Crystal structure of <i>Staphylococcus aureus </i>clumping factor A in complex with fibrinogen derived peptide and uses thereof

Abstract

The present invention discloses crystal structure of Staphylococcus aureus Clumping factor A (ClfA) in complex with fibrinogen (Fg) derived peptide. Also, the present invention also discloses the use of this structure in the design of ClfA targeted vaccines and therapeutic agents (including monoclonal antibodies). In addition, the present invention discloses isolated and purified engineered Staphylococcus clumping factor A protein (ClfA) with a stabilized, closed conformation and immunogenic compositions thereof including methods of treating a Staphylococcus infection in an individual.

Claims

1. A therapeutic agent comprising a binding agent that inhibits the binding of residues 521 to 529 of a clumping factor A protein (ClfA) of SEQ ID NO:35 to a gamma chain of a fibrinogen wherein the binding agent is a peptide selected from the group consisting of g.sup.1-17.sub.D16A (SEQ ID NO: 27), g.sup.1-17.sub.K12A (SEQ ID NO: 24), g.sup.1-17.sub.A11S (SEQ ID NO: 23) and g.sup.1-17.sub.V17A (SEQ ID NO: 31).

2. A therapeutic agent that blocks the interaction of microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) with fibrinogen comprising: a peptide consisting of residues 521-529 of ClfA (SEQ ID NO: 32), wherein the therapeutic agent reduces MSCRAMMs interactions with a gamma chain of a fibrinogen.

3. The therapeutic agent of claim 1, wherein the peptide is g.sup.1-17.sub.D16A (SEQ ID NO: 27).

4. The therapeutic agent of claim 1, wherein the peptide is g.sup.1-17.sub.K12A (SEQ ID NO: 24).

5. The therapeutic agent of claim 1, wherein the peptide is g.sup.1-17.sub.A11S (SEQ ID NO: 23).

6. The therapeutic agent of claim 1, wherein the peptide is g.sup.1-17.sub.V17A(SEQ ID NO: 31).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The appended drawings have been included herein so that the above-recited features, advantages and objects of the invention will become clear and can be understood in detail. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.

(2) FIGS. 1A-1D demonstrate that ClfA.sub.229-545 of SEQ ID NO:35 binds to Fg gamma (Fg γ) chain peptides. FIG. 1A shows a panel of Fg γ-chain peptides. The wild-type peptide corresponds to the 17 C-terminal residues of the Fg g-chain (395-411); the mutated peptides have individual amino acids replaced with Ala (or Ser). FIG. 1B shows that Fg γ peptides inhibit ClfA binding to immobilized GST-Fg γ in solid phase assays. Wells were coated with 1 mg GST-Fg γ peptide. ClfA.sub.229-545 (100 nM) was pre-incubated with wild-type Fg γ peptide (WT g.sup.1-17) or the P1 (G1A) to P17 (V17A) mutant peptide (50 mM) for 1 hr. FIG. 1C shows the binding of ClfA to immobilized GST-Fg γ and GST-Fg γ P16 using a solid-phase assay. Increasing concentrations of rClfA.sub.229-545 were incubated in microtiter wells containing 1 mg GST (circles), GST-Fg γ (triangles) or GST-Fg g P16 (squares). Bound ClfA was detected with anti-His monoclonal antibodies as described. FIG. 1D shows the binding of ClfA.sub.229-545 to Fg γ and Fg γ P16 peptides in solution using ITC.

(3) FIGS. 2A-2D illustrate Fg and Fg γ P16 peptide truncations binding to different forms of ClfA. FIG. 2A shows a panel of Fg γ P16 peptides with N- and C-terminal truncations. FIG. 2B shows N-terminal deletions of Fg γ P16 peptide bind ClfA.sub.229-545 with decreasing affinities. N- and C-terminal truncated Fg γ P16 peptides were tested for their ability to bind ClfA.sub.229-545 in solution using ITC. FIG. 2C shows a stable closed conformation ClfA.sub.229-545 was engineered by introducing a disulfide bridge. The left panel shows a ligand blot of rClfA.sub.D327C/K541C. Recombinant proteins were run in an SDS-PAGE in the presence or absence of 5 mM DTT and stained with Coomassie Blue (left panel) or transferred to a PDVF membrane (middle panel). Transferred proteins were probed with Fg (10 mg/ml) and detected with anti-Fg and AP-conjugated secondary antibodies. (Right panel) The purified closed form of ClfA.sub.327C/541C used for crystallization and ClfA.sub.229-545 were run in an SDS-PAGE and stained with Coomassie Blue (right panel). FIG. 2D shows the closed conformation of ClfA.sub.D327C/K541C binds immobilized Fg and GST-Fg γ P16. ClfA.sub.229-545 or ClfA.sub.D327C/K541C was incubated with wells coated with either Fg or GST-Fg γ P16 and detected with anti-His monoclonal antibodies as described below.

(4) FIGS. 3A-3D are a representation of ClfA.sub.D327C/K541C (N2-N3)-peptide complex. FIG. 3A is the ribbon representation of ClfA-peptide (Fg γ-chain analog) complex. The peptide is shown as ball and stick model. 2Fo-Fc map around the peptide contoured at 1.sigma. is shown in the close-up view. FIG. 3B is a stereo view of the superposition of the two complexes (A:C and B:D) in the asymmetric unit. FIG. 3C is a schematic representation of ClfA-Fg γ-peptide main-chain parallel β-complementation interaction. The anti-parallel β-complementation observed in SdrG.sub.273-597-Fg β-peptide complex is also shown for comparison. The residue numbers of both the Fg γ-chain sequence and the peptide numbering (1-17), in parenthesis, are shown. FIG. 3D is a stereo-view showing the side-chain interactions of the ClfA-Fg γ-peptide complex. Carbon atoms of the peptide are shown in grey; oxygen, red; nitrogen, blue. Side chain atoms of ClfA are shown as pink stick objects. Hydrogen bonds are shown as dotted lines.

(5) FIGS. 4A-4C illustrate the superposition of apo-ClfA, ClfA-peptide and SdrG-peptide structures. FIG. 4A shows the superposition of apo-ClfA.sub.221-559, ClfA.sub.D327C/K541C-peptide complex. The N3 domains of the two structures are superposed showing significant deviation in the inter-domain orientations. Apo-ClfA is shown as a cyan ribbon object and ClfA-peptide complex is shown in green. In FIG. 4B only N3 domain of apo-ClfA (cyan) is shown for clarity. The folded-back residues of the C-terminal residues of the apo-ClfA are shown in red. The Fg γ-chain peptide is shown as blue ribbon. FIG. 4C shows the superposition of ClfA-peptide and SdrG-peptide complexes. The peptide molecules corresponding to ClfA and SdrG complexes are shown as red and blue ribbon objects respectively. ClfA is colored by secondary structure and SdrG is shown as thin yellow uniform coil.

(6) FIGS. 5A-5C illustrate species specificity of ClfA-Fg binding. FIG. 5A shows that the closed conformation rClfA.sub.327C/541C binds immobilized Fg from different animal species with different apparent affinities in a solid-phase assay. FIG. 5B shows that the ClfA.sub.D327C/K541C binds human Fg γ P16 peptide with a higher affinity than bovine Fg γ peptide using ITC. FIG. 5C shows that the sequence comparison of human and bovine Fg γ-chain C-terminal residues (top). CPK representation of the binding pocket formed between the N2 and N3 domains bound to human versus bovine Fg γ peptide. ClfA is shown as grey CPK object and peptide atoms are shown in black (bottom).

(7) FIG. 6 illustrates that the γ.sup.1-17.sub.D16A and γ.sup.1-17.sub.K12A peptides bind weakly to platelet integrin α.sub.IIbβ.sub.3. Inhibition of Fg γ peptides (γ.sup.1-17.sub.D16A and γ.sup.1-17.sub.K12A and γ.sup.1-17; WT) on binding of full length Fg immobilized onto α.sub.IIbβ.sub.3. Wild-type Fg-γ.sup.1-17 peptide (square) inhibits Fg binding to α.sub.IIbβ.sub.3 whereas γ.sup.1-17.sub.D16A (triangle) and γ.sup.1-17.sub.K12A (inverted triangle) peptides have very little inhibitory effect.

