• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Feb 1991; 173(3): 937–946.
PMCID: PMC207209

Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella.


Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.5M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Blake MS, Johnston KH, Russell-Jones GJ, Gotschlich EC. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. [PubMed]
  • Blaser MJ. Helicobacter pylori and the pathogenesis of gastroduodenal inflammation. J Infect Dis. 1990 Apr;161(4):626–633. [PubMed]
  • Blaser MJ. Epidemiology and pathophysiology of Campylobacter pylori infections. Rev Infect Dis. 1990 Jan-Feb;12 (Suppl 1):S99–106. [PubMed]
  • Chandler HM, Gulasekharam J. The protective antigen of a highly immunogenic strain of clostridium chauvoei including an evaluation of its flagella as a protective antigen. J Gen Microbiol. 1974 Sep;84(1):128–134. [PubMed]
  • Cheng SC, Sanderson CR, Waters TE, Goodwin CS. Campylobacter pyloridis in patients with gastric carcinoma. Med J Aust. 1987 Aug 17;147(4):202–203. [PubMed]
  • Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed]
  • DeLange RJ, Chang JY, Shaper JH, Glazer AN. Amino acid sequence of flagellin of Bacillus subtilis 168. III. Tryptic peptides, N-bromosuccinimide peptides, and the complete amino acid sequence. J Biol Chem. 1976 Feb 10;251(3):705–711. [PubMed]
  • Dunn BE, Perez-Perez GI, Blaser MJ. Two-dimensional gel electrophoresis and immunoblotting of Campylobacter pylori proteins. Infect Immun. 1989 Jun;57(6):1825–1833. [PMC free article] [PubMed]
  • Fuerst JA, Perry JW. Demonstration of lipopolysaccharide on sheathed flagella of Vibrio cholerae O:1 by protein A-gold immunoelectron microscopy. J Bacteriol. 1988 Apr;170(4):1488–1494. [PMC free article] [PubMed]
  • Geis G, Leying H, Suerbaum S, Mai U, Opferkuch W. Ultrastructure and chemical analysis of Campylobacter pylori flagella. J Clin Microbiol. 1989 Mar;27(3):436–441. [PMC free article] [PubMed]
  • Gill PR, Agabian N. The nucleotide sequence of the Mr = 28,500 flagellin gene of Caulobacter crescentus. J Biol Chem. 1983 Jun 25;258(12):7395–7401. [PubMed]
  • Goodwin CS, McCulloch RK, Armstrong JA, Wee SH. Unusual cellular fatty acids and distinctive ultrastructure in a new spiral bacterium (Campylobacter pyloridis) from the human gastric mucosa. J Med Microbiol. 1985 Apr;19(2):257–267. [PubMed]
  • Guerry P, Logan SM, Thornton S, Trust TJ. Genomic organization and expression of Campylobacter flagellin genes. J Bacteriol. 1990 Apr;172(4):1853–1860. [PMC free article] [PubMed]
  • Harris LA, Logan SM, Guerry P, Trust TJ. Antigenic variation of Campylobacter flagella. J Bacteriol. 1987 Nov;169(11):5066–5071. [PMC free article] [PubMed]
  • Hazell SL, Lee A, Brady L, Hennessy W. Campylobacter pyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium. J Infect Dis. 1986 Apr;153(4):658–663. [PubMed]
  • Hu LT, Mobley HL. Purification and N-terminal analysis of urease from Helicobacter pylori. Infect Immun. 1990 Apr;58(4):992–998. [PMC free article] [PubMed]
  • Jiang SJ, Liu WZ, Zhang DZ, Shi Y, Xiao SD, Zhang ZH, Lu DY. Campylobacter-like organisms in chronic gastritis, peptic ulcer, and gastric carcinoma. Scand J Gastroenterol. 1987 Jun;22(5):553–558. [PubMed]
  • Joys TM, Rankis V. The primary structure of the phase-1 flagellar protein of Salmonella typhimurium. I. The tryptic peptides. J Biol Chem. 1972 Aug 25;247(16):5180–5193. [PubMed]
  • Krakowka S, Morgan DR, Kraft WG, Leunk RD. Establishment of gastric Campylobacter pylori infection in the neonatal gnotobiotic piglet. Infect Immun. 1987 Nov;55(11):2789–2796. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lambert JR, Borromeo M, Pinkard KJ, Turner H, Chapman CB, Smith ML. Colonization of gnotobiotic piglets with Campylobacter Pyloridis--an animal model? J Infect Dis. 1987 Jun;155(6):1344–1344. [PubMed]
  • LeGendre N, Matsudaira P. Direct protein microsequencing from Immobilon-P Transfer Membrane. Biotechniques. 1988 Feb;6(2):154–159. [PubMed]
  • Logan SM, Harris LA, Trust TJ. Isolation and characterization of Campylobacter flagellins. J Bacteriol. 1987 Nov;169(11):5072–5077. [PMC free article] [PubMed]
