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. 1995 May;63(5):1914–1920. doi: 10.1128/iai.63.5.1914-1920.1995

Cloning, expression, and nucleotide sequence of a Staphylococcus aureus gene (fbpA) encoding a fibrinogen-binding protein.

A I Cheung 1, S J Projan 1, R E Edelstein 1, V A Fischetti 1
PMCID: PMC173243  PMID: 7729902

Abstract

Septicemia due to Staphylococcus aureus often begins as a focal infection (e.g., colonized wounds or catheters) from which the organism gains access to the bloodstream. On the basis of recent data from this laboratory, it is likely that S. aureus colonizes catheters and endothelium by using a fibrinogen-binding protein to mediate adhesion to fibrinogen-coated surfaces. To characterize the fibrinogen-reactive protein, we screened a lambda Zap library of S. aureus DB, a clinical isolate, for clones that were reactive with fibrinogen. Of 100,000 plaques screened, 3 were found to react with fibrinogen on immunoblots. Plasmid DNA prepared from clones 14, 30, and 36, upon digestion with EcoR1, which released the insert, revealed fragments of 4.6, 3.6, and 3.2 kb, respectively. To identify the cloned protein expressed in E. coli, cells were fractionated into periplasmic, membrane, and cytoplasmic fractions. Expression studies of clone 14, which comprised approximately two-thirds of the mature molecule, including the C terminus, revealed a 34-kDa fibrinogen-reactive protein in both the periplasmic and membrane fractions. This protein, designated FbpA, could be partially purified on a fibrinogen column. By using both clones 14 and 36 as templates, the complete DNA sequence of the fibrinogen-binding protein was obtained, yielding a molecule with a predicted size of 69,991 Da. Although sequence analysis revealed a high degree of homology with coagulase, there is a unique sequence of 11 amino acids that is not found in three known coagulases as well as two recently cloned fibrinogen-binding proteins. This unique sequence shares homology with a cell wall anchor motif found in other gram-positive surface proteins.