(8) FIGS. 7A-7B illustrate FnbpA binding to GST-Fg γ chain peptides. FIG. 7A shows that Fg γ peptides inhibit FnbpA.sub.194-511 binding to immobilized GST-Fg γ. Wells were coated with 1 mg GST-Fg γ peptide. FnbpA.sub.194-511 (400 nM) was pre-incubated with wild-type Fg γ peptide (WT γ.sup.1-17) or the P1 (G1A) to P17 (V17A) mutant peptide (50 mM) for 1 hr. FIG. 7B is the ribbon representation of FnbpA.sub.194-511:Fg-γ-chain peptide binding model. N2 and N3 domains in FnbpA are shown as ribbons and peptide is shown as stick object.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

(9) The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

(10) The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”

(11) As used herein, the term “immunologically effective amount” refers to an amount that results in an improvement or remediation of the symptoms of the disease or condition due to induction of an immune response. Those of skill in the art understand that the effective amount may improve the patient's or subject's condition, but may not be a complete cure of the disease and/or condition.

(12) As used herein, “active immunization” is defined as the administration of a vaccine to stimulate the host immune system to develop immunity against a specific pathogen or toxin.

(13) As used herein, “passive immunization” is defined as the administration of antibodies to a host to provide immunity against a specific pathogen or toxin.

(14) As used herein, “CpG oligonucleotides” are defined by the presence of an unmethylated CG dinucleotide in a CpG motif.

(15) As used herein, “adjuvant” is defined as a substance which when included in a vaccine formulation non-specifically enhances the immune response to an antigen.

II. Present Invention

(16) In one embodiment of the present invention there is provided a therapeutic agent that binds Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMM) with higher binding affinity than native fibrinogen (Fg), the agent comprising amino acid sequence that differs from amino acid sequence of a native Fibrinogen in at least one amino acid residue. Examples of such an agent may include but is not limited to a peptide, a fusion protein, a small molecule inhibitor or a small molecule drug. Examples of the peptide may include but is not limited to a P16 peptide (Asp16.fwdarw.Ala), a P12 peptide (Lys12.fwdarw.Ala) or combination thereof. Further, examples of MSCRAMM may include but is not limited to a clumping factor A (ClfA), FnbpA, FnbpB or Fbl and the MSCRAMM may include but is not limited to those present on the surface of Staphylococcus aureus, Staphylococcus lugdunensis, or Staphylococcus epidermis.

(17) As used herein, “clumping factor A (ClfA) is identified with www.uniprot.ori/uniprot/Q53653, ENA Accession No. BAF67028.1, each of which is incorporated herein by reference in its entirety, which is a Staphylococcal clumping factor A, which is a cell surface-associated protein associated with virulence. ClfA is known to promote bacterial binding to the gamma-chain of human fibrinogen and induces formation of protective fibrinogen shield.

(18) As used herein, the term “homology” or “homolog” refers to the extent to which two amino acid sequences are identical. There may be partial homology (i.e., similarity) or complete homology (i.e., identity). An amino acid sequence that is “sequence homolog” to another amino acid sequence (e.g., the amino acid of SEQ ID NO:35) is defined herein as an amino sequence that exhibits greater than or equal to 70% identity to the second amino acid sequence (e.g., the amino acid sequence of SEQ ID NO:35), when a length of 8-20 (or more) amino acids are compared. An amino acid sequence that is “functional homolog” to another amino acid sequence (e.g. the amino acid of SEQ ID NO:35) is defined as an amino acid sequence that generates a peptide or protein that binds the same C-terminus of gamma chain of human fibrinogen as the ClfA. The skilled artisan will understand that certain amino acids may have conservative substitutions, which are substitutions where amino acids having similar side groups are substituted so-long as the activity of the protein remains the same. For example, conservative amino acid substitutions include amino acids within the native sequence selected from other members of a class to which the naturally occurring amino acid belongs. Representative amino acids within these various classes include, but are not limited to: hydrophobic side chains is alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, and tryptophan; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Other categories include, e.g., (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conserved substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs.

(19) In one non-limiting example, variations of the Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) include those proteins that are homologous in activity to ClfA (i.e., functional homologs), which have as an activity binding to the gamma chain of fibrinogen, as demonstrated herein at the molecular level via crystallography at a resolution of at least 2 angstroms. As such, a homology that is “a sequence homology” of ClfA is a peptide or protein that has greater than 70, 80, 90, 95, 98, or 99% identical amino acid sequence with ClfA, specifically, in the region that binds to the gamma chain of fibrinogen. As used herein, the term “functional homolog” refers to a peptide or protein that functions in a manner that is substantially homologous or identical to ClfA, e.g., by competing for and/or inhibit the binding of ClfA to the gamma chain of fibrinogen. Non-limiting examples of functional homologs include proteins from other bacteria or pathogens that bind to the gamma chain of fibrinogen, synthetic peptides that specifically bind to the gamma chain of fibrinogen, small molecules that bind to the gamma chain of fibrinogen, or antibodies or fragments thereof that also bind at the same binding site of gamma chain of fibrinogen as ClfA.

(20) Homologs of the proteins of the present invention can be identified by comparison of the amino acid sequence of the protein to amino acid sequences of proteins from the same or different bacteria, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. Thus, a homolog can be a protein from the same or a different organism that performs the same biological function as the polypeptide to which it is compared. An orthologous relation between two organisms is not necessarily manifest as a one-to-one correspondence between two genes, because a gene can be duplicated or deleted after organism phylogenetic separation, such as speciation. For a given protein, there may be no ortholog or more than one ortholog. Other complicating factors include alternatively spliced transcripts from the same gene, limited gene identification, redundant copies of the same gene with different sequence lengths or corrected sequence. A local sequence alignment program, e.g., BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal BLAST search is used in the present invention to filter hit sequences with significant E-values for ortholog identification. The reciprocal BLAST entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal BLAST's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation.

(21) In another embodiment of the present invention there is provided a method for inhibiting Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM):Fibrinogen (Fg) interaction, comprising: contacting an MSCRAMM with the above-described therapeutic agent, thereby inhibiting the MSCRAMM:Fibrinogen interaction. The therapeutic agent may not affect α.sub.IIbβ.sub.3 intergrin interaction.

(22) In yet another embodiment of the present invention there is provided a pharmaceutical composition, comprising: the above-described therapeutic agent and a pharmaceutically acceptable carrier.

(23) In yet another embodiment of the present invention there is provided a method of treating and/or preventing bacterial infection caused or due at least in part to a MSCRAMM: fibrinogen interaction in an individual, comprising: administering pharmacologically effective amounts of the pharmaceutical composition described supra such that administration of the composition inhibits binding of MSCRAMM to native fibrinogen and does not affect α.sub.IIbβ.sub.3 intergrin interaction. Examples of the bacteria may include but is not limited to Staphylococcus aureus, Staphylococcus lugdunensis, or Staphylococcus epidermis. Further, the routes of administration of the pharmaceutical composition may include but is not limited to subcutaneous, intravenous, intramuscular, intra nasal, vaginal, or oral routes. One of ordinary skill in the art is readily able to determine a suitable dosage. Additionally, the individual who may benefit from such a method may include but is not limited to one who is a healthy individual, an individual diagnosed with the bacterial infection, at risk of developing bacterial infection or suspected of suffering from the bacterial infection.

(24) In yet another embodiment of the present invention there is provided an anti-MSCRAMM:fibrinogen antibody effective to inhibit an MSCRAMM:fibrinogen interaction but not affecting binding of other proteins to fibrinogen. Such an antibody may be generated using peptides comprising MSCRAMM binding region on fibrinogen, the peptide differing from the native Fibrinogen in at least one amino acid residue. Examples of the peptide may include but is not limited to a P16 peptide (Asp16.fwdarw.Ala), a P12 peptide (Lys12.fwdarw.Ala) or combination thereof. Alternatively, the antibody may be generated using peptides derived from fibrinogen binding region of MSCRAMM, the peptide differing from the native MSCRAMM in at least one amino acid residue. Further, the antibody may be a monoclonal antibody, a polyclonal antibody or a chimeric antibody. Furthermore, the MSCRAMM may be present on Staphylococcus aureus, Staphylococcus lugdunensis, or Staphylococcus epidermis.

