1887

Abstract

Six strains of examined by electron microscopy were found to carry flagella. The flagella of these strains were extracted and the N-terminal sequences of the flagellin proteins were determined. Four of the strains carried the N-terminal sequence MRVNTNVSAL exhibiting up to 90% identity to numerous flagellins. Using degenerate primers based on the N-terminal sequence and the conserved C-terminal sequence of several flagellins, the gene encoding the flagellum subunit () was isolated and sequenced from two virulent strains. The two gene sequences exhibited 91% inter-strain identity. The gene consists of 870 nt encoding a protein of 290 amino acids with an estimated molecular mass of 31 kDa, while the extracted flagellin has an apparent molecular mass of 39 kDa on SDS-PAGE. The FliC protein displays a high degree of identity in the N- and C-terminal amino acids whereas the central region is variable. A second ORF is present downstream of displaying homology to glycosyltransferases. The gene was expressed in fusion with glutathione -transferase, purified and a polyclonal monospecific antiserum was obtained. Flagella of do not play a role in adherence, since the antiserum raised against the purified protein did not inhibit adherence to cultured cells. PCR-RFLP analysis of amplified flagellin gene products and Southern analysis revealed inter-strain heterogeneity; this could be useful for epidemiological and phylogenetic studies of this organism.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-4-957
2000-04-01
2019-10-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/4/1460957a.html?itemId=/content/journal/micro/10.1099/00221287-146-4-957&mimeType=html&fmt=ahah

References

  1. Arnold, F., Bedouet, L., Batina, G., Robreau, G., Talbot, F., Lecher, L. & Malcoste, R. ( 1998; ). Biochemical and immunological analysis of the flagellin of Clostridium tyrobutyricum ATCC 25755. Microbiol Immunol 42, 23-31.[CrossRef]
    [Google Scholar]
  2. Arora, S. K., Ritchings, B. W., Almira, E. C., Lory, S. & Ramphal, R. ( 1996; ). Cloning and characterization of Pseudomonas aeruginosa fliF, necessary for flagella assembly and bacterial adherence to mucin. Infect Immun 64, 2130-2136.
    [Google Scholar]
  3. Arora, S. K., Ritchings, B. W., Almira, E. C., Lory, S. & Ramphal, R. ( 1998; ). The Pseudomonas aeruginosa flagellar cap protein, FliD, is responsible for mucin adhesion. Infect Immun 66, 1000-1007.
    [Google Scholar]
  4. Bartlett, J. G., Chang, T. W., Gurwiyh, M., Gorbach, S. L. & Onderdonk, A. M. ( 1978; ). Antibiotic associated pseudomembranous colitis due to toxin producing clostridia. N Engl J Med 298, 531-534.[CrossRef]
    [Google Scholar]
  5. Borriello, S. P., Davies, H. A. & Barclay, F. E. ( 1988a; ). Detection of fimbriae amongst strains of Clostridium difficile. FEMS Microbiol Lett 49, 65-67.[CrossRef]
    [Google Scholar]
  6. Borriello, S. P., Welch, A. R., Barclay, F. E. & Davies, M. A. ( 1988b; ). Mucosal association by Clostridium difficile in the hamster gastrointestinal tract. J Med Microbiol 25, 191-196.[CrossRef]
    [Google Scholar]
  7. Bosshardt, S. C., Benson, R. F. & Field, B. S. ( 1997; ). Flagella are a positive predictor for virulence in Legionella. Microb Pathog 23, 107-112.[CrossRef]
    [Google Scholar]
  8. Brett, P. J., Mah, D. C. & Wood, D. E. ( 1994; ). Isolation and characterization of Pseudomonas pseudomallei flagellin proteins. Infect Immun 62, 1914-1918.
    [Google Scholar]
  9. 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, 45-148.
