of the native 25 kDa cell surface labeled EbpS with anti-rl~bpS antibodies Surface labeled extracts from S. aureus cells ~vere prepared by Iysotaphin digestion as described previously (20). Approximately 10' cpm of surface labeled extract was first 5 absorbed with 3 ml of pig IgG-Affi-Gel 10 resin for 2 h at room temperature. The unbound supernatant was collected by centrifugation and incubated with 1 ml of the anti-rEbpS IgG affinity resin in the absence or presence of 2 mg unlabeled rEbpS for 2 h at room temperature in 2 ml of binding buffer. The mixtures were transferred to disposable columns and washed with binding buffer until flow through reached 10 background radioactive levels. Bound cell surface-labeled molecules were eluted from the colurnn by 3 ml of 1% SDS buffer, spin-concentrated, and analyzed by 15%
SDS-P~GE and autoradiography.

Other procedures Purification and radiolabeling of full length recombinant human elastin. and cellular elastin binding assays were perforrned as described previously (20). Automated amino acid scquence and composition analyses were carried out in our laboratory by the Applied Biosystems 473A protein sequencer and Beckman System 6300 High 20 Performance Analyzer~ respectively. Electron spray mass spectrometry was perforrned by the Protein Chemistry Laboratory at Washington University School of Medicine, St. Louis, MO.

Results 25 Cloning of ebpS

The N-terminal sequence of native EbpS expressed on thc cell surface of S. aureu.s was determined previously to be ANNFKDDFEKNRQ (20). A degenerate oligonucleotide corresponding to residues '7- I I of the determined N-terminal 30 sequence was generated and used as a probe. Southern blot analysis was first W O 98/38312 PCTn~S97/03106 performed with S. aureus strain 12598 genomic DNA digested with restriction endonucleases to identify the hybridizing genomic fragment. As shown in Figure 1, the oligonucleotide probe hybridized to a 4.2 kb EcoR I fragment (lane A). On the basis of this observation, a size-selected genomic plasmid library in the 4.2 Icb region S was constructed from EcoR I-digested S. aureus genomic DNA and screened with the oligonucleotide probe by Southern blotting. 0~120 colonies screened, two positive clones with identical restriction enzyme digestion patterns were isolated. One of these clones, pEBPS-I, was used for further analysis.

10 To determine whether the correct 4.2 kb fragment was cloned, the radiolabeledoligonucleotide was hybridized to the pEBPS-I insert, and the cloned insert itself was used as a probe for Southern analyses with EcoR l-and EcoR I/Hind III/Hind II-digested genomic DNA. The oligonucleotide probe hybridized to the 4.2 kb pEBPS-.l insert (Fig. 1: lane B) and the insert recognized a 4.2 kb EcoR I genomic fragment 15 (Fig. 1: lane C). With EcoR I/Hind III/Hinc II-digested genomic DNA~ the radiolabeled pEBPS-1 insert hybridized to a 2.6 kb fragment (Fig. 1: lane D). The oligonucleotide and cloned insert probes consistentlv detected single fragments with identical size in Southern analyses using genomic DNA digested with various restriction endonucleases, indicating that ebpS is present as a single copy gene.
An~lysis of the primary sequence of ebpS

pEBPS-1 was digested with Hind III and Hinc II to yield a 2.6 kb fragment. This fragment was subcloned into pBluescript II KS+ to generate pKS-2.6 and sequenced25 to locate an open reading frarne cont~ining the N-terminal sequence o~cell surface EbpS. A 606 bp open reading frame which starts with an ATG codon was identified about 0.9 lcb 3' of the Hind III site. The physical map of pKS-2.6 and the primary sequence of the open reading frarne with up- and downstream sequences are shown in figures 2 and 3, respectively. Putative -10 and -35 hexamers were identified at positions -31 and -54, with a spacing of 17 bp. A third AT-rich promoter sequence W O 98/38312 PCTAJS97/~3106 has been proposed recently to exist in a region about 20 bp upstream of the -35 hexamer in E. coli (28), and this region for ebpS was 75% AT. A potential ribosome binding sequence, which complemented perfectly with the e7ctreme 3' region of , Bacillus subtilis 1 6S RNA (UCUUUCCUCC) (29), was found at position -7. Overall, 5 ebpS was G4% AT and 36% CG. Although two ATG codons were found in the r corrçct r~ading ~rame Qf e~S~ we have designated the second ATG as the initiation codon based on the location of the putative ribosome binding site. The N-terrninal sequence of cell surface EbpS determined from peptide sequencing was found to start at the second residue of the predicted sequence~ suggesting that the initial Met residue 10 is cleaved. The deduced sequence matched perfectly with the determined sequence of cell surface EbpS e:~cept for the first amino acid (Ala in native, Ser in deduced).
Since Ser residues are often misread because of its small peak in the peptide sequencing chromatogram~ we reexamined the original sequencing chromatogram of cell surface EbpS and have identified clear Ser and Ser' peaks indicating that the 15 residue in concem is a Ser and not Ala.

The mature protein has a predicted molecular mass of 23,344.7 daltons and an acidic pl of 4.9. Accordingly. the protein has a preponderance of acidic amino acids Asp (10.9%) and Glu (11.9%). but, is devoid of Cys residues. Garnier analysis predicts a 20 secondary structure that is 58.4% a helical and ~3.8% coiled coil. The BLAST
network ser- ice of the NI~l on the intemet was used to search for sequence homologies. The May 1, 1995 releases of the Brookhaven Protein Data Bank, GenBank, EMBL Data Library, SWISS-PROT protein sequence database~ and the trasnlated coding sequence of GenBank were used for comparison. No significant 25 homologies were found between reported sequences in these databases and the primary sequence of ebpS.

Fxpression of ebpS in E. coli -W O 98/38312 PCTnUS97J03106 We studies whether the cloned gene encodes an eiastin binding protein by expressing ebpS in E. coli. The PCR-arnplified ebpS open reading frame was expressed in E.
coli as a fusion protein cont~ininp: SiX His residues tagged to the N-terrninus of the protein. Recombinant EbpS (rEbpS) was purified from E. coli extracts by Ni++-NTA5 chromatography based on the high affinity binding interaction between Ni+l and His residues. As can be seen in figure 4, the three positive clones expressed large arnounts of homo~eneous rEbpS. However, purified rEbpS from all three clones migrated as a 45 kDa protein when fractionated by SDS-PAGE, which was a significant deviation from its predicted molecular mass of 26 kDa. Based on the size 10 difference and the fact that rEbpS was fractionated by reducing SDS-PAGE, it was unlikely that the observed abnormal migration was caused by dimerization of rEbpS.

To examine the fidelity of rEbpS. the N-terminal sequence of full length rEbpS, as well as internal secluences from a degradation product and two fragments generated by 15 CNBr cleavage were deterrnined by protein microsequencing. Altogether~ an unambiguous sequence was obtained for 58 residues, and they matched perfectly with the predicted sequence Fig. 2 underlined sequences). Furthermore. amino acid andmass spectrometry analyses indicated that the composition of rEbpS and actual molecular mass of rEbpS~ respectively, are in agreement with the predicted data.20 These results indicate that the correct protein has been expressed. and that overestimation of the molecular mass is due to aberrant migration in SDS-PAGE.

F.l~7~tin binding activities of rEbpS constructs 25 To investigate whether rEbpS interacts specifically with elastin, elastin peptide affinity chromato~raphy was performed with radiolabeled rEbpS. Iodinated rEbpS
was incubated with the elastin peptide affinity resin for ~ h at room temperature in the absence or presence of excess unlabeled elastin peptidcs. The mixture was then washed extensively with buffer until radioactivity of the flow through reached 30 background. The bound material was eluted with 1% SDS buffer and analyzed by W O 98138312 PCTrJS97/03106 SDS-PAGE and autoradiography. The starting material for this experiment was stored for one weelc at 4~C after purification with Ni'~-NTA chromatography. As can be seen in Figure 5. the starting material for this experiment was partially degraded (lane 13). Because the sequence of the 40 kDa major degradation product starts after 5 an Arg residue (Fi~. 2, starts at residue #60). a trypsin-like enzyme is the likely cause of degradation. This partially degraded material lacking the first 59 amino acids did not associate with the affinity resin. In contrast, the full length form of labeled rEbpS
interacted efficiently with the elastin peptide affinity resin in the absence (Fig. 5: lane C), but not in the presence, of excess unlabeled elastin peptides (lane D). These 10 results suggest that the 59 arnino acid region plays a critical role in elastin recognition.

