Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-11-25
2004-01-20
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023700
Reexamination Certificate
active
06680195
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the fields of microbiology and molecular biology. The invention includes the isolation and use of extracellular matrix-binding proteins and genes that express the proteins from
Staphylococcus aureus
to inhibit, prevent and diagnose
S. aureus
infection.
BACKGROUND OF THE INVENTION
In hospitalized patients
Staphylococcus aureus
is a major cause of infections associated with indwelling medical devices, such as catheters and prostheses, and related infections of surgical wounds. A significant increase in
Staphylococcus aureus
isolates that exhibit resistance to most known antibiotics has been observed in hospitals throughout the world. The recent emergence of resistance to vancomycin, the last remaining antibiotic for treating methicillin-resistant
Staphylococcus aureus
(MRSA) infections, has emphasized the need for alternative prophylactic or vaccine strategies to reduce the risk of nosocomial
S. aureus
infections.
Initial localized infections of wounds or indwelling medical devices can lead to serious invasive infections such as septicemia, osteomyelitis, and endocarditis. In infections associated with medical devices, plastic and metal surfaces become coated with host plasma and extracellular matrix proteins such as fibrinogen and fibronectin shortly after implantation. The ability of
S. aureus
to adhere to these proteins is of crucial importance for initiating infection. Vascular grafts, intravenous catheters, artificial heart valves, and cardiac assist devices are thrombogenic and prone to bacterial colonization.
S. aureus
is the most damaging pathogen to cause such infections.
Fibrin is the major component of blood clots, and fibrinogen/fibrin is one of the major plasma proteins deposited on implanted biomaterials. Considerable evidence exists to suggest that bacterial adherence to fibrinogen/fibrin is important in the initiation of device-related infection. For example, as shown by Vaudaux et al.,
S. aureus
adheres to in vitro plastic that has been coated with fibrinogen in a dose-dependent manner (
J. Infect. Dis
. 160:865-875 (1989)). In addition, in a model that mimics a blood clot or damage to a heart valve, Herrmann et al. demonstrated that
S. aureus
binds avidly via a fibrinogen bridge to platelets adhering to surfaces (
J. Infect. Dis
. 167: 312-322 (1993)).
S. aureus
can adhere directly to fibrinogen in blood clots formed in vitro, and can adhere to cultured endothelial cells via fibrinogen deposited from plasma acting as a bridge (Moreillon et al.,
Infect. Immun
. 63:4738-4743 (1995); Cheung et al.,
J. Clin. Invest
. 87:2236-2245 (1991)). As shown by Vaudaux et al. and Moreillon et al., mutants defective in the fibrinogen-binding protein clumping factor (ClfA) exhibit reduced adherence to fibrinogen in vitro, to explanted catheters, to blood clots, and to damaged heart valves in the rat model for endocarditis (Vaudaux et al.,
Infect. Immun
. 63:585-590 (1995); Moreillon et al.,
Infect. Immun
. 63: 4738-4743 (1995)).
An adhesin for fibrinogen, often referred to as “clumping factor,” is located on the surface of
S. aureus
cells. The interaction between the clumping factor on bacteria and fibrinogen in solution results in the instantaneous clumping of bacterial cells. The binding site on fibrinogen is located in the C-terminus of the gamma chain of the dimeric fibrinogen glycoprotein. The affinity is very high and clumping occurs in low concentrations of fibrinogen. Scientists have recently shown that clumping factor also promotes adherence to solid phase fibrinogen, to blood clots, and to damaged heart valves (McDevitt et al.,
Mol. Microbiol
. 11: 237-248 (1994); Vaudaux et al.,
Infect. Immun
. 63:585-590 (1995); Moreillon et al.,
Infect. Immun
. 63: 4738-4743 (1995)).