    [Google Scholar]
  10. Chung, C. T., Niemala, S. L. & Miller, H. R. ( 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]
  11. Davies, H. A. & Borriello, S. P. ( 1990; ). Detection of capsule in strains of Clostridium difficile. Microb Pathog 9, 141-146.[CrossRef]
    [Google Scholar]
  12. Delmée, M., Avesani, V., Delferriere, N. & Burtonboy, G. ( 1990; ). Characterization of flagella of Clostridium difficile and their role in serogrouping reactions. J Clin Microbiol 28, 2210-2214.
    [Google Scholar]
  13. Eaton, K. A., Suerbaum, S., Josenhams, C. & Krakowka, S. ( 1996; ). Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect Immun 64, 2445-2448.
    [Google Scholar]
  14. Edman, P. ( 1950; ). Preparation of phenylhydantoins from some natural amino acids. Acta Chem Scand 4, 277-282.[CrossRef]
    [Google Scholar]
  15. Eveillard, M., Fourel, V., Barc, M. C., Kerneis, S., Coconnier, M. H., Karjalainen, T., Bourlioux, P. & Servin, A. ( 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]
  16. Fedorov, O. V. & Efimov, A. V. ( 1990; ). Flagellin as an object for supramolecular engineering. Protein Eng 3, 411-413.[CrossRef]
    [Google Scholar]
  17. Feldman, M., Bryan, R., Rajan, S., Scheffler, L., Tang, B. S. L. & Prince, A. ( 1998; ). Role of flagella in pathogenesis of Pseudomonas aeruginosa pulmonary infection. Infect Immun 66, 43-51.
    [Google Scholar]
  18. Gardel, C. L. & Mekalanos, J. J. ( 1996; ). Alterations in Vibrio cholerae motility phenotypes correlate with changes in virulence factor expression. Infect Immun 64, 2246-2255.
    [Google Scholar]
  19. Ge, Y., Li, C., Slaughter, C. A. & Charon, N. W. ( 1988; ). Structure and expression of the FlaA periplasmic flagellar protein of Borrelia burgdorferi. J Bacteriol 180, 2418-2425.
    [Google Scholar]
  20. George, W. L. ( 1984; ). Antimicrobial agent associated colitis and diarrhea: historical background and clinical aspects. Rev Infect Dis 6, 208-213.[CrossRef]
    [Google Scholar]
  21. Gilman, M. Z. & Chamberlin, M. J. ( 1983; ). Development and genetic regulation of the Bacillus subtilis genes transcribed by σ28 RNA polymerase. Cell 35, 285-293.[CrossRef]
    [Google Scholar]
  22. Grant, C. C., Konkel, M. E., Cieplak, W. J. & Tompkins, L. S. ( 1993; ). Role of flagella in adherence, internalization and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures. Infect Immun 61, 1764-1771.
    [Google Scholar]
  23. Grossman, D. A., Witham, N. D., Burr, D., Lesmana, M., Rubin, F. A., Schoolnik, G. K. & Parsonnet, J. ( 1995; ). Flagellar serotypes of Salmonella typhi in Indonesia: relationship among motility, invasiveness, and clinical illness. J Infect Dis 171, 212-216.[CrossRef]
    [Google Scholar]
  24. Guerry, P. ( 1997; ). Nonlipopolysaccharide surface antigens of Campylobacter species. J Infect Dis 176 (suppl. 2), S122–S124.
    [Google Scholar]
  25. Heinzerling, H. F., Olivares, M. & Burne, R. A. ( 1997; ). Genetic and transcriptional analysis of flgB flagellar operon constituents in the oral spirochete Treponema denticola and their heterologous expression in enteric bacteria. Infect Immun 65, 2041-2051.