To further study the hypothesis that the N-terminal region of EbpS contains the ligand binding site, elastin binding properties of CNBr-cleaved rEbpS fragments were examined since the presence of a single internal Met residue is predicted from DNA
15 sequence data. In agreement with agreement with the predicted sequence two dominant bands were detected in the radiolabeled starting material of CNBr-cleaved rEbpS. Peptide microsequencing was employed to verify correct cleava~e. and to identify the N- and C-terminal fragments (Fig. 3: underlined). When elastin binding activities of these fragments were assaved with elastin peptide affinity 20 chromatography, only the N-terminal fragment bolmd tightly to the elastin peptide affinity resin. These findings are consistent with the elastin binding site contained in the first 59 amino acids of EbpS.

Fffects of rEbpS on S. aureus bindin~ to elastin If EbpS is the cell surface molecule responsible for elastin binding at the cellular level, then an active forrn of soluble EbpS should interfere with S. aureus ~inding to elastin. We tested this hypothesis by ex~mining the effects of rEbpS on S. aureus binding to elastin. Elastin labeled with 1'51 was incubated with S. aureus cells in the 30 absence or presence of varying concentrations of unlabeled rEbpS for I h at room W O 98/38312 PCT~US97/03106 temperature in 200 111 of TSB. After washing three times with TSB. radioactivityassociated with the cellular pellet was measured with a garnma counter. As can be seen in figure 6, rEbpS inhibited binding of labeled elastin in a concentration dependent manner. ~urthermore. S. atlreus binding to radiolabeled elastin was S abrogated at the highest concentration of rEbpS tested ( 19 ~lM). The controlpolyhistidine fusion protein mouse dihydrofolate reductase ~DHFR) did not affectbinding at 26 ~M. These results demonstrate that rEbpS inhibition of cellular elastin binding is specific and that the polyhistidine domain of rEbpS is not affecting binding.

10 F.xpression of EbpS on the cell surface of S. aureus The abilities of rEbpS to interact directly with elastin and to inhibit cellular elastin binding strongly suggest that EbpS is the cell surface protein me~ ting S. aureus binding to elastin. To provide further evidence that EbpS is a cell surface protein, 15 affinity chromatography was performed with surface labeled S. azlreus extracts and immobilized anti-rEbpS IgGs. S. aureus cells were surface labeled bv the IODOGENmethod and extracts were prepared by lysostaphin digestion. Approximately 10' cpm of this material was pre-absorbed to a pig IgG affinity resin to remove surface-labeled protein A, and the flow through was incubated with the anti-rEbpS IgG af~mity resin 20 for 2 h at 25 ~C. Af~er washing extensively with binding buffer, bound cell surface molecules were eluted with 1% SDS buffcr and anlayzed by SDS-PAGE and autoradiography. As shown in Figure 7, pre-absorption with the pig IgG resin removed surface labeled protein A from the starting material (compare 50 I;Da band in lanes A and B). Of the rem~ining numerous surface labeled proteins. a 35 and 25 kDa 25 protein associated with the anti-rEbpS IgG af~mity resin (lane C). ~o determine the specificity of binding, the sarne experiment was performed in the presence of excess unlabeled rEbpS As can be seen in lane D. binding ofthe surface 75 kDa protein, but not the 35 kDa protein, to immobilized anti-rEbpS IgG was inhibited by unlabeledrEbpS. Densitometric sc~nning of the bands revealed that the band intensity for the 30 25 and 35 kDa protein, but not the 35 kDa protein, to immobilized anti-rEbpS IgG

W O 98/38312 65 PCT~US97/03106 was inhibited by unlabeled rEbpS. Densitometric sc~nning of the bands revealed that the band intensity for the 25 and 35 kDa proteins decreased by 64 and 7%, respectively. in the presence of excess unlabeled rEbpS. These results indicate that the 25 kDa protein is cell surface EbpS and that the 35 kDa protein is interacting with ,, 5 the agarose affinit; support of the elastin peptide affinity resin.

- Because the anti-rEbpS antibody is polyclonal, we reasoned that some of the antibodies may recognize a region in cell surface EbpS that is critical for elastin binding. To test this idea, effects of anti-rEbpS IgG Fab fragments on S. aureusbinding to radiolabeled elastin binding were e~mine-l S aureu,~cells were inc~lb~te~
with labeled elastin in the absence or presence of imrnune or pre-immlln~ Fab fr~ments. As sho~n in Figure 8, Fab fragments from im~nune IgGs inhibited binding of S. aureus to radiolabeled elastin in a concentration-dependent manner. In contrast, Fab fragments from pre-immune antibodies did not affect binding at the two \\
y W O 98/38312 PCTrUS97/03106 DISCUSSION

Cell surface components of pathogenic bacteria play important roles in surviving the hostile environment of the host. For gram positive bacteria these surface molecules 5 are used in pathogenic processes such as evading host immune responses (30), digesting host carbohydrates to expose host attachment sites (31, 32), capturing host enzymes to digest host tissues (33), and binding host tissue determin~nt~ to establish a firm basis for colonization (34). Cell surface adhesins and MSCRAMMS interact with host E(~M components, and participate in the colonization of and extravasation 10 through tissues and organs. Previously it was demonstrated that S. aureus binds specifically to elastin. Results from binding assays at the cellular level suggested the existence of a single type of cell surface elastin binding protein that mediates the S.
aureus-elastin binding interaction. Accordingly, cell surface EbpS with similar binding properties to S. aureus binding to elastin was isolated. On the basis of these 15 findings, EpbS has been proposed to be the elastin MSCRAMM. The focus of thisstudy was to examine this hypothesis by studying the molecular structure and function of EbpS.

The present results demonstrate that the 606 bp ebpS exists as a single copy gene in 20 the S. aurez~s genome, and that the primary sequence of ebpS is novel. Mature EbpS
has a predicted molecular mass of 23 kDa and is highly charged. Furtherrnore, soluble EbpS expressed in E. coli interacts directly with elastin. Sequence, composition, and size fidelities of rEbpS were analyzed by peptide microsequencing, amino acid analysis, and mass spectrometry and deterrnined to be correct. The 25 recombinant protein with an actual size of 6 kDa, however, migratcs aberrantly as a 45 kDa protein in SDS-PAGE. Abnormal migration in SDS-PAGE has been observed frequently for proteins e~pressed as a polyhistidine fusion protein and also fornumerous gram positive cell surface proteins (35-39). In some cases~ the aberrant migration of gram positive surface proteins has been attributed to the presence of 30 multiple repetitive domains (35,36) and high Pro content (37,38) of these proteins.

W O 98/38312 PCT~US97/03106 The abnormal migration of rEbpS cannot be due to these factors because rEbpS lacks multiple repetitive domains and does not have a high Pro content (3.6% of total).
Since acidic amino acids are uniformly abundant in EbpS (pI=4.9), a likely explanation may be that the surface negative charge of rEbpS is hindering the uniform 5 attachment of anionic SDS and causing the observed abnormal migration in SDS-- PA5E.

Several independent criteria indicate that EbpS is the surface protein mediatingcellular elastin binding. First, rEbpS binds specificalty to immobilized elastin and 10 inhibits binding of S. aureus cells to elastin in a dose dependent manner. These results establish that EbpS is an elastin binding protein that is functionally active in a soluble form. Second. an antibody raised against rEbpS recognizes a 25 kDa protein expressed on the cell surface of S. aureus cells. In addition to the size similarity and antibody reactivity. further evidence that this 25 kDa protein is cell surface EbpS is 15 provided by the experiment showing that binding of the 25 kDa protein to immobilized anti-rEbpS IgG is inhibited in the presence of excess unlabeled rEbpS.
~inally~ Fab fragments prepared from the anti-rEbpS antibody, but not from its pre-immune control, inhibit binding of S. aureu~s to elastin This rcsult suggests that the topology of surface EbpS is such that the elastin binding site is accessible to interact 20 with ligands (i.e. elastin and the anti-rEbpS Fab fragment) and not embedded in the cell wall or membrane domains. The composite data demonstrate that l~bpS is the cell surface protein responsible for binding S. aureus to elastin.

