1887

Abstract

Previous results have demonstrated that adherence of to tissue culture cells is augmented by various stresses; this study focussed on whether the GroEL heat shock protein is implicated in this process. The 1940 bp operon of was isolated by PCR. The 1623 bp gene is highly conserved between various isolates as determined by RFLP-PCR and DNA sequencing, and the operon is present in one copy on the bacterial chromosome. The 58 kDa GroEL protein was expressed in in fusion with glutathione -transferase and the fusion protein was purified from IPTG-induced bacterial lysates by affinity chromatography on glutathione–Sepharose. A polyclonal, monospecific antiserum was obtained for GroEL which established by immunoelectron microscopy, indirect immunofluorescence and immunoblot analysis that GroEL is released extracellularly after heat shock and can be surface associated. Cell fractionation experiments suggest that GroEL is predominantly cytoplasmic and membrane bound. GroEL-specific antibodies as well as the purified protein partially inhibited cell attachment and expression of the protein was induced by cell contact, suggesting a role for GroEL in cell adherence.

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2001-01-01
2024-12-14
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References

  1. Bartlett, J. G., Chang, T. W., Gurwith, M., Gorbach, S. L. & Onderdonk, A. B.(1978). Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N Engl J Med 298, 531-534.[CrossRef] [Google Scholar]
  2. Borriello, S. P., Welch, A. R., Barclay, F. E. & Davies, H. A.(1988a). Mucosal association by Clostridium difficile in the hamster gastrointestinal tract. J Med Microbiol 25, 191-196.[CrossRef] [Google Scholar]
  3. Borriello, S. P., Davies, H. A. & Barclay, F. E.(1988b). Detection of fimbriae amongst strains of Clostridium difficile. FEMS Microbiol Lett 49, 65-67.[CrossRef] [Google Scholar]
  4. Bukau, B., Reilly, P., McCarty, J. & Walker, G. C.(1993). Immunogold localization of the DnaK heat shock protein in Escherichia coli cells. J Gen Microbiol 139, 95-99.[CrossRef] [Google Scholar]
  5. Cao, P., McClain, M. S., Forsyth, M. H. & Cover, T. L.(1998). Extracellular release of antigenic proteins by Helicobacter pylori. Infect Immun 66, 2984-2986. [Google Scholar]
  6. Chou, P. Y. & Fasman, G. D.(1978). Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol 47, 45-48. [Google Scholar]
  7. Chung, C. T., Niemela, S. L. & Miller, R. H.(1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA 86, 2172-2175.[CrossRef] [Google Scholar]
  8. Davies, H. A. & Borriello, S. P.(1990). Detection of capsule in strains of Clostridium difficile of varying virulence and toxigenicity. Microb Pathog 9, 141-146.[CrossRef] [Google Scholar]
  9. Dunn, B. E., Vakil, N. B., Schneider, B. G., Miller, M. M., Zitzer, J. B., Peutz, T. & Phadnis, S. H.(1997). Localization of Helicobacter pylori urease and heat shock protein in human gastric biopsies. Infect Immun 65, 1181-1188. [Google Scholar]
  10. Ensgraber, M. & Loos, M.(1992). A 66-kilodalton heat shock protein of Salmonella typhimurium is responsible for binding of the bacterium to intestinal mucus. Infect Immun 60, 3072-3078. [Google Scholar]
  11. Eschweiler, B., Bohrmann, B., Gerstenecker, B., Schiltz, E. & Kist, M.(1993).In situ localization of the 60 k protein of Helicobacter pylori, which belongs to the family of heat shock proteins, by immunoelectron microscopy. Zentbl Bakteriol 280, 73-85.[CrossRef] [Google Scholar]
  12. Eveillard, M., Fourel, V., Barc, M. C., Kerneis, S., Coconnier, M. H., Karjalainen, T., Bourlioux, P. & Servin, A. L.(1993). Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT29 cells in culture. Mol Microbiol 7, 371-381.[CrossRef] [Google Scholar]