    [Google Scholar]
  26. Hopp, T. P. & Woods, K. R. ( 1981; ). Prediction of protein antigenic determinants from aminoacid sequences. Proc Natl Acad Sci USA 78, 3824-3828.[CrossRef]
    [Google Scholar]
  27. Joys, T. M. ( 1988; ). The flagellar filament protein. Can J Microbiol 34, 452-458.[CrossRef]
    [Google Scholar]
  28. Karjalainen, T., Barc, M. C., Collignon, A., Trollé, S., Boureau, H., Cotte-Laffite, 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]
  29. Kennedy, M. J., Rosey, E. L. & Yancey, R. J. J. ( 1997; ). Characterization of flaA- and flaB- mutants of Serpulina hyodysenteriae: both flagellin subunits, FlaA and FlaB, are necessary for full motility and intestinal colonization. FEMS Microbiol Lett 153, 119-128.[CrossRef]
    [Google Scholar]
  30. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef]
    [Google Scholar]
  31. Larson, H. E., Honour, P., Price, A. B. & Borriello, S. P. ( 1978; ). Clostridium difficile and aetiology of pseudomembranous colitis. Lancet 1, 1063-1066.
    [Google Scholar]
  32. Liu, S. L., Ezaki, T., Miura, H., Matsui, K. & Yabuuchi, E. ( 1988; ). Intact motility as a Salmonella typhi invasion-related factor. Infect Immun 56, 1967-1973.
    [Google Scholar]
  33. Lyerly, D. M. H., Krivan, H. C. & Wilkins, T. D. ( 1988; ). Clostridium difficile: its disease and toxins. Clin Microbiol Rev 1, 1-12.
    [Google Scholar]
  34. McGee, K., Horstedt, P. & Milton, D. L. ( 1996; ). Identification and characterization of additional flagellin genes from Vibrio anguillarum. J Bacteriol 178, 1310-1319.
    [Google Scholar]
  35. Milton, D. L., Otoole, R., Horstedt, P. & Wolfwatz, P. ( 1996; ). Flagellin A is essential for the virulence of Vibrio anguillarum. J Bacteriol 178, 1310-1319.
    [Google Scholar]
  36. Mimori-Kiyosue, Y., Vonderviszt, F. & Namba, K. ( 1997; ). Locations of terminal segments of flagellin in the filament structure and their roles in polymerization and polymorphism. J Mol Biol 270, 222-237.[CrossRef]
    [Google Scholar]
  37. Mobley, H. L. ( 1996; ). Defining Helicobacter pylori as a pathogen: strain heterogeneity and virulence. Am J Med 100(5A), 2S–9S.[CrossRef]
    [Google Scholar]
  38. Mobley, H. L. T., Belas, R., Lockatell, V., Chippendale, G., Trifillis, A. L., Johnson, D. E. & Warren, J. W. ( 1996; ). Construction of a flagellum-negative mutant of Proteus mirabilis: effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection. Infect Immun 64, 5332-5340.
    [Google Scholar]
  39. Morooka, T., Umeda, A. & Amado, K. ( 1985; ). Motility as an intestinal colonization factor for Campylobacter jejuni. J Gen Microbiol 131, 1973-1980.
    [Google Scholar]
  40. Perelle, S., Gibert, M., Bourlioux, P., Corthier, G. & Popoff, M. ( 1997; ). Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196. Infect Immun 65, 1402-1407.
    [Google Scholar]
  41. 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]
  42. Pruckler, J. M., Benson, R. F., Moyenuddin, M., Martin, W. T. & Fields, B. S. ( 1995; ). Association of flagellum expression and intracellular growth of Legionella pneumophila. Infect Immun 63, 4928-4932.
    [Google Scholar]
  43. Richardson, K. ( 1991; ). Roles of motility and flagellar structure in pathogenicity of Vibrio cholerae: analysis of motility mutants in three animal models. Infect Immun 59, 2727-2736.
    [Google Scholar]
  44. Ritchings, B. W., Almira, E. C., Lory, S. & Ramphal, R. ( 1995; ). Cloning and phenotypic characterization of fleS and fleR, new response regulators of Pseudomonas aeruginosa which regulate motility and adhesion to mucin. Infect Immun 63, 4868-4876.