Although the present results indicate that EbpS is expressed on the cell surface. Iittle 25 is known regarding the mechanism involved in surfaced expression of the protein.
Several surface proteins of gram positive bacteria have been found to share common motifs important in sorting, transporting, and anchoring these proteins to the cell surface (40). The common features consist of a cleaved signal peptide. which is followed by the ligand binding extracellular N-terminal domain, a Pro-rich region 30 thought to span the cell wall, a conserved LPXTGX hexapeptide sequence, a hydrophobic membrane spanning domain, and a charged C-terrninal tail. A recent study by Schneewind et al. (41 ) have shown that at least for protein A of S. aurez~s, the common structure is cleaved after the Thr residue of the hexapeptide sequence and the protein is anchored to the cell wall via arnide linkage of the carboxyl group of Thr 5 and free amino group of the pentaglycine peptide moiety of the staphylococcal peptidoglycan. Apart from the putative localization of the elastin binding site to the extracellular N-terminal domain and identification of a charged C-terminal tail, our results show that EbpS does not contain other common motifs. This observation, however, is not unique for EbpS in that several other gram positive surface proteins 10 have been found to deviate from this conserved structure. Although we have not identified any sequence or structural similarities, EbpS and other proteins not conforrning to the common structure are generally smaller than the majority of proteins expressing the shared motifs. The list includes streptococcal proteins such as the fibronectin/fibrinogen binding protein (54 kDa) (42), albumin binding protein (36 kDa)(38), and the plasmin receptor (36 kDa) (43). Furthermore, similar to EbpS, the initial Met residue of the streptococcal plasmin receptor is cleaved in the mature protein (43). It is not known whether these correlations have a role in an alternative mechanism for surface expression.

20 The present and previous fin~ling.~ suggest the e~istence of a functionally active 40 kDa intracellular precursor form of EbpS that requires processing at the C-terrninus prior to surface expression. This notion is based on the following observations: i) there exists an intracellular 40 kDa elastin binding protein that is never detected during cell surface labeling experiments, ii) the 25 kDa EbpS and the 40 kDa elastin 25 binding protein have an identical N-terminal sequence. and iii) a single gene exists for EbpS. Because the size of the ebpS open reading frarne is not sufficient to encode a 40 kDa protein, at first we disregarded this hypothesis. However. our studies with rEbpS demonstrated that although the actual size of the recombinant protein is 26 kDa, it migrates aberrantly as a 45 kDa protein in SDS-PAGE. This finding suggests 30 that full length native EbpS, with a predicted size of 23 kDa. may be migrating in W O 98/38312 PCT~US97/03106 SDS-PAGE as the 10 kDa intracellular precursor, and that the 25 kDa surface form of EbpS is actually a smaller form of the molecule process~d at the C-terminus.
Although EbpS lacks an N-terrninal signal peptide and other known sorting and anchoring signals. this proposed intracellular processing event may explain some5 questions regarding how EbpS is targeted to the cell surface. In fact, C-terminal signal peptides have been identified in several bacterial proteins (44) and alternative means of anchoring proteins to the cells surface have been reported in gram positive bacteria (45). The C-terminus of EbpS may be processed intracellularly~ althoughwhere and how this cleavage occurs, and if this processing event is one of the signals 10 required for surface expression of EbpS, is yet to be ascertained.

It is now apparent that interactions between pathogenic bacteria and host ECM
components are plaving important roles in disease pathogenesis. However~ molecular structure:function analyses for most ECM adhesins have not been performed despite 15 the obvious potential of developing effective prophylactic and therapeutic agents based on information derived from these studies. In cases where investigations have been performed. the primary ligand binding site has been found to be contained in the C-terrninal end of the extracellular N-terminal domain (36,46,47). The present results showing lack of elastin binding activity by the C-terminal fragment of CNBr-cleaved 20 rEbpS and inhibition of cellular elastin binding by the anti-rEbpS Fab fragments suggest that the elastin binding site in EbpS is also contained in the e~;tracellular N-terminal domain. Further mapping of the putative elastin binding site within this region to the first 59 amino acids of EbpS is based on the finding that a degradation product of rEbpS lacking these residues failed to interact with elastin. Similar to other 25 domains in EbpS. the putative elastin binding region in EbpS lacks homology with sequences reported to various ~l~t~b~es. A truncated, recombinant construct of EbpS
cont~ining this 59 amino acid domain inhibits binding of S. aureus to elastin. These observations indicate that the 59 amino acid region contains the elastin binding site of EbpS.

CA 02247072 l998-08-27 W O 98/38312 PCT~US97/03106 7~

Cell-extracellular matrix (ECM) interactions are necessary events in various biological processes including embryonic development, infl~mm~tion. tumor cell 5 metastasis, homeostasis, and microbial infections [Bernfield, et al., Annu. I2ev. Cell Biol., 8:365-393 (1992); Liotta, etal., Annu. Rev. Biochem., 55:1037-1057 (1986), Patti, et al., Annu. Rev. Microbiol., 48:585-617 (1994)] Molecular interactions between ECM components and corresponding cell surface receptors instruct cells to differentiate, migrate, adhere, or proliferate in directing these biological processes.
10 Although there are exceptions~ mz~mm~ n cells typically use the integrins, an oc/,(~
heterodimeric receptor complex, to interact with the ECM [Hynes, Cell, 69:1 1-25(1992); Albelda, et al., FASEB J., 4:2868-2880 (1990)].

Bacterial pathogens also interact with the host matrix through specific cell surface 15 ECM binding molecules categorized collectively as adhesins or MSCRAMMS ~Patti, etal., 1994. supra: Hook, etal.,CellDiffer.De-~., 23:433-438(1990)]. Thegram positive bacterial pathogen Staphylococcus aureus (S. aureus) has been found to interact ~vith many ECM macromolecules such as collagen [Holderbaum. et al., Infect.
Inzmun., 5~:359-364(1986),Speziale.et al.. J. Bac~eriol., 167:77-81 (1986)], fibronectin [Kuusela, h'ature, 276:718-720 (1978)], l~minin [Lopes, et al., Science, 229: 75-277 (1985)], proteoglycans [Liang, et al., Infect. ~mmun., 60:899-906 (1992)], fibrinogen ~McDevitt, etal., A~lol. Microbiol., 11:237-248 (1994)], andelastin [Park, et al., J. Biol. Chem., 266:23399-23406 (1991)]. While S aureus adhesins bind host ECM, these interactions have different biological purposes and binding profiles from m:~mm~ ECM receptors In general, Staphylococcal ~CM
adhesins are used for pathological purposes such as in colonization of host tissues.
For example~ collagen [Patti, et al., Infcct. ~m m2~n., 62:152-161 (1994)1 and fibronectin [Baddour. Infect. Immun., 62: 2143-2148 ~1994)] adhesin mutants show a reduced capacity to cause disease in in ~ivo models. but are otherwise phenotypically normal.

W O 98/38312 PCT~US97/03106 At the molecular level, all characterized staphylococcal adhesins function as monomers. and endogenous bacterial ligands have not been identified. Available evidence suggests that ligand binding sites in staphylococcal ECM adhesins are contained within small regions of the extracellular domain. The ligand binding site in the staphylococcal fibronectin binding protein, for example, has been mapped to a repetitive 38 amino acid motif. and corresponding synthetic peptides have been found to possess direct binding activity and to inhibit bacterial binding to fibronectin ~Signas, et al., Proc. Natl. Acad. Sci. USA, 86:699-703 (1989); Raia, et al., Infect.
Immun., 58:2593-2598 (1990)~. Similarly, a synthetic 25 arnino acid peptide 10 corresponding to the region between Asp~Q9 and Tyr733 of the collagen adhesin has been shown to inhibit binding of type II collagen to S. aureus [Patti, et al., J. Biol.
Chem., 270:12005-12011 (1996)], suggesting that this short peptide sequence alone can mediate staphylococcal binding to collagen.

15 Specific binding between S. aureus and elastin is mediated by a 25 kDa elastin binding protein (EbpS) on the surface of S. aureus [Park. et al., 1991 ~ supra]. Elastin binding activity is localized to the extracellular~ arnino terminal end of EbpS within the first 59 amino acids ~Park~ et al., 1991, supra; Park, et al., J. Biol. Chem., 271:15803-15809 (1996)]. To betterdefine the amino acids in EbpS responsible for20 the elastin binding activity, overlapping synthetic peptides and truncatcd, recombinant EbpS constructs were used in elastin binding assays. Our results demonstrate that the critical elastin recognition sequence within the N-terminal domain resides between Gln~and Glu3~ SEQ ID NO: 13. Sequence comparison indicates that the minim~l sequence shared by all active EbpS constructs is the hexarner 25 Thr-Asn-Ser-His-Gln-Asp spanning residues 18-23 (SEQ ID NO: 16).