  13. Frisk, A., Ison, C. A. & Lagergard, T.(1998). GroEL heat shock protein of Haemophilus ducreyi: association with cell surface and capacity to bind to eukaryotic cells. Infect Immun 66, 1252-1257. [Google Scholar]
  14. Garduno, R. A., Faulkner, G., Trevors, M. A., Vats, N. & Hoffman, P. S.(1998a). Immunolocalization of Hsp60 in Legionella pneumophila. J Bacteriol 180, 505-513. [Google Scholar]
  15. Garduno, R. A., Garduno, E. & Hoffman, P. S.(1998b). Surface-associated Hsp60 chaperonin of Legionella pneumophila mediates invasion in a HeLa cell model. Infect Immun 66, 4602-4610. [Google Scholar]
  16. George, W. L. (1984). Antimicrobial agent-associated colitis and diarrhoea: historical background and clinical aspects. Rev Infect Dis 6 (Suppl 1), S208–S213.[CrossRef] [Google Scholar]
  17. Goulhen, F., Hafezi, A., Uitto, V. J., Hinode, D., Nakamura, R., Grenier, D. & Mayrand, D.(1998). Subcellular localization and cytotoxic activity of the GroEL-like protein isolated from Actinobacillus actinomycetemcomitans. Infect Immun 66, 5307-5313. [Google Scholar]
  18. Haldenwang, W. G.(1995). The sigma factors of Bacillus subtilis. Microbiol Rev 59, 1-30. [Google Scholar]
  19. Hecker, M., Schumann, W. & Volker, U.(1996). Heat-shock and general stress response in Bacillus subtilis. Mol Microbiol 19, 417-428.[CrossRef] [Google Scholar]
  20. Hoffman, P. S., Houston, L. & Butler, C. A.(1990).Legionella pneumophila htpAB heat shock operon: nucleotide sequence and expression of the 60-kilodalton antigen in L. pneumophila-infected HeLa cells. Infect Immun 58, 3380-3387. [Google Scholar]
  21. Huesca, M., Borgia, S., Hoffman, P. & Lingwood, C. A.(1996). Acidic pH changes receptor binding specificity of Helicobacter pylori: a binary adhesion model in which surface heat shock (stress) proteins mediate sulfatide recognition in gastric colonization. Infect Immun 64, 2643-2648. [Google Scholar]
  22. Huesca, M., Goodwin, A., Bhagwansingh, A., Hoffman, P. & Lingwood, C. A.(1998). Characterization of an acidic-pH-inducible stress protein (hsp70), a putative sulfatide binding adhesin, from Helicobacter pylori. Infect Immun 66, 4061-4067. [Google Scholar]
  23. Jonquières, R., Bierne, H., Fiedler, F., Gounon, P. & Cossart, P.(1999). Interaction between the protein InlB of Listeria monocytogenes and lipoteichoic acid: a novel mechanism of protein association at the surface of gram-positive bacteria. Mol Microbiol 34, 902-914.[CrossRef] [Google Scholar]
  24. Kamiya, S., Yamaguchi, H., Osaki, T. & Taguchi, H.(1998). A virulence factor of Helicobacter pylori: role of heat shock protein in mucosal inflammation after H. pylori infection. J Clin Gastroenterol 27, S35-S39.[CrossRef] [Google Scholar]
  25. Kaneda, K., Masuzawa, T., Yasugami, K., Suzuki, T., Suzuki, Y. & Yanagihara, Y.(1997). Glycosphingolipid-binding protein of Borrelia burgdorferi sensu lato. Infect Immun 65, 3180-3185. [Google Scholar]
  26. Karjalainen, T., Barc, M. C., Collignon, A., Trolle, S., Boureau, H., Cotte-Laffitte, J. & Bourlioux, P.(1994). Cloning of a genetic determinant from Clostridium difficile involved in adherence to tissue culture cells and mucus. Infect Immun 62, 4347-4355. [Google Scholar]
  27. Laemmli, U. K.(1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef] [Google Scholar]
  28. Lyerly, D. M., Krivan, H. C. & Wilkins, T. D.(1988).Clostridium difficile: its disease and toxins. Clin Microbiol Rev 1, 1-18. [Google Scholar]
  29. Matzura, O. & Wennborg, A.(1996). RNAdraw: an integrated program for RNA secondary structure calculation and analysis under 32-bit Microsoft Windows. Comput Appl Biosci 12, 247-249. [Google Scholar]
  30. Mogk, A., Homuth, G., Scholz, C., Kim, L., Schmid, F. X. & Schumann, W.(1997). The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis. EMBO J 16, 4579-4590.[CrossRef] [Google Scholar]