(25) In yet another embodiment of the present invention there is provided a method of treating a bacterial infection in an individual, comprising: administering immunologically effective amounts of the above-described anti-MSCRAMM:fibrinogen antibody to the individual, thereby treating the bacterial infection in the individual. Such an antibody may inhibits interaction between MSCRAMM and Fibrinogen may not affect the α.sub.IIbβ.sub.3 intergrin interaction. Examples of the individual who may benefit from this method may include but is not limited to one who is diagnosed with the infection, is at risk of developing the infection or is suspected of suffering from the infection. One of ordinary skill in the art is readily able to determine a suitable dosage. Further, examples of the routes of administration of the antibody may include subcutaneous, intramuscular, intravenous, intranasal, vaginal, oral, or other mucosal routes.

(26) In yet another embodiment of the present invention there is provided a method for determining structure of MSCRAMM in complex with fibrinogen, comprising: determining amino acid residue in the MSCRAMM binding region of native fibrinogen that is critical for the MSCRAMM:fibrinogen interaction; determining amino acid residue of the MSCRAMM that binds to the MSCRAMM binding region of native fibrinogen; and performing computational modeling of the MSCRAMM sequence that binds to the MSCRAMM binding region of native fibrinogen, thereby determining the structure of the MSCRAMM in complex with the fibrinogen. This method may further comprise identifying potential agents that inhibit MSCRAMM:fibrinogen interaction without affecting binding of other proteins to fibrinogen. Such a potential agent may include one that comprises amino acid sequence of MSCRAMM binding region on fibrinogen, the amino acid sequence differing from the fibrinogen in at least one amino acid residue or an amino acid sequence of fibrinogen binding region of MSCRAMM, the amino acid sequence differing from the MSCRAMM in at least one amino acid residue.

(27) Additionally, the amino acid residue in the MSCRAMM binding region of native fibrinogen may be determined by: synthesizing control peptides that comprise the native fibrinogen sequence that binds MSCRAMM; synthesizing substituted peptides that differ from the control peptide in one or more amino acid residues; and comparing binding of MSCRAMM to native fibrinogen in presence of control peptide or in presence of substituted peptide, where less potent inhibition of MSCRAMM binding to native fibrinogen in presence of substituted peptide compared to control peptide indicates that the amino acid residue(s) that were substituted are less important for the MSCRAMM:fibrinogen interaction, where extensive inhibition of MSCRAMM binding to native fibrinogen in presence of substituted peptide compared to control peptide indicates that the amino acid residue(s) that were substituted are critical for the MSCRAMM:fibrinogen interaction.

(28) Further, the amino acid residue in the MSCRAMM may be determined by comparing the stability of a native MSCRAMM:fibrinogen complex with the stability of a mutated MSCRAMM:fibrinogen complex, where said fibrinogen in the complex comprises peptide derived from MSCRAMM binding region of native fibrinogen. Examples of the MSCRAMM may include but is not limited to a clumping factor A (ClfA), FnbpA, FnbpB or Fbl. Further, the MSCRAMM may include but is not limited to one that is present on the surface of Staphylococcus aureus, Staphylococcus lugdunensis, or Staphylococcus epidermis.

(29) In yet another embodiment of the present invention there is provided a crystal structure of a Staphylococcus clumping factor A protein (ClfA):fibrinogen derived peptide complex that diffracts x-rays for determining atomic coordinates of the complex with a resolution of about 1.95 angstroms. The Staphylococcus may be those species described supra. In the crystal structure the fibrinogen derived peptide may be a P16 peptide (Asp16.fwdarw.Ala) or a P12 peptide (Lys12.fwdarw.Ala) having an N-terminal truncation−2Nt, −4Nt or −6Nt. Particularly, the crystal structure may be a ClfA/P16-4Nt complex.

(30) In yet another embodiment of the present invention there is provided an isolated and purified engineered Staphylococcus clumping factor A protein (ClfA) with a stabilized, closed conformation. For example, the ClfA protein may be ClfA.sub.D327C/K541C protein.

(31) In a related embodiment there is provided an immunogenic composition comprising the ClfA protein described supra and an immunologically acceptable adjuvant or diluent. The immunogenic composition may comprise a vaccine.

(32) In another related embodiment there is provided a method of vaccinating an individual against a Staphylococcus infection comprising administering an immunologically effective amount of the immunogenic composition described supra to the individual.

(33) The general purpose of the present invention is to provide a detailed structural characterization of how ClfA binds Fg and subsequently use this structural information in the design of vaccines and therapeutic compounds for the prevention and treatment of staphyloccocal infections. Several of the peptides have been shown to have enhanced binding to Fg but show decreased binding to host proteins that target the same region of Fg. The two extensively studied linear peptide binding MSCRAMMs SdrG and ClfA use very similar pockets between the N2 and N3 domains for ligand binding but show significant differences in mechanism of binding. Based on the results presented here, it is postulated that the mechanism of interaction between ClfA and Fg is a variation of the “Dock, Lock and Latch (DLL)” model of SdrG binding to Fg. In the DLL model of binding, the apo-form of the SdrG is in an open conformation to allow the ligand access to the binding cleft. A closed conformation of SdrG is unable to bind Fg. In the ClfA model, it is believed that the peptide may thread into the cavity formed in a stabilized closed configuration and therefore the ClfA-Fg binding mechanism could be called “Latch and Dock”.

(34) In the case of CNA, a collagen binding MSCRAMM from S. aureus, the collagen molecule binds to CNA through a “collagen hug” model (29) which represents yet another variant of the DLL binding mechanism. All three MSCRAMM-ligand structures determined so far, SdrG, CNA and the ClfA have different ligand binding characteristics and mechanisms, although the overall structures of the ligand binding regions of these MSCRAMMs are very similar. These observations suggest that an ancestral MSCRAMM has evolved to accommodate different ligands without greatly altering the overall organization of the proteins.

(35) Although there are many antibiotics available in the market to treat S. aureus infections, the strategy discussed herein is a novel approach targeting ClfA on S. aureus. The primary disadvantage of using a small segment (peptide) of the interacting protein molecule is non-specificity and undesirable binding and adverse effects. The process of modified peptide by variations in amino acid will be effective and easier to achieve the much desired specificity. These peptides can be significantly efficient over any small molecule or any other antibiotic treatment. Based on the structure disclosed herein, two peptides, P16 (Asp16.fwdarw.Ala) peptide and P12 (Lys12.fwdarw.Ala) peptide are synthesized and can be used as inhibitors of ClfA. To further enhance the specificity towards ClfA and decrease undesirable activation of platelets, a combination of two variants such as double mutant analog (P12+P16) will be synthesized and tested. The present invention contemplates attempting further variations in the sequence to achieve additional affinity towards ClfA. These peptides are assessed in a mouse model of S. aureus induced septic death.

(36) Alternate formulations may include the design of small molecule inhibitors that specifically bind to ClfA and/or tailoring/modify existing small molecule drugs. Several small molecule drugs are available that mimic the same region of Fg that bind to integrin. The features/amino acid differences that contribute to the specificity of the peptide can be incorporated in the existing anti-platelet drug molecules to achieve the specificity for ClfA.

(37) Overall, the present invention provides the Fg/ClfA complex structure that can be (1) used to develop therapeutics that specifically will inhibit ClfA:Fg interaction but will not affect α.sub.IIbβ.sub.3 integrin interactions; (2) to design ClfA constructs that will be optimal vaccine candidates and can be used for the generation and screening of therapeutic monoclonal antibodies; and (3) to model other MSCRAMM Fg interactions with similar substrate specificities such as FnbpA, FnbpB and FbI.

(38) Treatment methods involve treating and/preventing an infection in an individual with a pharmacologically effective or an immunologically effective amount of a pharmaceutical composition containing therapeutic agents described herein. Such therapeutic agent may comprise a peptide, fusion peptide, small molecule inhibitor, small molecule drug or an antibody. A pharmacologically effective amount is described, generally, as that amount sufficient to detectably and repeatedly inhibit MSCRAMM:fibrinogen interaction so as to prevent, ameliorate, reduce, minimize or limit the extent of a disease or its symptoms. An immunologically effective amount is described, generally, as that amount sufficient to detectably and repeatedly induce an immune response so as to prevent, ameliorate, reduce, minimize or limit the extent of a disease or its symptoms. More specifically, it is envisioned that the treatment with the pharmaceutical composition or an immunogenic composition enhances antibody response, reduces the level of inflammatory cytokines and the levels of endotoxins and decreases the bacterial load in the individual to prevent the infection caused by the bacteria.