Materials and Methods Materials _ Restriction enzvmes, calf intestinal alkal;ne phosphatase, T4 DNA ligase, isopropyl-,B-thiogalactoside, 5-bromo-4-chloro-3-indolyl-~-D-galactoside, and Hind III-digested ~ DNA markers were purchased from Promega (Madison, WI).
Luria-Bertani medium and Luria-Bertani agar-medium capsules were from BIO 101 (La Jolla, CA). Tryptic soy broth (TSB) was obtained from Remel (Lenexa, KS~.
Na'2sI was from ICN (Costa Mesa, CA). Papain and protein A immobilized to cross-linked agarose, Immunopure Sulfo-NHS-Biotinylation kit, and IODOGEN were purchased from Pierce (Rockford, IL). QIAexpress vector kit type IV and the midi-prep plasmid purification kit were obtained from Qiagen (Chatsworth, CA).
10 Nitrocellulose membrane and blotting paper were from Schleicher & Schuell (Keene, NH). Affi-Gel-10 affinity support was from Bio-Rad (Melville, NY). All other materials were purchased from Sigma Chemical (St. Louis, MO).

Synthetic peptides Synthetic peptides cont~ining the deduced primary sequence of EbpS were preparedby conventional solid phase synthesis on an Applicd Biosystems model 431 A
synthesizer using FastMoc chemistry. Peptides were purifled by reverse phase high performance liquid chromatography (Beckman Cl 8~ 0-80% linear water-acetonitrile20 gradient containing 0.05% trifluoroacetic acid). Purity of the peptides were confirrned by either amino terminal sequencing or electron spray mass spectrometry. All synthetic peptides were soluble in the assay buffer at the concentrations tested.

Fxpression of recombinant F.bpS proteins Expression of full length recombinant EbpS (rEbpS) as a fusion protein with a polyhistidine tag at the N-terminus was as previously described ~Park. et al., 1996, supra]. Essentially the sarne protocol was followed to make truncated recombinant EbpS- I and 2 (trEbpS-I and trEbpS-2), except that different PCR primers were used.
30 For the generation of trEbpS-1 cont~inin~ EbpS residues 1-78, a 2.6 kb Hind III/Hinc W O 98/38312 PCT~US97/03106 Il fragment in pBluescript KS+ which contains full length ebpS was PCR-amplifiedusing the oligonucleotide 5'-TGTGGATCCATAGAAAGGAAGGTGGCTGTG-3' as the forward primer and the oligonucleotide S'-CATTGAGCTCAGATGTTTGTGATTC-3' as the reverse primer. To make 5 trEbpS-2 corresponding to amino acid residues 1-34 of EbpS, the same template and forward primer were used, whereas the oligonucleotide 5'-GTTCGAGCTCTGATTGGTC~ 1 l l C-3' served as the reverse primer. The forward prirner contained a BamH I site (underlined), and A of the two ATG codons was changed to G (in bold letters) to avoid internal initiation of translation as 10 recommended by Qiagen. The reverse primers contained a Sac I cleavage site (underlined). PCR amplification was performed with a Perkin Elmer thermocycler using standard reagents. Conditions for PCR amplification. and subsequent restriction enzyme di~estion. Iigation, transformation, expression~ and purification of recombinant proteins were as previously described [Park, et al., 1996, supra].
Direct binding of trEbpS proteins to elastin trEbpS-1 and trEbpS-2 were biotinylated usin a commercially available l;it (Pierce).
Recombinant proteins ( 1 mg) were incubated with sulfo-NHS-biotin reagent ( mg) in 20 1 ml of PBS for 2 h at 4~C. Biotinylated proteins were then separated from free biotinylating reagent by PD-10 gel filtration chromatography.

Three micrograms of recombinant human tropoelastin. bovine serum albumin (BSA), and ovalbumin were fractionated by 10% SDS-PAGE and transferred to nitrocellulose 25 membranes by Western blotting. Transferred blots were blocked overnight at 4~C
with blocking buffer which contained 0.5% (w/v) BSA and 0.05% (v/v) Tween-20 in Tris-buffered saline (TBS: 50 mM Tris, 150 mM NaCl, p~ 7.5). The blots were washed twice with blocking buffer and incubated for 2 hours at room temperature with either S ~lM biotinylated trEbpS-l or trEbpS-2 in the absence or presence of 3 30 mg/ml elastin peptides in blocking buffer. After washing twice with blocking buffer, W O 98/38312 PCTfUS97/03106 the blots were incubated with a 1:1000 dilution of avidin conjugated to horseradish peroxidase. Membranes were developed by 4-chloro-naphthol.

EbpS polvclonal antibodies s Immunization protocols and characterization of the anti-EbpS polyclonal antibodies have been described previously [Park, et al., 1996, supra]. To subtract the population of antibodies reco~nizing EbpS residues 1-34, anti-EbpS IgGs (25 mg) purified bycaprylic acid precipitation [McKinney, etal., J. Immunol. Methods, 96:271-278 (1987)] were incubated overnight at 4~C with trEbpS-2 (8 mg) coupled to 3 ml of Affi-Gel- 10. On the following day, the mixture was transferred to a disposable polypropylene colurnn, and unbound IgGs were collected by gravity flow. The efficiency of the absorption was tested by Western immunoblotting against the full length and two truncated constructs of recombinant EbpS (Fig. 2c). Fab fragments15 from both the original and trEbpS-2-absorbed IgG fractions were prepared by digestion with imrnobilized papain as described previously [Park, et al., 1996, sz~pra].

Other procedures 20 Purification and radiolabeling of full length recombinant human soluble tropoelastin, generation of elastin peptides. cellular elastin binding assays, SDS-PAGE, and Western blotting were performed as described previously [Park, et al., 1996, szlpra].
Automated arnino acid seqn~ncing was performed using an Applied Biosystems 473A
protein sequencer. Electron spray mass spectrometry was perforrned by the Protein 25 Chemistry Laboratory at Washington University School of Medicine (St. Louis, MO).
The PROTEAN program ~DNAStar, Madison, WI) was used to predict the secondary structure of EbpS constructs.

Results CA 02247072 l998-08-27 W O 98/38312 PCT~US97/03106 Truncated recombinant EbpS constructs bind to el~stin and inhibit S at~reus binding to elastin Results from studies of elastin binding properties of various EbpS fragments ~Park, et S al., 1996, supra] and recombinant constructs are surnmarized in Figure 9. A
cyanogen bromide fragment cont~inin~ the first 125 amino acids of EbpS showed binding activity, whereas an EbpS de~radation product lacking the first 59 aminoacids, a cyanogen bromide fragment cont~ining the C-terminal one-third of the protein and a synthetic peptide corresponding to residues 1- 13 did not interact with elastin.
10 These results suggested that the elastin binding site in EbpS is contained in arnino acid residues 14-59 SEQ ID NO:15 (shaded area in Figure 1).

To identify the amino acids critical for binding? two truncated recombinant constructs of EbpS (trEbpS-I having a nucleic sequence of SEQ ID NO:9, and trEbpS-2 having a 15 nucleic acid sequence of SEQ ID NO: 11) were generated and the corresponding polypeptides were tested both for their ability to directly bind elastin and to inhibit bindin~: of S aureus to elastin. trEbpS- I . with a predicted molecular mass of 12.8 kDa. contains residues 1 -78 of EbpS (SEQ ID NO: 10), whereas trEbpS- 2 spans residues 1-34 (SEQ ID NO:12) and has a predicted mass of 7.5 kDa. Characterization 20 of the truncated constructs by mass spectrometry. peptide n1icrosequencing. and imrnunoblotting with the anti-rEbpS antibody (Fig. I OB, lanes C and D) confirmed that correct truncated proteins have been expressed. Both proteins migrated at higher than predicted molecular weights when fractionated by SDS-PAGE (Fig. 10). This behavior has also been observed with fi~ll length recombinant EbpS (rEbpS) SEQ ID
25 NO:2. Aberrant migration in SDS-PAGE appears to be a cornrnon charactcristic of gram positive cell surface proteins ~McDevitt. et al., 1994, supra; Signas, et al., 1989 supra; Murphy, et al., Biochem. J., 277: 777-279 (1991); Sela, et al., Mol. Microbiol., 10:1049-1055 (1993); Sjobring, Infect. Im mtm., 60:3601-3608 (1992); Talay, et al..
Mol. A~licrobiol., 13:531 -539 (1994)]

WO 98/38~12 PCT~US97/03106 To examine whether trEbpS-1 and trEbpS-2 bind directly to elastin, the truncatedproteins were biotinylated and reacted with tropoelastin that was transferred tonitrocellulose membranes. As shown in Figure 11~ both truncated constructs bound to the 67 kDa tropoelastin in the absence (lanes A and C), but not in the presence (lanes 5 B and D)~ of competing soluble elastin peptides. The biotinylated proteins did not bind to either ovalburnin or BSA under similar conditions, demonstrating that the binding interaction between the two polypeptides, i.e., trEbpS-I and trEbpS-2, and tropoelastin is specific.

l O Effects of trEbpS constructs on elastin binding at the cellular level were tested by incubating S. aureus cells with radiolabeled elastin in the absence or presence of increasing amounts of either soluble full length (rEbpS) or truncated forms of the receptor. All three polypeptides inhibited binding of S. aureus cells to elastin in a concentration dependent manner (Fig. 12). rEbpS and trEbpS-I completely inhibited 15 elastin binding at the highest concentration tested trEbpS-2 was somewhat less effective as an inhibitor, with about 20% residual elastin binding activity at the highest inhibitor concentration.

