Biochemical and immunological characterization of the flagella protein of Campylobacter Pylori
Date
1990
Authors
Betts, Janice Deborah
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Abstract
In 1987, Lee and coworkers (Infect. Immun. 55:828-831) reported that a Campylobacter pylori protein of subunit molecular weight (Mr) 57-59,000 (57-59 kDa) reacted in Western blots with an antiserum (SML2) directed against the flagellin of Campylobacter jejuni . Subsequent reports by Newell (J. Gen. Microbiol. 133:163-170) and Mills et. al. (J. Clin. Microbiol. 26: 1411-1413) of a cross reacting antigen among campylobacters supported the suggestion that the protein being recognized by the anti-C. jejuni flagellin antiserum was the C. pylori flagellin, while reports by Geis et al. (J. Clin. Microbiol. 27:436-441), Perez-Perez and Blaser (Infect. Immun. 55:1256-1955)and Dunn et. al. (Infect. Immun. 57:1825-1833) disputed the findings . This study was initiated to clarify whether or not the 57-59 kDa antigenically cross-reactive protein of C. pylori was flagellin, or whether the antigenic cross reactivity was due to determinants shared by an unrelated protein.
To answer this question, flagellar filaments were isolated from C. pylori 5294 using two principles of separation: CsCl density gradient ultracentrifugation, and pH 2.0 acid dissociation and differential ultracentrifugation. In both cases a 58 kDa subunit was the predominant protein recovered and in both cases this protein was shown to react by western blotting with antiserum (SML2) directed against the flagellin protein of C. jejuni. The 58 kDa subunit protein was purified to homogeneity using a Mono Q anion exchange column, and N-terminal amino acid sequence analysis showed significant identity with the N-terminal amino acid sequence of the 59.5 kDa flagellin of C. coli VC167B, and sequence relatedness with the N-terminal sequences of flagellins of Salmonella species and Bacillus species. The amino acid composition of the 58 kDa protein subunit of C. pylori was also typical of a flagellin molecule. Particularly significant was the absence of cysteine and tryptophan residues and the low content of proline residues which are features characteristic of other flagellins.
Polyclonal antisera and monoclonal antibodies directed against the 58 kDa protein were produced, and indirect immunofluorescence and immunogold electron microscopic studies showed that the antibodies bound to the surface of the inner flagellin filament. Cell absorption studies with polyclonal antisera confirmed that the sheath did not contain the 58 kDa protein. Immunochemical analysis with polyclonal antiserum against C. pylori flagellin verified the presence of cross reactive epitopes among Campylobacter flagellins. This antigenic cross-reactivity did not however extend to the flagellins of other gram-negative bacteria. Monoclonal antibody (Mab) 72c was also specific for a linear epitope shared by Campylobacter flagellins . This epitope was exposed on the surface of the inner flagellin filament of C. pylori, but was not surface-exposed on the unsheathed flagella filament of C. coli VC167B, indicating that the flagella filaments of the two species were assembled in a different fashion. In addition to this shared epitope, western blotting, ELISA, immunogold electron microscopy, and indirect immunofluorescence studies showed that the 58 kDa flagellin protein of C. pylori carried two additional species-specific epitopes. Both epitopes were exposed on the surface of the inner flagellin filament. The epitope for MAb 220a was linear, while the epitope for MAb 104a appeared to be topographically assembled.