(39) The pharmacologically or immunologically effective amount of the composition or antibody, respectively to be used are those amounts effective to produce beneficial results, particularly with respect to preventing the infection caused by the bacteria, in the recipient animal or patient. Such amounts may be initially determined by reviewing the published literature, by conducting in vitro tests or by conducting metabolic studies in healthy experimental animals. Before use in a clinical setting, it may be beneficial to conduct confirmatory studies in an animal model, preferably a widely accepted animal model of the particular disease to be treated. Preferred animal models for use in certain embodiments are rodent models, which are preferred because they are economical to use and, particularly, because the results gained are widely accepted as predictive of clinical value.

(40) The pharmaceutical composition disclosed herein and the antibody generated thereof may be administered either alone or in combination with another drug, a compound, or an antibiotic. Such a drug, compound or antibiotic may be administered concurrently or sequentially with the immunogenic composition or antibody disclosed herein. The effect of co-administration with the pharmaceutical composition or antibody is to lower the dosage of the drug, the compound or the antibiotic normally required that is known to have at least a minimal pharmacological or therapeutic effect against the disease that is being treated. Concomitantly, toxicity of the drug, the compound or the antibiotic to normal cells, tissues and organs is reduced without reducing, ameliorating, eliminating or otherwise interfering with any cytotoxic, cytostatic, apoptotic or other killing or inhibitory therapeutic effect of the drug, compound or antibiotic.

(41) The composition described herein and the drug, compound, or antibiotic may be administered independently, either systemically or locally, by any method standard in the art, for example, subcutaneously, intravenously, parenterally, intraperitoneally, intradermally, intramuscularly, topically, enterally, rectally, nasally, buccally, vaginally or by inhalation spray, by drug pump or contained within transdermal patch or an implant. Dosage formulations of the composition described herein may comprise conventional non-toxic, physiologically or pharmaceutically acceptable carriers or vehicles suitable for the method of administration.

(42) The pharmaceutical composition or antibody described herein and the drug, compound or antibiotic may be administered independently one or more times to achieve, maintain or improve upon a therapeutic effect. It is well within the skill of an artisan to determine dosage or whether a suitable dosage of either or both of the immunogenic composition or antibody and the drug, compound or antibiotic comprises a single administered dose or multiple administered doses.

(43) As is well known in the art, a specific dose level of such a pharmaceutical composition or antibody for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy. The person responsible for administration will determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

(44) One of skill in the art realizes that the pharmacologically effective amount of the immunogenic composition or the antibody can be the amount that is required to achieve the desired result: enhance antibody response, reduce the level of inflammatory cytokines and levels of endotoxins, decrease the bacterial load, etc.

(45) Administration of the pharmaceutical composition of the present invention and the antibody to a patient or subject will follow general protocols for the administration of therapies used in treatment of bacterial infections taking into account the toxicity, if any, of the components in the immunogenic composition, the antibody and/or, in embodiments of combination therapy, the toxicity of the antibiotic. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy.

(46) As is known to one of skill in the art the pharmaceutical composition described herein may be administered along with any of the known pharmacologically acceptable carriers. Additionally the pharmaceutical composition can be administered via any of the known routes of administration such as subcutaneous, intranasal or mucosal. Furthermore, the dosage of the composition to be administered can be determined by performing experiments as is known to one of skill in the art.

(47) The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1

(48) Bacterial Strains, Plasmids and Culture Conditions

(49) Escherichia coli XL-1 Blue (Stratagene) was used as the host for plasmid cloning and protein expression. Chromosomal DNA from S. aureus strain Newman was used to amplify the ClfA DNA sequence. All E. coli strains containing plasmids were grown on LB media with ampicillin (100 μg/ml).

Example 2

(50) Manipulation of DNA

(51) DNA restriction enzymes were used according to the manufacturer's protocols (New England Biolabs) and DNA manipulations were performed using standard procedures (30) (Sambrook and Gething, 1989). Plasmid DNA used for cloning and sequencing was purified using the Qiagen Miniprep kit (Qiagen). DNA was sequenced by the dideoxy chain termination method with an ABI 373A DNA Sequencer (Perkin Elmer, Applied Biosystems Division). DNA containing the N-terminal ClfA sequences were amplified by PCR (Applied Biosystems) using Newman strain chromosomal DNA as previously described (31). The synthetic oligonucleotides (IDT) used for amplifying clfA gene products and for cysteine mutations are listed in Table I.

(52) TABLE-US-00001 TABLE 1 ClfA229 5′-CCCGGATCCGGCACAGATATTACGAAT-3′ (SEQ ID NO: 1) ClfA545 5′-CCCGGTACCTCAAGGAACAACTGGTTTATC-3′ (SEQ ID NO: 2) For disulfide mutant: rClfA327 5′-TGCTTTTACATCACATTTAGTATTTAC-3′ (SEQ ID NO: 3) fClfA327 5′-GTAAATACTAAATGTGATGTAAAAGCA-3′ (SEQ ID NO: 4) ClfA541 5′-CCCGGTACCTCAAGGAACAACTGGACAATCGATACCGTC-3′ (SEQ ID NO: 5) Peptides: Wild-type Fg γ 395-411 GEGQQHHLGGAKQAGDV (SEQ ID NO: 6) Fg γ 395-411 D410A: GEGQQHHLGGAKQAGAV (SEQ ID NO: 28) P16 -2Nt 397-411   GQQHHLGGAKQAGAV (SEQ ID NO: 7) P16 -4Nt 399-411     QHHLGGAKQAGAV (SEQ ID NO: 8) P16 -6Nt 401-411       HLGGAKQAGAV (SEQ ID NO: 9) P16 -8Nt 403-411         GGAKQAGAV (SEQ ID NO: 10) P16 -2Ct 395-409 GEGQQHHLGGAKQAG (SEQ ID NO: 11) P16 -4Ct 395-407 GEGQQHHLGGAKQ (SEQ ID NO: 12)

Example 3

(53) Construction of Disulphide Mutants (Stable Form of ClfA)

(54) Cysteine mutations were predicted by comparing ClfA.sub.221-559 to SdrG.sub.(273-597) disulfide mutant with stable closed conformations (32) and by computer modeling. A model of ClfA in closed conformation was built based on the closed conformation of the SdrG-peptide complex (25). The Cβ-Cβ distances were calculated for a few residues at the C-terminal end of the latch and strand E in the N2 domain. Residue pairs with Cβ-Cβ distance less than 3 Å were changed to cysteines to identify residues that could form optimum disulfide bond geometry. The D327C/K541C mutant was found to form a disulfide bond at the end of the latch. The cysteine mutations in ClfA.sub.D327C/K541C were generated by overlap PCR (33-34). The forward primer for PCR extension contained a BamHI restriction site and the reverse primer contained a KpnI restriction site. The mutagenesis primers contained complementary overlapping sequences. The final PCR product was digested with BamHI and KpnI and was ligated into same site in the expression vector pQE30 (Qiagen). All mutations were confirmed by sequencing. The primers used are listed in Table I.

Example 4

(55) Expression and Purification of Recombinant Proteins

(56) E. coli lysates containing recombinant ClfA and GST-Fg γ-chain fusion proteins were purified as previously described (35). PCR products were subcloned into expression vector pQE-30 (Qiagen) to generate recombinant proteins containing an N-terminal histidine (His) tag as previously described (9). The recombinant ClfA His-tag fusion proteins were purified by metal chelation chromatography and anion exchange chromatography as previously described (23). To generate recombinant ClfA.sub.229-545 and ClfA.sub.221-559 proteins, PCR-amplified fragments were digested with BamHI and KpnI and cloned into BamHI/KpnI digested PQE-30. The primers used to generate the recombinant constructs are listed in Table I. The reactions contained 50 ng of strain Newman DNA, 100 pmol of each forward and reverse primers, 250 nM of each dNTP, 2 units of Pfu DNA polymerase (Stratagene) and 5 ml Pfu buffer in a total volume of 50 ml. The DNA was amplified at 94° C. for 1 min, 48° C. for 45 sec; 72° C. for 2 min for 30 cycles, followed by 72° C. for 10 min. The PCR products were analyzed by agarose gel electrophoresis using standard methods (30) and purified as described above.