Pre-absor~tion of the anti-rEbpS antibodv with trEbpS-2 neutralizes its inhibitorv 20 ~

Fab fragments of a polyclonal antibody raised against rEbpS inhibit binding of S.
aureus to elastin ~Park, et al., 1996, supra], suggesting that a population of antibodies in the immune serum recognize a region in EbpS critical for elastin binding. To test 25 this possibility, anti-rEbpS IgGs were absorbed to the trEbpS- ' construct coupled to Affi-Gel 10, and unbound IgGs were collected. Irnrnunoblotting revealed that thetrEbpS-2-absorbed immunoglobulins retained the ability to interact with both trEbpS
and the full length rEbpS-1 (Fig. lOC, lanes B and C), although with reduced activity.
The imrnunoglobulin fraction that was not absorbed to the trEbpS-2 construct did not 30 react with trEbpS-2 on Western blot (Fig. IOC, lane D~.

W O 98/38312 PCTnU~97/03106 Fab fragments from both the nonabsorbed and trEbpS-2-absorbed immunoglobulins were generated by papain digestion and tested for their effects on staphylococcal elastin binding. Consistent with previous findings, Fab fragments from the original anti-rEbpS IgGs abrogated the binding of S. aure~s to elastin (Fig. 13). In contrast, serum preabsorbed with trEbpS-2 inhibited the binding of S. aureus to elastin by only 30~/0 at the highest concentration tested (300 ~lg/ml).

Conti~uous svnthetic F.bpS peptides inhibit.S. aureus binding to elastin 10 The fin~ings described above suggest that the elastin recognition domain in EbpS is contained within residues 14-34. To more precisely define the elastin binding site7 overlapping synthetic peptides within this region were generated (Figure 14) andtested for their ability to inhibit binding to staphylococcal elastin. Repetitive sequences in EbpS were searched to find a candidate elastin binding site. since several 15 staphylococcal and streptococcal ECM adhesins have been shown to use repetitive domains for ligand recognition ~Signas, et c 1., 1989. supra; Sela, et al. 1993, supra].
Although no identical repetitive sequences were identified. there are two related sequences, '~HQDHTEDVE'9. SEQ ID NO:20 and 37HQDTIENTE~5, SEQ ID NO:23.
in the amino-terrninal end of the molecule. The sequence 7'HQD~ITEDVE'9 is within 20 the putative amino terrninal elastin binding site and is contained in all active EbpS
constructs. The second sequence. 37HQDTIENTEJs. is present only in full length EbpS, and in trEbpS-1, which does not actively bind tropoelastin.

To deterrnine whether the E~QDHTEDVE sequence might participate in elastin 25 binding, we generated two synthetic 17 amino acid peptides. Pl (SEQ ID NO:18) and P2 (SEQ ID NO: 19), corresponding to residues 18-34 (Fig. 14). The P I peptide was made according to the deduced sequence of EbpS. In the P~ peptide. Asp~3, Glu76.and Glu79 were substituted with Asn, Pro, and Gln, respectively. The charged amino acids were targeted for substitution because staphylococcal elastin binding has been 30 shown to involve electrostatic interactions ~Park, Cell Biology, 1-161 (1993)]. As _ CA 02247072 l998-08-27 W O 98/38312 PCT~US97/03106 shown in figure 15. the Pl peptide inhibited the binding of S. aureus to elastin in a concentration dependent manner. Elastin binding was abrogated at the highest concentration of Pl . whereas minim~l inhibition (~15%) was observed with the P2peptide.
s To define the elastin binding activity more completely, three overlapping 1 0-mers spanning arnino acid residues 14-36 were generated (Fig. 14) and tested for their ability to inhibit staphylococcal binding to elastin. Peptide P4 (SEQ lD NO:21 residues 21-30 cont;~inin~ the HQDH~EDVE sequence) and the P5 peptide (SEQ ID
10 NO:21 residues 27-36) reduced elastin binding by approximately 35% at the highest concentration tested, whereas the P3 peptide (SEQ ID NO: 14 residues 1~-23) inhibited binding of S. aureus to radiolabeled elastin by more than 95% in a concentration-dependent manner (Fig. 15). Sequence comparison of P3 and other active EbpS constructs revealed that the he~apeptide '8~NSHQD'3 (SEQ ID NO:16) is 15 the only sequence shared by all members. However, the he~apeptide TNSHQD (P6)and its control TNSHQS (SEQ ID NO:17, P7) did not inhibit staphylococcal elastinbindin& at anv concentration tested (0.075-2.00 mg/ml). These findings indicate that the region around the HQDHTEDVE sequence participates in elastin recognition.

20 Discussion ECM adhesins are important for bacterial colonization of and dissemination through host tissues. The identification of the elastin binding site of EbpS is required for understanding the mechanism of S. aur~us adhesion to elastin. Using overlapping 25 EbpS fragments and recombinant constructs the elastin binding site in EbpS was mapped to the amino terminal domain of the molecule. Overlapping synthetic peptides spanning amino acids 14-34 were then used to better define the binding domain. Among these, only peptides corresponding to residues 14-~3 and 18-34 specifically inhibited elastin binding by more than 95%. Common to all active 30 synthetic peptides and proteolytic and recombinant fragments of EbpS is the CA 02247072 l998-08-27 W O 98/38312 PCT~US97/03106 hexameric sequence '8Thr-Asn-Ser-His-Gln-Asp'3. Further evidence that this sequence is important for elastin binding was the loss of activity when Asp23 was substituted with Asn in the synthetic peptide corresponding to residues 18-34.
However, the synthetic hexamer TNSHQD by itself did not inhibit staphylococcal 5 binding to elastin. These findings indicate that although the presence of the TNSHQD
,. sequence is essential for EbpS activity, flanking amino acids in the N- or C-terrninal direction and the carboxyl side chain of Asp'3 are required for elastin recognition.

The minim~l requirements for elastin recognition by EbpS are unexpectedly similar to 10 what has been observed for the interaction between S. aureus and fibronectin.Fibronectin binding to S. aureus is mediated by a surface fibronectin binding protein, and the fibronectin binding site in this adhesin has been mapped to an extracellular 38 amino acid motif repeated three times and partially a fourth time [Signas. et al.. 1989, supra]. A subsequent investigation by McGavin et al. LJ. Biol. Chem., 266:8343-8347 (1991)] has shown that essential amino acids are contained within residues 21-33 of the 38 amino acid motif and that flanking N- and C-terminal amino acids are required for activit~ . The carboxyl side chains of acidic amino acids are also essential. The flanking residues are required to acquire a conformation that is favorable for fibronectin binding.
Similar to the proposed ligand recognition mechanism for the staphylococcal fibronectin binding protein, the TNSHQD synthetic peptide by itself could be inactive because it folds improperly and flanking residues are required to form a secondary structure that is necessary for activity. The N-terminal region of full length EbpS is 25 predicted to fold into amphipathic c~ helices except for regions including residues 14-73 which is where the TNSHQD sequence resides. These predictions indicate that although the TNSHQD sequence defines the elastin contact site, presentation of this region in an active manner requires fl~nking secondary sequences which would stabilize the structure of the elastin binding site in EbpS. This is one explanation for W O 98/38312 PCTnJS97/03106 why the binding afflnities of active EbpS synthetic polypeptides for elastin areconsiderably lower than those of the larger elastin binding EbpS polypeptides.

