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.

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2000-04-01
2022-01-20
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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. 2S122–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 100:5A2S–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;
    [Google Scholar]
  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. 1104–1207Edited by Sneath P. H. A., Mair N. S., Sharpe M. E., Holt J. G. Baltimore: Williams & Wilkins;
    [Google Scholar]
  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 BacteriaSociety for General Microbiology Symposium 52 pp. 217–245Edited by Baumberg S., Young J. P. W., Wellington E. M. H., Saunders J. R. Cambridge: Cambridge University Press;
    [Google Scholar]
  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]
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