Example 5

(57) Enzyme-Linked Immunosorbent Assay

(58) The ability of the wild-type ClfA.sub.229-545 and disulfide ClfA mutants to bind Fg was analyzed by ELISA-type binding assays. Immulon 4HBX Microtiter plates (Thermo) were coated with human Fg (1 μg/well) in HBS (10 mM HEPES, 100 mM NaCl, 3 mM EDTA, pH 7.4) over-night at 4° C. The wells were washed with HBS containing 0.05% (w/v) Tween-20 (HBST) and blocked with 5% (w/v) BSA in HBS for 1 h at 25° C. The wells were washed 3 times with HBST and recombinant ClfA proteins in HBS were added and the plates were incubated at 25° C. for 1 h. After incubation, the plates were washed 3 times with HBST. Anti-His antibodies (GE Healthcare) were added (1:3000 in HBS) and the plates were incubated at 25° C. for 1 h. The wells were subsequently washed 3 times with HBST and incubated with Goat anti-mouse-AP secondary antibodies (diluted 1:3000 in HBS; Bio-Rad) at 25° C. for 1 h. The wells were washed 3 times with HBST and AP-conjugated polyclonal antibodies were detected by addition of p-nitrophenyl phosphate (Sigma) in 1 M diethanolamine (0.5 mM MgCl.sub.2, pH 9.8) and incubated at 25° C. for 30-60 min. The plates were read at 405 nm in an ELISA plate reader (Themomax, Molecular Devices). For the inhibition assays, recombinant ClfA.sub.229-545 was pre-incubated with Fg γ peptides in HBS for 1 h at 37° C. The recombinant protein-peptide solutions were then added to plates coated with 1 mg/well GST fusion protein containing the native human Fg γ 395-411 sequence (called GST-Fg γ.sup.1-17) and bound protein was detected as described above. If the peptide binds ClfA it would inhibit binding of the GST-Fg γ.sup.1-17 to the MSCRAMM.

Example 6

(59) Synthesis of Gamma Chain Peptides

(60) The wild-type and mutated peptides corresponding to the 17 C-terminal residues of the fibrinogen γ-chain (residues 395-411) and truncated versions of this peptide (listed in Table I) were synthesized as previously described and purified using HPLC (9).

Example 7

(61) Isothermal Titration Calorimetry

(62) The interaction between ClfA proteins and soluble Fg peptides was analyzed by Isothermal titration calorimetry (ITC) using a VP-ITC microcalorimeter (MicroCal). The cell contained 30 mM ClfA and the syringe contained 500-600 mM peptide in HBS buffer (10 mM HEPES, 150 mM NaCl, pH 7.4). All samples were degassed for 5 min. The titration was performed at 30° C. using a preliminary injection of 5 ml followed by 30 injections of 10 ml with an injection speed of 0.5 ml/sec. The stirring speed was 300 rpm. Data were fitted to a single binding site model and analyzed using Origin version 5 (MicroCal) software.

Example 8

(63) Crystallization

(64) The ClfA.sub.D327C/K541C protein was purified as described and concentrated to 30 mg/ml. The synthetic γ-chain peptide analogs, P16 and N-terminal truncations of P16 (P16-2Nt, P16-4Nt and P16-6Nt) were mixed with the protein at 1:20 molar ratio and left for 30 min at 5° C. This mixture was screened for crystallization conditions. Small needles of the ClfA/P16-2Nt, -4Nt and -6Nt were obtained during initial search of the crystallization condition, but we could only successfully optimize ClfA/P16-4Nt and ClfA/P16-6Nt. Diffraction quality crystals were obtained by mixing 2 μl of protein solution with 2 μl of reservoir solution containing 16-20% PEG 8K, 110 mM succinic acid pH 6.0.

Example 9

(65) X-Ray Data Collection, Structure Solution and Refinement

(66) Crystals of ClfA/P16-4Nt were flash frozen with a stabilizing solution containing 20% glycerol. Diffraction data were measured on Rigaku R-Axis IV.sup.++ detector. A total of 180 frames were collected at a detector distance of 120 mm with 1° oscillation. Data were indexed, integrated and scaled using d*terk (47) (Pflugrath, 1999). The crystals diffracted to 1.95 Å and the data statistics were listed in Table 2. Calculation of the Matthews coefficient suggested the presence of 2 copies of the molecule in the unit cell of the triclinic cell. The structure was solved by molecular replacement (MR) with the program PHASER (36) using individual N2 and N3 domains of ClfA as search model. Solutions for the N3 domain were obtained for the two copies followed by the solutions of N2 domains. Data covering 2.5-15 Å were used for the molecular replacement solution. Electron density maps calculated during the initial rounds of refinement showed interpretable density for 11 out of 13 peptide residues in both the copies of the complex. Modeling building of the peptide and rebuilding of a few loop regions were performed using the program COOT (37). A few cycles of ARP/WARP (38) were performed to improve the map and for the building of water model. After a few cycles of refinement using Refmac5.0 (39), electron density was clear for only the backbone atoms for two remaining N-terminal residues of the peptide molecule D and one residue for peptide C. The final model of ClfA included residues 230-299, 303-452, 456-476 and 479-545 in molecule A and 230-438, 440-476 and 479-542 in molecule B. The structure was refined to a final R-factor of 20.9% and R-free of 27.8%. Stereochemical quality of the model was validated using PROCHECK (40).

(67) TABLE-US-00002 TABLE 2 Crystallographic data measurement and refinement data Cell dimensions a, b, c (Å) 35.43, 61.84, 81.78 α, β, γ (°) 85.44, 81.84, 82.45 Space group P1 Resolution (Å) 1.95-15.0 Reflections total/unique 86051/46090 Completeness (%) 93.9 R.sub.merge* 0.074 Number of molecules in the asymmetric unit 2 Rfactor/R.sub.free.sup.+ 0.211/0.279 Bond rms deviation (Å) 0.015 Angle rms deviation (°) 1.64 Average B value (Å) 29.9 No of non-hydrogen atoms 5226 Protein 4558 Peptide 141 Water 527 Rms deviations from ideal values Bond lengths (Å) 0.22 Bond Angles (°) 1.95 PDB ID 2vr3 *R.sub.merge = Σ|I.sub.j − [I]|/ΣI.sub.i; where I.sub.j is the measured and [I] is the mean intensity of reflection hkl; .sup.+R.sub.free is calculated over 2% of randomly selected reflections not included in the refinement.

Example 10

(68) Integrin (α.sub.IIbβ.sub.3) Inhibition Assay

(69) For α.sub.IIbβ.sub.3 inhibition assay. α.sub.IIbβ.sub.3Immulon 4HBX Microtiter 96-well plates (Thermo) were coated with α.sub.IIbβ.sub.3 (0.25 mg/well) in TBS (25 mM Tris, 3 mM KCl, 140 mM NaCl, pH 7.4) over night at 4° C. The wells were washed with TBS containing 0.05% (w/v) Tween-20 (TBST). After blocking with 3% (w/v) BSA dissolved in TBS for 1 h at RT, 10 nM of full length Fg was applied in the presence of either WT γ.sup.1-17, γ.sup.1-17.sub.D16A or γ.sup.1-17.sub.K12A peptides and plates were incubated at RT for another hour. The bound full length Fg was then detected by goat anti human Fg (1:1000 dilution, Sigma) antibody followed by horseradish peroxidase-conjugated rabbit anti-goat IgG antibody (1:1000 dilution, Cappel). After incubation with 0.4 mg/ml of substrate, o-phenylenediamine dihydrochloride (OPD, Sigma) dissolved in phosphate-citrate buffer, pH 5.0, bound antibodies were determined in an ELISA reader at 450 nm. The proteins, antibodies and peptides were diluted in TBST containing 1% (w/v) BSA, 2 mM MgCl.sub.2, 1 mM of CaCl.sub.2 and MnCl.sub.2.

Example 11

(70) Molecular Modeling

(71) All molecular modeling studies were performed using InsightII software (Accrelys Inc). Modeling of FnbpA-peptide complex was performed using “Homology” module available in InsightII using ClfA.sub.229-545 peptide complex as a template. Prior to model building, the amino acid sequence of ClfA.sub.229-545 was aligned with FnbpA (GENBANK® ID: CA077272) using Lalign (41). The aligned sequences were manually checked for any gaps in the core β-sheet forming regions of ClfA. The final model was subjected to molecular dynamics simulation followed by conjugate gradient energy minimization. Figures were made using RIBBONS (42). The atomic coordinates and structure factors of the complex structure have been deposited in Protein Data Bank with accession number 2vr3.