The properties of staphylococcal elastin and fibronectin recognition mech~ni.sm.~ are S opposite that of corresponding m~mm~ n receptors. ~mmf~ n receptors bind to their respective li~ands through the interaction of structural domains in the receptor and a short contiguous peptide sequence in the ligand. Structural domains forrned by both the oc and F3 integrin subunits. for example, interact with short peptide sequences such as RGD [Pierschbacher, et al., Nature ~Lond.), 309:30-33 (1984)]. LDV [Mould~
10 et al., J. Biol. Chem., 265:4020-4024 (1990)], REDV [Mould, et al., J. Biol. Chem., 266:3579-3585 (1991)], and IDAPS [Mould, e~ al., ~MBO J., 10:4089-4095 (1991)3.
Sirnilarly, the 67 kDa m~mm~lian elastin binding protein recognizes the hydrophobic VGVAPG he:Yapeptide se~uence rHinek, et al., Science, 239: 1539- 1541 (1988)] . In contrast, staphylococcal elastin and fibronectin adhesins interact with their ligands by 15 fitting a small region of the adhesin into a structural binding pocket formed by the ligand. These differences in ligand recognition mech~nism~ can be important in both assuring a lack of binding competition between the staphylococcal and m~mm~ n elastin and fibronectin binding systems, and in promoting efficient binding of ~staphylococci to host tissue components for colonization and disscmination purposes.
The different strategv in receptor-ligand recognition used by bacteria indicates that other elastin binding proteins rnay use a similar approach for interacting with elastin.
Sequence comparisons, however, failed to detect the TNSHQD sequence in several known elastin-binding proteins. including pancreatic and neutrophil elastases 25 rFletcher, et al., Biochemistry, 26:7256-7261 (1987): Shotton, et al., Na-ure, 225:811-816 ~1970); Steffensen, et al., J. Biol. Chem., 270: 11555-11566 (1995)], Iysostaphin ~Park, et al., Int. J. Biochem. Cell Biol., 27: 139- 146 (1995)], microfibril-associated glycoprotein [Bro~-n-Augsburger, et al., ~. Biol. Chenl., 269:28443-28449 (1994)]. or Iysozyme [Park. et c~l., J. Inl~est. Derma~ol., 106: 1075- 1080 (1996)~ . Furthermore.

W O 98/38312 PCT~US97/03106 none of these proteins contained significant homology to the extended EbpS sequence corresponding to residues 14-34.

The amino terminal third of the elastin protein is the sight of EbpS binding. An5 antibody generated against a peptide encoded by exons 9 and 10 of elastin specifically inhibits staphylococcal elastin binding indicating that the EbpS binding site islocalized to this particular region.

Abbreviations as used herein~
EbpS: elastin binding protein of Staphylococcus azlreus, ECM: extracellular matrix, MSCRAMMS: microbial surface components recognizing adhesive matrix molecules.
rEbpS: full length recombinant EbpS, trEbpS: truncated recombinant EbpS. TBS:
Tris-buffered saline. and TSB: tryptic soy broth.
Summary A cell surface 25 kDa elastin binding protein of Staphylococcus allrel~.s (EbpS)mediates binding of this pathogen to elastin. Results from binding assavs examining 20 the activity of fragments of EbpS suggested that residues 1-59 contain the elastin recognition site. Functional analysis of rccombinant truncated forms of ~bpS andsynthetic peptides have been used to localize the elastin binding site to within a 21 amino acid region contained within residues 14-34 ofthe binding protein. Furtherevidence for the importance of this site was obtained by demonstrating that the 25 inhibitory activity of anti-EbpS antibodies on staphylococcal elastin binding were neutralized whcn these antibodies were pre-absorbed with a truncated recombinantEbpS construct containing residues 1-34. Studies testing effects of overlapping synthetic peptides showed that sequences spanning amino acids Gln'J-Asp'; and Thr'g-Glui~ inhibit binding of Staphvlococcus aurezls to elastin. Substitution of Asp~;
30 with Asn abrogated the blocking activity of the peptide, demonstrating the requirement ~or a charged amino acid at this location. The composite data indicate that staphylococcal elastin binding is mediated by a discrete domain defined by short peptide sequences in the N-terrninal e~tracellular region of EbpS

S While the invention has been described and illustrated herein by references to various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art.

The following is a list of documents related to the above disclosure and particularly to the experimental procedures and discussions. The documents should be considered as incorporated by reference in their entirety.

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38. Sjobring, U (199 ), Infecl. Immlln. 60~ 3601-3608 39. Talay, S.R.. Valentin-Weigand, P.. Tirnmis, K.N.~ and Chhatwal~ G.S. (1994),A~lol. Microbiol. 13, 531 -539.

40. Schneewind. O., Model, P.~ and Fischetti, V.A. (1992), Cell 70, 267-281 41. Schneewind, O., Fowler, A., and Faull, K.F. (1995), Science 268, 103-106 W O 98/38312 PCT~US97/03106 42. Courtney, H.S., Li, Y., Dale, J.B., and ~asty, D.L. (1994), Infect. Immun. 62, 3937-3946.

43. LoKenberg~ R., Broder, C.C., Boyle, M.P., Kain, S.J., Schroeder, B.L., and Curtiss III, R. (1992), J. Bacteriol. 174, 5204-5210.

44 Fath, M.J. and Kolter, R. (1993), Microbiol. ~el~., 57, 995-1017.

45. Yother, J., and White, J.M. (1994), J Bacteriol. 176, 2976-2985.

46. McGavin, M.J. Gurusiddappa~ S., Lindgren, P., Lindberg, M.. Raucii, G., and Hook, M. (1993), J. Biol. Chem. 268, 23946-23953.

47. Patti, J.M, House-Pompeo, K., Boles, J.O., Garza, N., Gurusiddappa, S., and Hook, M. (1995), J Biol. Cl~em. 270, 12005-1201 1.

W O 98/38312 PCT~US97/03106 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Mecha~, Robert P.
Park, Pyong W.
(ii) TITLE OF lNv~ ON: BACTERIAL ELASTIN BINDING PROTEIN, NUCLEIC ACID SEQUENCE ENCODING SAME AND DIAGNOSTIC AND
THERAPEUTIC MEHTODS OF USE THEREOF
~iii) NUMBER OF SEQUENCES: 23 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: David A. Jackson, Esq.
(B) STREET: 411 Hackensack Ave, Continental Pla~a, 4th Floor (C) CITY: Hackensack (D) STATE: New Jersey (E) COUNTRY: USA
(F) ZIP: 07601 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
~D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE: 27-FEB-1997 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/609,134 (B) FILING DATE: 29-FEB-1996 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Jackson Esq., David A.
(B) REGISTRATION NUMBER: 26,742 (C) REFERENCE/DOCKET NUMBER: 1113-1-003 CIP
(ix) TELECOMMUNICATICN INFORMATION:
(A) TELEPHONE: 201-487-5800 (B) TELEFAX: 201-343-1684 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 789 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (geno~ic) W O 98/38312 PCT~US97/03106 (ix) EEATURE:
(A) NAME/~EY. CDS
(B) LOCATION: 136. 744 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GAAAGGAAGA TGGCT ATG TCT AAT AAT TTT AAA GAT GAC TTT GAA AAA AAT 171 Met Ser Asn Asn Phe Lys Asp Asp Phe Glu Lys Asn l 5 10 Arg Gln Ser Ile Asp Thr Asn Ser His Gln Asp His Thr Glu Asp-Val Glu Lys Asp Gln Ser Glu Leu Glu His Gln Asp Thr Ile Glu Asn Thr Glu Gln Gln Phe Pro Pro Arg Asn Ala Gln Arg Arg Lys Arg Arg Arg GAT TTA GCA ACG AAT CAT AAT A~A CAA GTT CAC AAT GAA TCA CAA ACA 363 Asp Leu Ala Thr Asn His Asn Lys Gln Val His Asn Glu Ser Gln Thr Ser Glu Asp Asn Val Gln Asn Glu Ala Gly Thr Ile Asp Asp Arg Gln Val Glu Ser Ser His Ser Thr Glu Ser Gln Glu Pro 5er His Gln Asp Ser Thr Pro Gln His Glu Glu Gly Tyr Tyr Asn Lys Asn Ala Phe Ala Met Asp Lys Ser His Pro Glu Pro Ile Glu Asp Asn Asp Lys His Glu 125 130 135 . 140 Thr Ile Lys Glu Ala Glu Asn Asn Thr Glu His Ser Thr Val Ser Asp Lys Ser Glu Ala Glu Gln Ser Gln Gln Pro Lys Pro Tyr Phe Ala Thr Gly Ala Asn Gln Ala Asn Thr Ser Lys Asp Lys His Asp Asp Val Thr Val Lys Gln Asp Lys Asp Glu Ser Lys Asp His His Lys Trp AAAAGGCG Q G QATTGGTG CTGGAA QGC GG~l~llGCA GGTGC 789 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 202 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Asn Asn Phe Lys Asp A5p Phe Glu Lys Asn Arg Gln Ser Ile ~sp Thr Asn Ser His Gln Asp His Thr Glu Asp Val Glu Lys Asp Gln Ser Glu Leu Glu His Gln Asp Thr Ile Glu Asn Thr Glu Gln Gln Phe Pro Pro Arg Asn Ala Gln Arg Arg Lys Arg Arg Arg Asp Leu Ala Thr Asn His Asn Lys Gln Val His Asn Glu Ser Gln Thr Ser Glu Asp Asn ~al Gln Asn Glu Ala Gly Thr Ile Asp Asp Arg Gln Val Glu Ser Ser ~is Ser Thr Glu Ser Gln Glu Pro Ser His Gln Asp Ser Thr Pro Gln His Glu Glu Gly Tyr Tyr Asn Lys Asn Ala Phe Ala Met Asp Lys Ser His Pro Glu Pro Ile Glu Asp Asn Asp Lys His Glu Thr Ile Lys Glu Ala Glu Asn Asn Thr Glu His Ser Thr Val Ser Asp Lys Ser Glu Ala Glu Gln Ser Gln Gln Pro Lys Pro Tyr Phe Ala Thr Gly Ala Asn Gln Ala Asn Thr Ser Lys Asp Lys His Asp Asp Val Thr Val Lys Gln Asp Lys Asp Glu Ser Lys Asp His His Lys Trp -W O 98/38312 PCT~US97/03106 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:

