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Campylobacter jejuni is a leading bacterial cause of human enterocolitis and part of the normal flora in both wild and domesticated animals. High genetic diversity exists in Campylobacter strains, and lack of simple and efficient typing schemes creates a challenge for understanding the pathobiology and epidemiology of Campylobacter infections. In addition, the high incidence in humans, the common presence in animals, and the complexity of transmission highlights the need for development of effective and safe vaccines to control C. jejuni infections. The major outer membrane protein (MOMP, encoded by the cmp gene), a predominant surface protein of C. jejuni, consists of both conserved and variable regions, and is believed to be a promising candidate for the design of molecular typing tool and effective vaccines. In this project, multiple experiments were conducted to determine the nature of MOMP sequence variation, the feasibility of cmp-based typing tools, and the antigenic features of MOMP. The cmp alleles from different Campylobacter strains were amplified and sequenced. Alignment of the cmp sequences revealed existence of structural mosaicism, suggesting the possibility of intragenic recombination in cmp. To test the contention that the mosaic structures of cmp resulted from recombination events, both computerized statistical method, Sawyer’s sequence substitution and distribution test, and an experimental natural transformation model were applied. Results indicated that mosaic structures indeed exist in cmp of C. jejuni and intragenic recombination mediated by natural transformation is a mechanism responsible for the MOMP mosaicism. Feasibility of a cmp-based typing tool was evaluated using pulsed-field gel electrophoresis (PFGE) as a “gold” standard. The cmp alleles (n=60) were sequenced from multiple strains, grouped, and compared with the PFGE profiles of the strains using Bionumerics. Results showed that typeability for the cmp-based method and PFGE are both 100%. The discriminatory index is 0.973 for the cmp-based method and 0.969 for PFGE. The cmp sequence types are well correlated with PFGE types. These results indicate that the cmp-based typing is a simple and highly discriminatory approach for molecular differentiation of C. jejuni strains. To analyze the antigenic features of MOMP, different detergents and liposome reconstitution were used to restore the native conformation of recombinant MOMP (rMOMP). Results showed that reconstitution of rMOMP into liposomes efficiently refolded rMOMP and greatly enhanced the reactivity of rMOMP to antibodies against conformational epitopes. Serum samples from chickens infected by C. jejuni produced greater reactivity to liposome-reconstituted rMOMP than to denatured rMOMP, suggesting that the in vivo antibody response to MOMP is predominantly against conformational epitopes. These findings indicate that liposome-reconstituted rMOMP is a useful means for functional characterization of MOMP and may be exploited for use as a vaccinogen. |