    [Google Scholar]
  45. Rosalski, A., Sidorczyk, Z. & Kotelko, K. ( 1997; ). Potential virulence factors of Proteus bacilli. Microbiol Mol Biol Rev 6, 65-89.
    [Google Scholar]
  46. Sakamoto, Y., Sutherland, K. J., Tamaoka, J., Kobayashi, T., Kudo, T. & Horikoshi, K. ( 1992; ). Analysis of the flagellin (hag) gene of alkalophilic Bacillus sp. C-125. J Gen Microbiol 138, 2159-2166.[CrossRef]
    [Google Scholar]
  47. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  48. Scherer, D. C., DeBurron-Connors, C. I. & Minnick, M. F. ( 1993; ). Characterization of Bartonella bacilliformis flagella and effect of antiflagellin antibodies on invasion of human erythrocytes. Infect Immun 61, 4962-4971.
    [Google Scholar]
  49. Seddon, S. V. & Borriello, S. P. ( 1992; ). Proteolytic activity of Clostridium difficile. J Med Microbiol 36, 307-311.[CrossRef]
    [Google Scholar]
  50. Sneath, P. H. A. ( 1986; ). Endospore-forming Gram-positive rods and cocci. In Bergey’s Manual of Systematic Bacteriology, pp. 1104-1207. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
  51. Szymanski, C. M., King, M., Haardt, M. & Armstrong, G. T. ( 1995; ). Campylobacter jejuni motility and invasion of Caco-2 cells. Infect Immun 63, 4295-4300.
    [Google Scholar]
  52. Tamura, Y., Kijima-Tanaka, M., Aoki, A., Ogikubo, Y. & Takahashi, T. ( 1995; ). Reversible expression of motility and flagella in Clostridium chauvoei and their relationship to virulence. Microbiology 141, 605-610.[CrossRef]
    [Google Scholar]
  53. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. ( 1997; ). The clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24, 4876-4882.
    [Google Scholar]
  54. West, N. P., Fitter, J. T., Jakubzik, U., Rohde, M., Guzman, C. A. & Walker, M. J. ( 1997; ). Non-motile mini-transposon mutants of Bordetella bronchiseptica exhibit altered abilities to invade and survive in eukaryotic cells. FEMS Microbiol Lett 146, 263-269.[CrossRef]
    [Google Scholar]
  55. Whittam, T. S. ( 1995; ). Genetic population structure and pathogenicity in enteric bacteria. In Population Genetics of Bacteria (Society for General Microbiology Symposium 52), pp. 217-245. Edited by S. Baumberg, J. P. W. Young, E. M. H. Wellington & J. R. Saunders. Cambridge: Cambridge University Press.
  56. Wilson, D. R. & Beveridge, T. J. ( 1993; ). Bacterial flagellar filaments and their component flagellins. Can J Microbiol 39, 451-472.[CrossRef]
    [Google Scholar]
  57. Winstanley, C. & Morgan, J. A. ( 1997; ). The bacterial flagellin gene as a biomarker for detection, population genetics and epidemiological analysis. Microbiology 143, 3071-3084.[CrossRef]
    [Google Scholar]
  58. Winstanley, C., Coulson, M., Wepner, B., Morgan, J. A. & Hart, C. ( 1996; ). Flagellin gene and protein variation amongst clinical isolates of Pseudomonas aeruginosa. Microbiology 142, 2145-2151.[CrossRef]
    [Google Scholar]
  59. Zhang, M. Y., Lovgren, A., Low, M. G. & Landen, R. ( 1993; ). Characterization of an avirulent pleiotropic mutant of the insect pathogen Bacillus thuringiensis: reduced expression of flagellin and phospholipases. Infect Immun 61, 4947-4954.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-4-957
Loading
/content/journal/micro/10.1099/00221287-146-4-957
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error