Example 12

(72) Identification of Critical Residues in Fg Required for Binding to ClfA

(73) In previous studies, a segment of ClfA composed of residues 221-559 was shown to bind to the C-terminal end of the human Fg γ-chain (9). Based on structural similarities with SdrG, a smaller ClfA construct (229-545) predicted to be composed only of the N2 N3 domains was designed and it was shown that ClfA.sub.229-545 retained the Fg-binding activity. To identify specific residues in Fg that are important for binding to ClfA.sub.229-545, a panel of peptides (FIG. 1A; SEQ ID NOS: 14-29, and 31) based on the Fg γ-chain sequence 395-411 (referred to as g.sup.1-17) were synthesized in which each position was sequentially substituted with an alanine residue (alanines 11 and 14 changed to serines). These peptides were tested as inhibitors in solid-phase binding assays. Peptides g.sup.1-17.sub.H6A, g.sup.1-17.sub.H7A, g.sup.1-17.sub.G10A, g.sup.1-17.sub.Q13A, g.sup.1-17.sub.A14S and g.sup.1-17.sub.G15A were significantly less potent inhibitors than the native sequence suggesting that the Fg residues H6, H7, G10, Q13, A14 and G15 interact with ClfA (FIG. 1B). Remarkably, peptides g.sup.1-17.sub.A11S, g.sup.1-17.sub.D16A and g.sup.1-17.sub.V17A showed an enhanced inhibition of ClfA binding to a recombinant form of residues 395-411 of the Fg γ chain fused to a GST protein (GST-Fg g.sub.1-17) compared to a peptide with the wild-type sequence, indicating a higher affinity of the peptide variants for ClfA. This shows that are sequence variations in the fibrinogen peptide that act as “functional homologs” of fibrinogen peptide, which demonstrates that the sequence variations in the binding sequence of i.e. ClfA (SEQ ID NO:35) could also serve as functional homologs to ClfA.

(74) The ability of ClfA.sub.229-545 to bind to the peptide containing the g.sup.1-17.sub.D16A mutation was further characterized. In solid-phase assays, ClfA binds to immobilized GST-Fg g.sup.1-17 fusion protein with a lower affinity (K.sub.d=657 nM) compared to the mutated GST-Fg g.sup.1-17.sub.D16A (K.sub.d=35 nM) (FIG. 1C). In solution, using isothermal titration calorimetry (ITC) assays, (FIG. 1D), ClfA also binds with a lower affinity to the native g.sup.1-17 peptide (K.sub.d of 5.8 mM) compared to the mutant Fg g.sup.1-17.sub.D16A (K.sub.d of 3 mM). Thus, although the apparent dissociation constants differ according to the assays used to estimate them, similar trends in affinity between the wild-type and the D16A mutation were observed. It is currently unknown why the difference between the K.sub.ds was much greater in the solid phase binding assays compared to the ITC analysis.

(75) The present invention demonstrates that alanine substitution at the C-terminal region of the peptide affected MSCRAMM binding suggesting that the ClfA binding site is located at the very C-terminus of the Fg γ-chain (FIGS. 1A-1D). Results also show that certain amino acid changes in the g.sup.1-17 sequence enhance ClfA binding compared to the wild-type Fg sequence indicating that the human Fg γ C-terminal 17 residues may not be the optimum ligand for ClfA.

(76) Analysis of the previously solved SdrG-Fg peptide complex crystal structure showed that only 11 out of the 18 peptide residues interacted with the MSCRAMM. Similarly, only a part of the 17-residue sequence may be required for binding to ClfA. In order to establish the minimum Fg peptide required for binding to ClfA.sub.229-545, a series of N- and C-terminal truncations of the g.sup.1-17.sub.D16A peptide were synthesized (FIG. 2A; SEQ ID NOS: 7-12 & 28). Truncations of 2, 4, 6 or 8 amino acids at the N-terminus of the Fg g-peptide resulted in a reduced but detectable binding affinity when tested using ITC. There was a direct relationship between the length of the peptide and its affinity for ClfA. The smaller the peptide, the lower was the observed affinity for the MSCRAMM (FIG. 2B). Thus, the N-terminal residues of the Fg peptide (residues 1-8) may either contribute to or stabilize the binding of the peptide to ClfA, but are not critical for the interaction. On the other hand, deletions of 2 or 4 residues from the C-terminal end of the g.sup.1-17.sub.D16A peptide abolished binding (data not shown). These results indicate that the C-terminal amino acids of Fg are critical for binding to ClfA; these data correlate with a previous report that showed that Fg lacking the C-terminal residues AGDV in the g chain (corresponding to residues 14-17 in the peptide) or a variant that replaces the last four g-chain residues with 20 amino acids lacks the ability to bind recombinant ClfA.sub.221-550 and or to induce S. aureus clumping (9).

Example 13

(77) A Stabilized Closed Confirmation of ClfA.sub.229-545 Binds Fg with a Higher Affinity than the Open Form

(78) The Fg binding mechanism of SdrG.sub.276-596 involves a transition from an open conformation, where the peptide binding trench between the N2 and N3 domains is exposed for ligand docking, to a closed conformation of the SdrG.sub.276-596 in complex with the ligand. The insertion of the N3 extension into the latching trench on N2 stabilizes the closed conformation (32). The closed conformation of apo SdrG N2N3, stabilized by introducing a disulfide bond between the end of the N3 latch and the “bottom” of N2, no longer binds Fg (32) demonstrating that the dynamics of the latch is critical for the SdrG ligand interaction. To explore if the binding of ClfA to Fg is also dependent on a movement of the latch, a ClfA construct containing two cysteine substitutions was constructed. The locations of the cysteine mutations were determined using computer modeling and by sequence alignment to corresponding mutations in SdrG (32). The mutant ClfA.sub.D327C/K541C generated a stable, closed conformation form. This recombinant His-tag fusion protein was purified by Ni.sup.+ chelating chromatography; ion-exchange and gel permeation chromatography. The ClfA.sub.D327C/K541C open and closed conformation forms were examined by SDS-PAGE analysis (FIG. 2C).

(79) Under non reducing conditions, the disulfide bonded closed form of ClfA.sub.D327C/K541C migrated faster on SDS-PAGE than its non-disulfide bonded open form. Presumably, under non-reducing conditions, closed conformation mutants are more compact and migrate faster on SDS-PAGE than open conformation constructs. Under reducing conditions, the disulfide mutant and the wild-type protein migrate at the same rate. Surprisingly, the disulfide mutant ClfA.sub.D327C/K541C was able to bind Fg both in the open and closed conformations (FIG. 2C). Elisa-type binding assays where Fg or GST Fg γ.sup.1-17 peptide were coated in microtiter wells and incubated with ClfA showed that the closed conformation ClfA.sub.D327C/K541C bound the ligand with a much lower apparent K.sub.d(34 nM Fg; 20 nM GST-Fg γ.sup.1-17) compared to the wild-type ClfA.sub.229-545 (apparent K.sub.d 305 nM Fg; 222 nM GST-Fg γ.sup.1-17) (FIG. 2C). These results demonstrate that an open conformation may not be required for Fg binding to ClfA and that Fg binding by ClfA involves a mechanism that is different from the DLL mechanism employed by SdrG.

Example 14

(80) Crystal Structure of ClfA.sub.(229-545)/D327C/K541C) in Complex with a 13-Residue Fg-Derived Peptide

