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

Flagellar filaments were isolated from Helicohacter 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 (M(r)s) of 57,000 and 56,000 were obtained. N-terminal amino acidanalysis 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-M(r) flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-M(r) 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-M(r) flalellin species were located in different regions of the assembled flagellar filament. The minor 57,000-M(r) species was located proximal to the hook, and the major 56,000-M(r) 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-M(r) flagellin carried sequences antigenically cross-reactive with the 57,000-M(r) 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-M(r) 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.