WO 98/38312 PCT~US97/03106 (A) LENGTH: 13 amino acids tB) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ala Asn Asn Phe Lys Asp Asp Phe Glu Lys Asn Arg Gln l 5 lO
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Leu Pro Xaa Thr Gly Xaa l 5 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Z34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear CA 02247072 l998-08-27 W O 98/38312 PCT~US97/03106 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATGTCTAATA ATTTTAAAGA TGACTTTGAA A~AAATCGTC AATCGATAGA CACAAATTCA 60 CATCAAGACC ATACGGAAGA TGTTGA~AAA GACCAATCAG AATTAGAACA TCAGGATACA 120 ATAGAGAATA CGGAGCAACA GTTTCCGCCA AGA~ATGCCC A~AGAAGAAA AAGACGCCGT 180 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Ser ~sn Asn Phe Lys Asp Asp Phe Glu Lys Asn Arg Gln Ser Ile Asp Thr Asn Ser His Gln Asp His Thr Glu Asp Val Glu Lys Asp Gln Ser Glu Leu Glu His Gln Asp Thr Ile Glu Asn Thr Glu Gln Gln Phe Pro Pro Arg Asn Ala Gln Arg Arg Lys Arg Arg Arg Asp Leu Ala Thr Asn His Asn Lys Gln Val His Asn Glu Ser Gln Thr Ser Glu (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 base pairs CA 02247072 l998-08-27 WO 98/38312 PCT~US97/03l06 (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Ser Asn Asn Phe Lys Asp Asp Phe Glu Lys Asn Arg Gln Ser Ile Asp Thr Asn Ser His Gln Asp His Thr Glu Asp Val Glu Lys Asp Gln Ser Glu (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide CA 02247072 l998-08-27 WO 98/38312 PCT~US97/03106 (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Gln Ser Ile Asp Thr Asn Ser His Gln Asp His Thr Glu Asp Val Glu 1 5 lO 15 Lys Asp Gln Ser Glu (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Gln Ser Ile Asp Thr Asn Ser His Gln Asp l 5 lO
(2) INFORMATION FOR SEQ ID NO :15:
(i) SEQUENC~ CHARACTERISTICS:
(A) LENGTH: 46 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULr TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO :15:
Gln Ser Ile Asp Thr Asn Ser His Gln Asp His Thr Glu Asp Val Glu W O 98/38312 PCTrUS97/03106 1 5 lO 15 Lys Asp Gln Ser Glu Leu Glu His Gln Asp Thr Ile Glu Asn Thr Glu Gln Gln Phe Pro Pro Arg Asn Ala Gln Arg Arg Lys Arg Arg (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Thr Asn Ser His Gln Asp (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Thr Asn Ser His Gln Ser (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid W O 98/38312 PCT~US97/03106 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Thr Asn Ser His Gln Asp His Thr Glu Asp Val Glu Lys Asp Gln Ser Glu (2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Thr Asn Ser His Gln Asn His Thr Pro Asp Val Gln Lys Asp Gln Ser l 5 10 15 Glu (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

W O 98/38312 PCT~US97/03106 (v) FRAGMENT TYPE: ;nt~rn~l (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
His Gln Asp His Thr Glu Asp Val Glu l 5 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
His Gln Asp His Thr Glu Asp Val Glu Lys l 5 10 (2) INFORMATION FOP~ SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 0 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Asp Val Glu Lys Asp Gln Ser Glu Leu Glu l 5 10 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
-WO 98/38312 PcTnus97/o3lo6 (A) LENGT~: 9 amino acids (B) TYPE: amino acid (C) STR~M~N~.~S: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
His Gln Asp Thr Ile Glu Asn Thr Glu

Claims (64)

WHAT IS CLAIMED IS:

1 . A microbial surface component recognizing adhesive matrix molecules (MSCRAMM), comprising a material selected from the group consisting of a protein, active fragments thereof, agonists thereof, mimics thereof, and combinations thereof, said MSCRAMM having the following characteristics:
a) binding to elastin;
b) inhibited activity in the presence of SDS, and c) enhanced activity in the presence of thiol reductants.

2. The MSCRAMM of Claim 1 which is a member of the family of elastin binding proteins.

3. The MSCRAMM of Claim 1 which is bacterial in origin.

4. The MSCRAMM of Claim 1 which is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2, and active fragmentsthereof.

5. The MSCRAMM of Claim 1 which is derived from mammalian cells.

6. The MSCRAMM of Claim 1 labeled with a detectable label.

7. The MSCRAMM of Claim 6 wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.

8. An antibody to a MSCRAMM, the MSCRAMM to which said antibody is raised having the following characteristics:
a) binding to elastin;
b) inhibited activity in the presence of SDS; and c) enhanced activity in the presence of thiol reductants.

9. The antibody of Claim 8 which is selected from the group consisting of polyclonal antibodies, monoclonal antibodies and chimeric antibodies

10. The antibody of Claim 8 which is a monoclonal antibody.

11. An immortal cell line that produces a monoclonal antibody according to Claim10.

12. The antibody of Claim 8 labeled with a detectable label.

13. The antibody of Claim 12 wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.

14. A DNA sequence or degenerate variant thereof, which encodes a MSCRAMM~ or a fragment thereof, selected from the group consisting of:
(A) the DNA sequence of SEQ ID NO: 1;
(B) DNA sequences that hybridize to the foregoing DNA sequence under standard hybridization conditions; and (C) DNA sequences that code on expression for an amino acid sequence encoded by the foregoing DNA sequence.

15. A recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes a MSCRAMM, or a fragment thereof, selected from the group consisting of:
(A) the DNA sequence of SEQ ID NO: 1;
(B) DNA sequences that hybridize to the foregoing DNA sequence under standard hybridization conditions; and (C) DNA sequences that code on expression for an amino acid sequence encoded by the foregoing DNA sequence.

16. The recombinant DNA molecule of either of Claims 14 or 15, wherein said DNA sequence is operatively linked to an expression control sequence.

17. The recombinant DNA molecule of Claim 16, wherein said expression control sequence is selected from the group consisting of the early or late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage .lambda., the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase and the promoters of the yeast .alpha.-mating factors.

18. A probe capable of screening for the MSCRAMM in alternate species prepared from the DNA sequence of Claim 14, or from SEQ ID NO:3.

19. A unicellular host transformed with a recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes a MSCRAMM, or a fragment thereof, selected from the group consisting of:
(A) the DNA sequence of SEQ ID NO: 1;
(B) DNA sequences that hybridize to the foregoing DNA sequence under standard hybridization conditions; and (C) DNA sequences that code on expression for an amino acid sequence encoded by the foregoing DNA sequence;
wherein said DNA sequence is operatively linked to an expression control sequence.

20. The unicellular host of Claim 19 wherein the unicellular host is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeasts, CHO, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

21. A method for detecting the presence or activity of a MSCRAMM, said MSCRAMM having the following characteristics: binding to elastin: inhibited activity in the presence of SDS; and enhanced activity in the presence of thiol reductants, wherein said MSCRAMM is measured by:
A. contacting a biological sample from a mammal in which the presence or activity of said MSCRAMM is suspected with a binding partner of saidMSCRAMM under conditions that allow binding of said MSCRAMM to said binding partner to occur; and B. detecting whether binding has occurred between said MSCRAMM from said sample and the binding partner;
wherein the detection of binding indicates that presence or activity of said MSCRAMM in said sample.