  • Logan SM, Trust TJ. Location of epitopes on Campylobacter jejuni flagella. J Bacteriol. 1986 Nov;168(2):739–745. [PMC free article] [PubMed]
  • Logan SM, Trust TJ, Guerry P. Evidence for posttranslational modification and gene duplication of Campylobacter flagellin. J Bacteriol. 1989 Jun;171(6):3031–3038. [PMC free article] [PubMed]
  • Luke CJ, Kubiak E, Cockayne A, Elliott TS, Penn CW. Identification of flagellar and associated polypeptides of Helicobacter (formerly Campylobacter) pylori. FEMS Microbiol Lett. 1990 Sep 1;59(1-2):225–230. [PubMed]
  • Marshall BJ. Campylobacter pylori: its link to gastritis and peptic ulcer disease. Rev Infect Dis. 1990 Jan-Feb;12 (Suppl 1):S87–S93. [PubMed]
  • Marshall BJ, Armstrong JA, McGechie DB, Glancy RJ. Attempt to fulfil Koch's postulates for pyloric Campylobacter. Med J Aust. 1985 Apr 15;142(8):436–439. [PubMed]
  • Marshall T, Williams KM, Vesterberg O. Two-dimensional electrophoresis of proteins in human serum: improved resolution by use of narrow pH gradients and prolonged electrophoresis. Clin Chem. 1984 Dec;30(12 Pt 1):2008–2013. [PubMed]
  • Metzger H, Shapiro MB, Mosimann JE, Vinton JE. Assessment of compositional relatedness between proteins. Nature. 1968 Sep 14;219(5159):1166–1168. [PubMed]
  • Mills SD, Kurjanczyk LA, Penner JL. Identification of an antigen common to different species of the genus Campylobacter. J Clin Microbiol. 1988 Jul;26(7):1411–1413. [PMC free article] [PubMed]
  • Minnich SA, Newton A. Promoter mapping and cell cycle regulation of flagellin gene transcription in Caulobacter crescentus. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1142–1146. [PMC free article] [PubMed]
  • Morris A, Nicholson G. Ingestion of Campylobacter pyloridis causes gastritis and raised fasting gastric pH. Am J Gastroenterol. 1987 Mar;82(3):192–199. [PubMed]
  • Newell DG. Identification of the outer membrane proteins of Campylobacter pyloridis and antigenic cross-reactivity between C. pyloridis and C. jejuni. J Gen Microbiol. 1987 Jan;133(1):163–170. [PubMed]
  • O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed]
  • Pearson TW, Pinder M, Roelants GE, Kar SK, Lundin LB, Mayor-Withey KS, Hwett RS. Methods for derivation and detection of anti-parasite monoclonal antibodies. J Immunol Methods. 1980;34(2):141–154. [PubMed]
  • Penner JL. The genus Campylobacter: a decade of progress. Clin Microbiol Rev. 1988 Apr;1(2):157–172. [PMC free article] [PubMed]
  • Pleier E, Schmitt R. Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti. J Bacteriol. 1989 Mar;171(3):1467–1475. [PMC free article] [PubMed]
  • Robertson EF, Dannelly HK, Malloy PJ, Reeves HC. Rapid isoelectric focusing in a vertical polyacrylamide minigel system. Anal Biochem. 1987 Dec;167(2):290–294. [PubMed]
  • Romaniuk PJ, Zoltowska B, Trust TJ, Lane DJ, Olsen GJ, Pace NR, Stahl DA. Campylobacter pylori, the spiral bacterium associated with human gastritis, is not a true Campylobacter sp. J Bacteriol. 1987 May;169(5):2137–2141. [PMC free article] [PubMed]
  • Thomashow LS, Rittenberg SC. Isolation and composition of sheathed flagella from Bdellovibrio bacteriovorus 109J. J Bacteriol. 1985 Sep;163(3):1047–1054. [PMC free article] [PubMed]
  • Thornton SA, Logan SM, Trust TJ, Guerry P. Polynucleotide sequence relationships among flagellin genes of Campylobacter jejuni and Campylobacter coli. Infect Immun. 1990 Aug;58(8):2686–2689. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet. 1983 Jun 4;1(8336):1273–1275. [PubMed]
  • Weissborn A, Steinmann HM, Shapiro L. Characterization of the proteins of the Caulobacter crescentus flagellar filament. Peptide analysis and filament organization. J Biol Chem. 1982 Feb 25;257(4):2066–2074. [PubMed]
  • Wyatt JI. The role of Campylobacter pylori in the pathogenesis of peptic ulcer disease. Scand J Gastroenterol Suppl. 1989;157:7–22. [PubMed]
  • Yokote Y, Arai KM, Akahane K. Recovery of tryptophan from 25-minute acid hydrolysates of protein. Anal Biochem. 1986 Feb 1;152(2):245–249. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...