(81) Crystallization screens were carried out with ClfA.sub.D327C/K541C in complex with several N-terminal truncations of the g.sup.1-17.sub.D16A peptide that were shown to bind the MSCRAMM. Crystals of the stable closed conformation of ClfA.sub.229-545 in complex with several peptides were obtained, but structure determination was attempted for only the ClfA.sub.(229-545)D327C/K541C-g.sup.5-17.sub.D16A peptide. The crystals of the ClfA-peptide complex diffracted to a 1.95 Å resolution. Two copies of ClfA-peptide complex were found in the asymmetric part of the unit cell and are referred to as A:C and B:D. Although the 13 residues of the Fg g.sup.5-17 chain synthetic peptide were used for crystallization, only 11 residues were completely observed in both copies. The two molecules of ClfA.sub.D327C/K541C (A and B) are nearly identical with rms deviation of 0.3 Å for 312 Cα atoms and 0.55 Å for backbone atoms. As observed in the apo-ClfA.sub.221-559 structure, the ClfA.sub.(229-545)D327C/K541C N2 and N3 domains adopt a DE-variant IgG fold (24). The overall structure of the ClfA.sub.D327C/K541C peptide complex (A:C) and the two copies of the complexes A:C and B:D superimposed are shown in FIGS. 3A and 3B, respectively. The C-terminal extension of the N3 domain makes a β-sheet complementation with strand E of the N2 domain. This conformation is locked by the engineered disulfide bond as predicted by SDS-PAGE analysis (FIG. 2C) and confirmed by the crystal structure. The two copies of the Fg γ-peptide molecules are nearly identical with rms deviation of 0.5 Å for 11 Ca atoms and 0.89 Å for backbone atoms. The interaction between the ClfA.sub.D327C/K541C and the peptide buries a total surface area of 1849 Å.sup.2 and 1826 Å.sup.2 in the A:C and B:D complex, respectively. The interaction of the peptide with the N2 domain is predominantly hydrophobic in nature, in addition to a few main-chain hydrogen bonds (FIG. 3C). Interactions between the Fg peptide and the N3 domain are both hydrophobic and electrostatic with the electrostatic contribution coming almost entirely from the main chain-main chain hydrogen bonds due to the parallel β-sheet formation of the peptide with strand G of the N3 domain (FIG. 3C). The side-chain interactions between the peptide and ClfA are predominantly hydrophobic. The 11 C-terminal residues of the Fg g-chain peptide sequence that interact with ClfA are composed of only two polar residues, Lys12 and Gln13. Side chain atoms of Lys12 point away and do not interact with the ClfA protein whereas Gln13 makes two hydrogen bonds with the main chain atoms of Ile 384 in ClfA (FIG. 3D). A water-mediated interaction is also observed between Gln13 of the peptide and Asn525 of ClfA. Tyr338 in the N2 domain and Trp523 in the N3 domain play an important role in anchoring the peptide molecule. Tyr338 and Trp523 are stacked with residues Gly15 and Gly10, respectively. In addition, Met521 and Phe529 make hydrophobic interactions with Ala7 and Val117, respectively. The C-terminal residues of the peptide Ala14, Gly15, Ala16, and Val117 are buried between the N2-N3 domain interface with the terminal Val residue, presumably threaded through a preformed ligand binding tunnel after ClfA.sub.D327C/K541C adopted its closed conformation. A hydrogen bond is observed between Lys389 of ClfA and the C-terminal carboxyl group of the peptide (FIG. 6B).

Example 15

(82) Structural Differences Between the Closed Confirmation ClfA.sub.(D327C/541C)-Peptide Complex and the Apo-CIFA.sub.221-559 Protein

(83) The individual N2 and N3 domains in the apo-CIFA.sub.221-559 and the closed form of ClfA.sub.D327C/K541C are almost identical with rms deviations of 0.33 and 0. Å for molecule A and 0.35 and 0.42 Å for molecule B, but the relative orientation of the N2 and N3 domains are significantly different (FIG. 4A). This difference affects the association of the N2 and N3 domains. In the apo conformation, the buried surface area between the N2 and N3 domains is 87 Å.sup.2 compared to 367 Å.sup.2 in the closed form of the ClfA.sub.(221-559)D327/K541-peptide complex. In the apo-ClfA.sub.221-559, the C-terminal residues (Ala528-Glu559) of the N3 domain fold back and do not interact with the N2 domain.

(84) To understand if the altered N2-N3 orientation of the apo-form of ClfA (FIG. 4A) is due to the folded-back conformation, a model of the apo-ClfA.sub.221-559 was constructed with the folded-back N3 domain and the N2 domain adopting an N2-N3 orientation similar to that observed in the closed form of the ClfA-peptide complex. This model shows that Tyr338 in the N2 domain makes severe clashes with residues Ser535 and Gly534 of the folded back segment. An alternate conformation for these residues is unlikely due to spatial constraints. Thus, it is unlikely that the two domains in the folded-back conformation could adopt an orientation similar to their orientation in the ClfA-peptide complex. Moreover the folded-back segment completely occupies the binding site (FIG. 4B). Therefore, in the folded-back conformation, the ligand binding site appear not to be accessible to the peptide.

(85) It is presently unclear what the spatial rearrangements of the N2N3 domains are in intact ClfA expressed on the surface of a staphylococcal cell. The two structures of these domains solved so far where one is active and the other inactive provide a structural basis for the possible regulation of ClfA's Fg binding activity by external factors. One such factor may be Ca.sup.2+ which has been shown to inhibit ClfA-Fg binding (O'Connell et al., 1998). Alternatively, it is possible that the folded-back conformation (which is a larger protein construct) is only one of the many possible conformations adopted by the unbound protein. Most likely, MSCRAMMs proceed from the unbound to the bound forms in a very dynamic mechanism where different intermediate forms could be achieved.

Example 16

(86) Structural Similarities/Differences Between the Closed Form of the ClfA-Peptide and SdrG-Peptide Complexes

(87) The major difference between Fg-binding to ClfA and SdrG is that the directionality of the bound ligand peptide is reversed (FIG. 4C). The C-terminal residues of the ligand is docked between the N2 and N3 in ClfA and makes a parallel β-sheet complementation with strand G of the N3 domain, whereas in SdrG, the N-terminal residues of the ligand are docked between the N2 and N3 domains and form an anti-parallel β-sheet with the G strand. In both cases there are 11 ligand residues that make extensive contact with the MSCRAMM but with one residue shift towards the N3 domain in ClfA. Of these 11 residues, 7 and 11 residues participate in the β-strand complementation of SdrG and ClfA, respectively. Although the peptide binding model of ClfA is different to that of SdrG, the inter-domain orientations of the two MSCRAMMS are very similar (25). Superposition of 302 corresponding atoms in the N2 and N3 domains of ClfA and SdrG showed a small rms deviation of 0.65 Å indicating the high structural similarity between the two MSCRAMMS. Another striking difference is that ClfA does not require an open-conformation for ligand binding, whereas Fg cannot bind to a stabilized closed conformation of SdrG. ClfA binds the C-terminal end of Fg and the last few residues of the γ-chain can be threaded in to the binding pocket. In the SdrG-Fg interaction, the binding segment in Fg does not involve the seven N-terminal residues of the ligand and therefore an open conformation is required for ligand binding.

Example 17

(88) A Structural Model for Fg Binding to FnbpA

(89) FnbpA, like ClfA, has been shown to bind the Fg γ-chain at the C-terminus. The panel of peptides with alanine substitutions (FIG. 1A) was tested as inhibitors of FnpA binding to Fg in a solid phase assay. The pattern of inhibition was similar to that measured for ClfA (FIG. 7A). In addition, earlier mutational studies on FnbpA showed that two residues, N304 and F306, were required for full Fg binding (43). The corresponding residues in ClfA are P336 and Y338. Tyr 338 plays a key role in anchoring Gly15 of the γ-chain peptide. Together, these results indicate that the FnbpA Fg binding mechanism could be similar to that of ClfA. The availability of the now determined ClfA-peptide complex prompted us to model an FnbpA-Fg complex (FIG. 7B). The homology model of the FnbpA-peptide complex showed that FnbpA can adopt a structure similar to that of the ClfA-ligand complex. Although there is only 25% sequence identity between ClfA and FnbpA, this model shows that almost 50% of the residues that interact with the γ-chain peptide are conserved between FnbpA and ClfA and many others are similar. Together, the binding data and the modeling studies suggest that ClfA and FnbpA bind Fg by a similar mechanism.