The gene for a clumping factor protein, designated ClfA, has been cloned, sequenced and analyzed in detail at the molecular level (McDevitt et al.,
Mol. Microbiol
. 11: 237-248 (1994); McDevitt et al.,
Mol. Microbiol
. 16:895-907 (1995)). The predicted protein is composed of 933 amino acids. A signal sequence of 39 residues occurs at the N-terminus followed by a 520 residue region (region A), which contains the fibrinogen binding domain. A 308 residue region (region R), composed of 154 repeats of the dipeptide serine-aspartate, follows. The R region sequence is encoded by the 18 basepair repeat GAYTCNGAYT CNGAYAGY (SEQ ID NO: 9) in which Y equals pyrimidines and N equals any base. The C-terminus of ClfA has features present in many surface proteins of Gram-positive bacteria such as an LPDTG (SEQ ID NO: 10) motif, which is responsible for anchoring the protein to the cell wall, a membrane anchor, and positive charged residues at the extreme C-terminus.
The platelet integrin alpha IIb&bgr;3 recognizes the C-terminus of the gamma chain of fibrinogen. This is a crucial event in the initiation of blood clotting during coagulation. ClfA and alpha IIb&bgr;3 appear to recognize precisely the same sites on fibrinogen gamma chain because ClfA can block platelet aggregation, and a peptide corresponding to the C-terminus of the gamma chain (198-411) can block both the integrin and ClfA interacting with fibrinogen (McDevitt et al.,
Eur. J. Biochem
. 247:416-424 (1997)). The fibrinogen binding site of alpha IIb&bgr;3 is close to, or overlaps, a Ca
2+
binding determinant referred to as an “EF hand”. ClfA region A carries several EF hand-like motifs. A concentration of Ca
2+
in the range of 3-5 mM blocks these ClfA-fibrinogen interactions and changes the secondary structure of the ClfA protein. Mutations affecting the ClfA EF hand reduce or prevent interactions with fibrinogen. Ca
2+
and the fibrinogen gamma chain seem to bind to the same, or to overlapping, sites in ClfA region A.
The alpha chain of the leucocyte integrin, alpha M&bgr;2, has an insertion of 200 amino acids (A or I domain) which is responsible for ligand binding activities. A novel metal ion-dependent adhesion site (MIDAS) motif in the I domain is required for ligand binding. Among the ligands recognized is fibrinogen. The binding site on fibrinogen is in the gamma chain (residues 190-202). It was recently reported that
Candida albicans
has a surface protein, alpha Int1p, having properties reminiscent of eukaryotic integrins. The surface protein has amino acid sequence homology with the I domain of alpha M&bgr;2, including the MIDAS motif. Furthermore, alpha Int1p binds to fibrinogen.
ClfA region A also exhibits some degree of sequence homology with alpha Int1p. Examination of the ClfA region A sequence has revealed a potential MIDAS motif. Mutations in supposed cation coordinating residues in the DXSXS (SEQ ID NO: 13) portion of the MIDAS motif in ClfA results in a significant reduction in fibrinogen binding. A peptide corresponding to the gamma-chain binding site for alpha M&bgr;2 (190-202) has been shown by O'Connell et al. to inhibit ClfA-fibrinogen interactions (O'Connell et al.,
J. Biol. Chem
., in press). Thus it appears that ClfA can bind to the gamma-chain of fibrinogen at two separate sites. The ligand binding sites on the ClfA are similar to those employed by eukaryotic integrins and involve divalent cation binding EF-hand and MIDAS motifs.
Scientists have recently shown that
S. aureus
expresses proteins other than ClfA that may bind fibrinogen (Boden and Flock,
Mol. Microbiol
. 12:599-606 (1994)). One of these proteins is probably the same as the broad spectrum ligand-binding protein reported by Homonylo-McGavin et al.,
Infect. Immun
. 61:2479-2485 (1993). Another is coagulase, as reported by Boden and Flock,
Infect. Immun
. 57:2358-2363 (1989), a predominantly extracellular protein that activates the plasma clotting activity of prothrombin. Coagulase binds prothrombin at its N-terminus and also interacts with soluble fibrinogen at its C-terminus. Cheung et al.,
Infect. Immun
. 63:1914-1920 (1995) have described a variant of coagulase that binds fibrinogen. There is some evidence that coagulase can contribute, in a min
Eidhin Deidre Ni
Foster Timothy J.
Hook Magnus A. O.
Josefsson Elisabet
Patti Joseph M.
Duffy Patricia A.
Inhibitex, Inc.
Larson & Taylor PLC
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