22. A method for detecting the presence and activity of a polypeptide ligand associated with a given invasive stimulus in mammals comprising detecting the presence or activity of a MSCRAMM according to the method of Claim 21. wherein detection of the presence or activity of the MSCRAMM indicates the presence and activity of a polypeptide ligand associated with a given invasive stimulus in mammals.

23. The method of Claim 22 wherein said invasive stimulus is an infection.

24. The method of Claim 23 wherein said infection is bacterial in origin.

25. A method for detecting the binding sites for a MSCRAMM, said MSCRAMM
having the following characteristics:
A) binding to elastin;

B) inhibited activity in the presence of SDS; and C) enhanced activity in the presence of thiol reductants; said method comprising:
i. placing a labeled MSCRAMM sample in contact with a biological sample from a mammal in which binding sites for said MSCRAMM are suspected;
ii. examining said biological sample in binding studies for the presence of said labeled MSCRAMM;
wherein the presence of said labeled MSCRAMM indicates a binding site for a MSCRAMM.

26. A method of testing the ability of a drug or other entity to modulate the activity of a MSCRAMM which comprises A. culturing a colony of test cells which has a receptor for the MSCRAMM in a growth medium containing the MSCRAMM;
B. adding the drug under test; and C. measuring the reactivity of said MSCRAMM with the receptor on said colony of test cells.
wherein said MSCRAMM has the following characteristics:
i) binding to elastin;
ii) inhibited activity in the presence of SDS; and iii) enhanced activity in the presence of thiol reductants.

27. An assay system for screening drugs and other agents for ability to modulatethe production of a MSCRAMM, comprising:
A. culturing an observable cellular test colony inoculated with a drug or agent;
B. harvesting a supernatant from said cellular test colony; and C. examining said supernatant for the presence of said MSCRAMM
wherein an increase or a decrease in a level of said MSCRAMM indicates the ability of a drug to modulate the activity of said MSCRAMM, said MSCRAMM having the following characteristics:
i) binding to elastin;
ii) inhibited activity in the presence of SDS; and iii) enhanced activity in the presence of thiol reductants.

28 A test kit for the demonstration of a MSCRAMM in a eukaryotic cellular sample, comprising:
A. a predetermined amount of a detectably labelled specific binding partner of a MSCRAMM, said MSCRAMM having the following characteristics:
binding to elastin; inhibited activity in the presence of SDS; and enhanced activity in the presence of thiol reductants;
B. other reagents; and C. directions for use of said kit.

29. A test kit for demonstrating the presence of a MSCRAMM in a eukaryotic cellular sample, comprising:
A. a predetermined amount of a MSCRAMM, said MSCRAMM
having the following characteristics: binding to elastin; inhibited activity in the presence of SDS; and enhanced activity in the presence of thiol reductants;
B. a predetermined amount of a specific binding partner of said MSCRAMM;
C. other reagents; and D. directions for use of said kit;
wherein either said MSCRAMM or said specific binding partner are detectably labelled.

30. The test kit of Claim 28 or 29 wherein said labeled immunochemically reactive component is selected from the group consisting of polyclonal antibodies to the MSCRAMM, monoclonal antibodies to the MSCRAMM, fragments thereof, and mixtures thereof.

31. A method of preventing and/or treating cellular debilitations, derangements and/or dysfunctions and/or other disease states in mammals, comprising administering to a mammal a therapeutically effective amount of a material selected from the group consisting of a MSCRAMM, an agent capable of promoting the production and/or activity of said MSCRAMM, an agent capable of mimicking the activity of said MSCRAMM, an agent capable of inhibiting the production of said MSCRAMM, and mixtures thereof, or a specific binding partner thereto, said MSCRAMM having thefollowing characteristics:
a) binding to elastin;
b) inhibited activity in the presence of SDS; and c) enhanced activity in the presence of thiol reductants.

32. The method of Claim 31 wherein said disease states include those caused at least in part by bacterial infection.

33. The method of Claim 31 wherein said MSCRAMM is administered to modulate the course of therapy where MSCRAMM is being administered as the primary therapeutic agent.

34. The method of Claim 31 wherein said MSCRAMM is administered to modulate the course of therapy where MSCRAMM is being co-administered with one or more additional therapeutic agents.

35. A pharmaceutical composition for the treatment of cellular debilitation, derangement and/or dysfunction in mammals, comprising:
A. a therapeutically effective amount of a material selected from the group consisting of a MSCRAMM, an agent capable of promoting the production and/or activity of said MSCRAMM, an agent capable of mimicking the activity of said MSCRAMM, an agent capable of inhibiting the production of said MSCRAMM, and mixtures thereof, or a specific binding partner thereto, said MSCRAMM havingthe following characteristics: binding to elastin; inhibited activity in the presence of SDS; and enhanced activity in the presence of thiol reductants; and B. a pharmaceutically acceptable carrier.

36. The MSCRAMM of Claim 17 wherein said MSCRAMM has a predicted molecular weight of about 25 KD, and a predicted PI of about 4.9.

37. The MSCRAMM of Claim 3, wherein said MSCRAMM is isolated from Staphylococcus aureus.

38. A polypeptide comprising an amino acid sequence that corresponds to the elastin binding site of a microbial surface component recognizing adhesive matrix molecules (MSCRAMM), wherein said polypeptide consists of between 8 and 80 amino acids, and binds elastin.

39. The polypeptide of Claim 38, wherein the MSCRAMM has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:2 comprising a conservative substitution thereof.

40. The polypeptide of Claim 38 having the amino acid sequence consisting of theN-terminal 59 amino acids of the MSCRAMM.

41. The polypeptide of Claim 38 that further inhibits the binding of S. aureus to elastin.

42. A pharmaceutical composition comprising the polypeptide of Claim 41 and a pharmaceutically acceptable carrier.

43. A method of treating a Staphylococcus aureus infection by administering a therapeutically effective amount of the pharmaceutical composition of Claim 42.

44. The polypeptide of Claim 38 in which said elastin binding site comprises theamino acid sequence of SEQ ID NO:16 or SEQ ID NO:16 comprising a conservative substitution thereof.

45. The polypeptide of Claim 44 that has the amino acid sequence of SEQ ID
NO: 10 or SEQ ID NO: 10 comprising a conservative substitution thereof.

46. The polypeptide of Claim 44 that contains between 10 and 46 amino acids.

47. The polypeptide of Claim 46 comprising the amino acid sequence of SEQ ID
NO:14 or SEQ ID NO:14 comprising a conservative substitution thereof.

48. The polypeptide of Claim 46 that contains about 10 amino acids.

49. The polypeptide of Claim 46 comprising the amino acid sequence of SEQ ID
NO: 18 or SEQ ID NO: 18 comprising a conservative substitution thereof.

50. The polypeptide of Claim 49 that has the amino acid sequence of SEQ ID
NO:12 or SEQ ID NO:12 comprising a conservative substitution thereof.

51. An immunogenic composition comprising the polypeptide of Claim 38 in an admixture with an adjuvant.

52. The immunogenic composition of Claim 51 wherein the polypeptide is conjugated to a carrier molecule.

53. A method for generating an antibody to the elastin-binding domain of a MSCRAMM comprising immunizing an animal with the composition of Claim 51.

54. The antibody of Claim 53 which is selected from the group consisting of a polyclonal antibody and a monoclonal antibody.

55. A nucleic acid encoding a polypeptide that is between 8 and 80 amino acids, wherein said polypeptide binds elastin and comprises an amino acid sequence thatcorresponds to the elastin binding site of a microbial surface component recognizing adhesive matrix molecules (MSCRAMM).

56. The nucleic acid of Claim 55 having the nucleic acid sequence of SEQ ID
NO:9.

57. The nucleic acid of Claim 55 which is DNA.

58. A cloning vector which comprises the DNA of Claim 57.

59. An expression vector which comprises the DNA of Claim 57 operatively associated with an expression control sequence.

60. A unicellular host transfected or transformed with the expression vector of Claim 59.

61. The unicellular host of Claim 60 that is a bacterium.

62. A mammalian cell transfected or transformed with the expression vector of Claim 59.

63. A method of expressing the polypeptide of Claim 38 comprising culturing a cell containing an expression vector comprising a nucleic acid encoding the polypeptide under conditions that allow the polypeptide to be expressed.

64. The method of Claim 63 further comprising the step of purifying the expressed polypeptide.