Example 18

(90) Species Variations in Fg-Binding to ClfA

(91) There is a significant variation in the C-terminal sequences of the Fg g-chain among different animal species. The binding of ClfA.sub.327C/541C to Fg isolated from different animal species was explored using a solid-phase binding assay. ClfA.sub.327C/541C binds bovine Fg with significantly lower apparent affinity than human Fg; binding of the MSCRAMM to sheep Fg could not be detected (FIG. 5A). The bovine Fg γ sequence is available and the binding data obtained in the ELISA type assay was corroborated by measuring the affinity of ClfA.sub.327C/541C for the Fg g.sup.1-17.sub.D16A peptide and a peptide representing the bovine Fg γ chain sequence using ITC (FIG. 5B). A closer examination of the ClfA-peptide interaction and the sequence variations between the human and the bovine Fg γ-chain C-terminal segment suggests that two of the four amino acid variations, at positions 14 and 16, could potentially explain the difference in the affinity (FIG. 5C Upper panel). In the ClfA-peptide crystal structure, Ala14 and Ala16 are completely buried between the N2 and N3 domains (binding trench). Replacement of Ala with Val at either position would impose steric conflicts between ClfA and Fg. However, Asp, and not Ala, is the natural sequence at position 16 of the peptide in human Fg. Modeling shows that Asp could adopt a conformation that could allow the side chain to point towards the solvent with minimal steric conflict with the ClfA. The less bulky Ala would fit better in the binding site than Asp, which explains the higher affinity of ClfA for the γ.sup.1-17.sub.D16A peptide compared to the WT peptide. Valine is branched at the Cβ atom and this residue would make steric clashes with the residues lining the binding trench in ClfA independent of the side-chain conformation of the Val residue. The other two non-contributing variations in the bovine compared to the human Fg sequence are His6.fwdarw.Gln and Val17.fwdarw.Glu. The electron density for the side chain of His6 in the peptide is not interpretable indicating that the side-chain of the His6 and its corresponding residue, Gln, in bovine Fg do not participate significantly in the interaction. Molecular modeling shows that even a bulkier Glu residue instead of Val at this position 17 is unlikely to sterically clash with ClfA. Therefore H.fwdarw.Q and V.fwdarw.Q variations at positions 6 and 17 may not contribute to the difference in affinity. A specific linear sequence often appears to be recognized by a staphylococcal MSCRAMM, which raises the possibility that the MSCRAMMs can differentiate between the ligand analogs from different species. This hypothesis is illustrated herein where it can also explain in structural terms the preferential binding of ClfA to human over bovine fibrinogen. The observed species specificity of MSCRAMM ligand interaction potentially could contribute to the observed species tropism of many staphylococcal strains.

Example 19

(92) Comparison of Fg Binding to ClfA and the Platelet Integrin α.sub.IIbβ.sub.3

(93) The C-terminus of Fg γ-chain, which is targeted by ClfA, is also important for platelet aggregation mediated by the α.sub.IIbβ.sub.3 integrin, a vital step in thrombosis (9, 44). The Fg γ-chain complex with α.sub.IIbβ.sub.3 structure is not available but structures of related complexes provide clues on how α.sub.IIbβ.sub.3 likely interact with Fg (45). In addition, the crystal structure of the α.sub.Vβ.sub.3 integrin in complex with an RGD ligand provided a structural model of a similar ligand-integrin interaction (46). In this structure, the Asp (D) residue of the RGD sequence coordinates with the metal ion in the Metal Ion Dependent Adhesion Site (MIDAS) of the integrin and thus plays a key role in the interaction. The platelet specific integrin α.sub.IIbβ.sub.3 recognizes ligands with an RGD sequence or the sequence Lys-Gln-Ala-Gly-Asp-Val (SEQ ID NO: 30) found in Fg (45). Structural studies with drug molecules that antagonize the integrin-RGD or -Fg interaction showed that each of the drug molecules contains a carboxyl group moiety that mimics the aspartic acid and a basic group that mimics the Arg (or Lys in the case of Fg) in the ligand (45). These results suggest that the Lys and Asp residues in the C-terminal γ-chain sequence are critical for the interaction with integrin. Interestingly, the present invention shows that these Lys and Asp residues in Fg are not critical for ClfA binding (FIG. 1B). In fact, substitution of Asp with Ala (γ.sup.1-17.sub.D16A) results in a higher binding affinity. Absence of a strong interaction with Lys12 in the ClfA-peptide complex structure also correlates with the biochemical data, suggesting that Arg is not a key player in the ClfA-Fg interaction. In general, the present invention shows that K406 and D410, which are essential for platelet integrin α.sub.IIbβ.sub.3-Fg interaction, are dispensable for the ClfA-Fg interaction. Thus, although ClfA and α.sub.IIbβ.sub.3 target the same stretch of amino acids in Fg, there are significant differences in the binding interactions.

Example 20

(94) The g.sup.1-17.sub.D16A and g.sup.1-17.sub.K12A Peptides are Selective Antagonists of Fg-ClfA Interaction.

(95) Although ClfA and α.sub.IIbβ.sub.3 target the same stretch of amino acids in Fg, there are significant differences in the binding interactions. Two of the series of peptides, g.sup.1-17.sub.D16A and g.sup.1-17.sub.K12A, synthesized earlier for the characterization of WT g.sup.1-17 peptide, lack Asp and Lys residues respectively at positions 416 and 410. These residues are quintessential for Fg binding to plate integrin α.sub.IIbβ.sub.3. The, g.sup.1-17.sub.D16A and g.sup.1-17.sub.K12A peptides either shows similar or enhanced binding to ClfA (FIGS. 1B, 1D) but are expected to bind weakly to platelet integrin. Therefore, g.sup.1-17.sub.D16 and g.sup.1-17.sub.K12A peptides could serve as selective antagonist of Fg-ClfA interaction.

(96) To examine this possibility, the ability of the synthesized Fg WT g.sup.1-17 and mutated peptides (g.sup.1-17.sub.D16A and g.sup.1-17.sub.K12A) to inhibit full length Fg binding to α.sub.IIbβ.sub.3 was analyzed by inhibitory ELISA type assay (FIG. 6). The WT, g.sup.1-17 peptide completely inhibited the binding of full-length fibrinogen to α.sub.IIbβ.sub.3 whereas, g.sup.1-17.sub.D16A and g.sup.1-17.sub.K12A weakly inhibited Fg binding α.sub.IIbβ.sub.3. These results clearly demonstrated that the g.sup.1-17.sub.D16A and g.sup.1-17.sub.K12A peptides bind weakly to platelet integrin and therefore could serve as an antagonist of Fg-ClfA interaction.

(97) TABLE-US-00003 ClfA Protein Sequence, SEQ ID NO: 35         10         20         30         40         50 MNMKKKEKHA IRKKSIGVAS VLVGTLIGFG LLSSKEADAS ENSVTQSDSA         60         70         80         90        100 SNESKSNDSS SVSAAPKTDD TNVSDTKTSS NTNNGETSVA QNPAQQETTQ        110        120        130        140        150 SSSTNATTEE TPVTGEATTT TTNQANTPAT TQSSNTNAEE LVNQTSNETT        160        170        180        190        200 FNDTNTVSSV NSPQNSTNAE NVSTTQDTST EATPSNNESA PQSTDASNKD        210        220        230        240        250 VVNQAVNTSA PRMRAFSLAA VAADAPAAGT DITNQLTNVT VGIDSGTTVY        260        270        280        290        300 PHQAGYVKLN YGFSVPNSAV KGDTFKITVP KELNLNGVTS TAKVPPIMAG        310        320        330        340        350 DQVLANGVID SDGNVIYTFT DYVNTKDDVK ATLTMPAYID PENVKKTGNV        360        370        380        390        400 TLATGIGSTT ANKTVLVDYE KYGKFYNLSI KGTIDQIDKT NNTYRQTIYV        410        420        430        440        450 NPSGDNVIAP VLTGNLKPNT DSNALIDQQN TSIKVYKVDN AADLSESYFV        460        470        480        490        500 NPENFEDVTN SVNITFPNPN QYKVEFNTPD DQITTPYIVV VNGHIDPNSK        510        520        530        540        550 GDLALRSTLY GYNSNIIWRS MSWDNEVAFN NGSGSGDGID KPVVPEQPDE        560        570        580        590        600 PGEIEPIPED SDSDPGSDSG SDSNSDSGSD SGSDSTSDSG SDSASDSDSA        610        620        630        640        650 SDSDSASDSD SASDSDSASD SDSDNDSDSD SDSDSDSDSD SDSDSDSDSD        660        670        680        690        700 SDSDSDSDSD SDSDSDSDSD SDSDSDSDSD SDSDSDSDSD SDSDSDSDSD        710        720        730        740        750 SDSDSDSDSD SDSDSDSDSD SDSDSDSDSD SDSDSDSDSD SDSDSDSDSD        760        770        780        790        800 SDSDSDSDSD SDSDSDSASD SDSDSDSDSD SDSDSDSDSD SDSDSDSDSD        810        820        830        840        850 SDSDSDSESD SDSESDSDSD SDSDSDSDSD SDSDSDSASD SDSGSDSDSS        860        870        880        890        900 SDSDSESDSN SDSESGSNNN VVPPNSPKNG TNASNKNEAK DSKEPLPDTG        910        920        930 SEDEANTSLI WGLLASIGSL LLFRRKKENK DKK

(98) It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

(99) It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

(100) All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

(101) The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

(102) As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the phrase “consisting essentially of” requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.

(103) The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

(104) As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least +1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

(105) All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

(106) To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

(107) For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.

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