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Selected References

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  1. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  2. Bodén M. K., Flock J. I. Cloning and characterization of a gene for a 19 kDa fibrinogen-binding protein from Staphylococcus aureus. Mol Microbiol. 1994 May;12(4):599–606. doi: 10.1111/j.1365-2958.1994.tb01046.x. [DOI] [PubMed] [Google Scholar]
  3. Bodén M. K., Flock J. I. Evidence for three different fibrinogen-binding proteins with unique properties from Staphylococcus aureus strain Newman. Microb Pathog. 1992 Apr;12(4):289–298. doi: 10.1016/0882-4010(92)90047-r. [DOI] [PubMed] [Google Scholar]
  4. Bodén M. K., Flock J. I. Fibrinogen-binding protein/clumping factor from Staphylococcus aureus. Infect Immun. 1989 Aug;57(8):2358–2363. doi: 10.1128/iai.57.8.2358-2363.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cheung A. L., Fischetti V. A. Role of surface proteins in staphylococcal adherence to fibers in vitro. J Clin Invest. 1989 Jun;83(6):2041–2049. doi: 10.1172/JCI114115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheung A. L., Fischetti V. A. The role of fibrinogen in staphylococcal adherence to catheters in vitro. J Infect Dis. 1990 Jun;161(6):1177–1186. doi: 10.1093/infdis/161.6.1177. [DOI] [PubMed] [Google Scholar]
  7. Cheung A. L., Koomey J. M., Butler C. A., Projan S. J., Fischetti V. A. Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6462–6466. doi: 10.1073/pnas.89.14.6462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheung A. L., Koomey J. M., Lee S., Jaffe E. A., Fischetti V. A. Recombinant human tumor necrosis factor alpha promotes adherence of Staphylococcus aureus to cultured human endothelial cells. Infect Immun. 1991 Oct;59(10):3827–3831. doi: 10.1128/iai.59.10.3827-3831.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheung A. L., Krishnan M., Jaffe E. A., Fischetti V. A. Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial cells. J Clin Invest. 1991 Jun;87(6):2236–2245. doi: 10.1172/JCI115259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen M. L. Epidemiology of drug resistance: implications for a post-antimicrobial era. Science. 1992 Aug 21;257(5073):1050–1055. doi: 10.1126/science.257.5073.1050. [DOI] [PubMed] [Google Scholar]
  11. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Espersen F., Clemmensen I., Barkholt V. Isolation of Staphylococcus aureus clumping factor. Infect Immun. 1985 Sep;49(3):700–708. doi: 10.1128/iai.49.3.700-708.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  14. Fischetti V. A., Jones K. F., Manjula B. N., Scott J. R. Streptococcal M6 protein expressed in Escherichia coli. Localization, purification, and comparison with streptococcal-derived M protein. J Exp Med. 1984 Apr 1;159(4):1083–1095. doi: 10.1084/jem.159.4.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fischetti V. A., Pancholi V., Schneewind O. Conservation of a hexapeptide sequence in the anchor region of surface proteins from gram-positive cocci. Mol Microbiol. 1990 Sep;4(9):1603–1605. doi: 10.1111/j.1365-2958.1990.tb02072.x. [DOI] [PubMed] [Google Scholar]
  16. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  17. Kaida S., Miyata T., Yoshizawa Y., Igarashi H., Iwanaga S. Nucleotide and deduced amino acid sequences of staphylocoagulase gene from Staphylococcus aureus strain 213. Nucleic Acids Res. 1989 Nov 11;17(21):8871–8871. doi: 10.1093/nar/17.21.8871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaida S., Miyata T., Yoshizawa Y., Kawabata S., Morita T., Igarashi H., Iwanaga S. Nucleotide sequence of the staphylocoagulase gene: its unique COOH-terminal 8 tandem repeats. J Biochem. 1987 Nov;102(5):1177–1186. doi: 10.1093/oxfordjournals.jbchem.a122156. [DOI] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. McDevitt D., Francois P., Vaudaux P., Foster T. J. Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus. Mol Microbiol. 1994 Jan;11(2):237–248. doi: 10.1111/j.1365-2958.1994.tb00304.x. [DOI] [PubMed] [Google Scholar]
  21. Neu H. C. The crisis in antibiotic resistance. Science. 1992 Aug 21;257(5073):1064–1073. doi: 10.1126/science.257.5073.1064. [DOI] [PubMed] [Google Scholar]
  22. Perlman D., Halvorson H. O. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J Mol Biol. 1983 Jun 25;167(2):391–409. doi: 10.1016/s0022-2836(83)80341-6. [DOI] [PubMed] [Google Scholar]
  23. Phonimdaeng P., O'Reilly M., Nowlan P., Bramley A. J., Foster T. J. The coagulase of Staphylococcus aureus 8325-4. Sequence analysis and virulence of site-specific coagulase-deficient mutants. Mol Microbiol. 1990 Mar;4(3):393–404. doi: 10.1111/j.1365-2958.1990.tb00606.x. [DOI] [PubMed] [Google Scholar]
  24. Phonimdaeng P., O'Reilly M., O'Toole P. W., Foster T. J. Molecular cloning and expression of the coagulase gene of Staphylococcus aureus 8325-4. J Gen Microbiol. 1988 Jan;134(1):75–83. doi: 10.1099/00221287-134-1-75. [DOI] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schneewind O., Model P., Fischetti V. A. Sorting of protein A to the staphylococcal cell wall. Cell. 1992 Jul 24;70(2):267–281. doi: 10.1016/0092-8674(92)90101-h. [DOI] [PubMed] [Google Scholar]
  27. So M., Crosa J. H., Falkow S. Polynucleotide sequence relationships among Ent plasmids and the relationship between Ent and other plasmids. J Bacteriol. 1975 Jan;121(1):234–238. doi: 10.1128/jb.121.1.234-238.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Usui Y. Biochemical properties of fibrinogen binding protein (clumping factor) of the staphylococcal cell surface. Zentralbl Bakteriol Mikrobiol Hyg A. 1986 Sep;262(3):287–297. doi: 10.1016/s0176-6724(86)80001-3. [DOI] [PubMed] [Google Scholar]
  30. Voller A., Bidwell D. E., Bartlett A. Enzyme immunoassays in diagnostic medicine. Theory and practice. Bull World Health Organ. 1976;53(1):55–65. [PMC free article] [PubMed] [Google Scholar]
  31. Watanakunakorn C., Baird I. M. Staphylococcus aureus bacteremia and endocarditis associated with a removable infected intravenous device. Am J Med. 1977 Aug;63(2):253–256. doi: 10.1016/0002-9343(77)90239-x. [DOI] [PubMed] [Google Scholar]
  32. Yother J., Briles D. E. Structural properties and evolutionary relationships of PspA, a surface protein of Streptococcus pneumoniae, as revealed by sequence analysis. J Bacteriol. 1992 Jan;174(2):601–609. doi: 10.1128/jb.174.2.601-609.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yother J., White J. M. Novel surface attachment mechanism of the Streptococcus pneumoniae protein PspA. J Bacteriol. 1994 May;176(10):2976–2985. doi: 10.1128/jb.176.10.2976-2985.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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