  31. Parsons, L. M., Limberger, R. J. & Shayegani, M.(1997). Alterations in levels of DnaK and GroEL result in diminished survival and adherence of stressed Haemophilus ducreyi. Infect Immun 65, 2413-2419. [Google Scholar]
  32. Phadnis, S. H., Parlow, M. H., Levy, M., Ilver, D., Caulkins, C. M., Connors, J. B. & Dunn, B. E.(1996). Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis. Infect Immun 64, 905-912. [Google Scholar]
  33. Poilane, I., Karjalainen, T., Barc, M. C., Bourlioux, P. & Collignon, A.(1998). Protease activity of Clostridium difficile strains. Can J Microbiol 44, 157-161.[CrossRef] [Google Scholar]
  34. Qoronfleh, M. W., Bortner, C. A., Schwartzenberg, P. & Wilkinson, B. J.(1998). Enhanced levels of Staphylococcus aureus stress protein GroEL and DnaK homologs early in infection of human epithelial cells. Infect Immun 66, 3024-3027. [Google Scholar]
  35. Rao, S. P., Ogata, K., Morris, S. L. & Catanzaro, A.(1994). Identification of a 68 kD surface antigen of Mycobacterium avium that binds to human macrophages. J Lab Clin Med 123, 526-535. [Google Scholar]
  36. Rusanganwa, E., Singh, B. & Gupta, R. S.(1992). Cloning of HSP60 (GroEL) operon from Clostridium perfringens using a polymerase chain reaction based approach. Biochim Biophys Acta 1130, 90-94.[CrossRef] [Google Scholar]
  37. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  38. Schubert, K., Bichlmaier, A. M., Mager, E., Wolff, K., Ruhland, G. & Fiedler, F.(2000). P45, an extracellular 45 kDa protein of Listeria monocytogenes with similarity to protein p60 and exhibiting peptidoglycan lytic activity. Arch Microbiol 173, 21-28.[CrossRef] [Google Scholar]
  39. Seddon, S. V. & Borriello, S. P.(1992). Proteolytic activity of Clostridium difficile. J Med Microbiol 36, 307-311.[CrossRef] [Google Scholar]
  40. Tasteyre, A., Barc, M.-C., Karjalainen, T., Dodson, P., Hyde, S., Bourlioux, P. & Borriello, P.(2000). A Clostridium difficile gene encoding flagellin. Microbiology 146, 957-966. [Google Scholar]
  41. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.(1997). The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882.[CrossRef] [Google Scholar]
  42. Vanet, A. & Labigne, A.(1998). Evidence for specific secretion rather than autolysis in the release of some Helicobacter pylori proteins. Infect Immun 66, 1023-1027. [Google Scholar]
  43. Waligora, A. J., Barc, M.-C., Bourlioux, P., Collignon, A. & Karjalainen, T.(1999).Clostridium difficile cell attachment is modified by environmental factors. Appl Environ Microbiol 65, 4234-4238. [Google Scholar]
  44. Yamaguchi, H., Osaki, T., Taguchi, H., Hanawa, T., Yamamoto, T. & Kamiya, S. (1996a). Induction and epitope analysis of Helicobacter pylori heat shock protein. J Gastroenterol 31 (Suppl 9), 12–15. [Google Scholar]
  45. Yamaguchi, H., Osaki, T., Taguchi, H., Hanawa, T., Yamamoto, T. & Kamiya, S.(1996b). Flow cytometric analysis of the heat shock protein 60 expressed on the cell surface of Helicobacter pylori. J Med Microbiol 45, 270-277.[CrossRef] [Google Scholar]
  46. Yamaguchi, H., Osaki, T., Kurihara, N., Taguchi, H., Hanawa, T., Yamamoto, T. & Kamiya, S.(1997). Heat-shock protein 60 homologue of Helicobacter pylori is associated with adhesion of H. pylori to human gastric epithelial cells. J Med Microbiol 46, 825-831.[CrossRef] [Google Scholar]
  47. Yamaguchi, H., Osaki, T., Taguchi, H., Hanawa, T., Yamamoto, T. & Kamiya, S.(1998). Relationship between expression of HSP60, urease activity, production of vacuolating toxin, and adherence activity of Helicobacter pylori. J Gastroenterol 33, 6-9.[CrossRef] [Google Scholar]
  48. Zugel, U. & Kaufmann, S. H.(1999). Role of heat shock proteins in protection from and pathogenesis of infectious diseases. Clin Microbiol Rev 12, 19-39. [Google Scholar]
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