Glycosylation of Campylobacter flagella

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DESCRIPTION (provided by applicant): Campylobacter jejuni is the leading cause of foodborne illness in North America and is among the major causes of bacterial diarrhea worldwide. Flagella and motility are required for intestinal colonization and invasion of intestinal epithelial cells by C. jejuni, and flagellin is an immunodominant and possibly a protective antigen. Flagellin from C. jejuni strain 81-176 and Campylobacter coli strain VC167 are glycosylated at 19 and 16 serine or threonine residues, respectively, with a 9 carbon sugar called pseudaminic acid and derivatives of pseudaminic acid. The modifications, which account for approximately 10% of the weight of these glycoproteins, are surface exposed on the flagella filament and are likely involved in interaction of flagellin with the eukaryotic host. Genetic analyses indicate that the pathway for biosynthesis of pseudaminic acid is conserved in both 81-176 and VC167. Flagellins from both strains contain minor modifications that are acetamidino forms of pseudaminic acid (mass 315 Da). However, the 315 Da group synthesized by 81-176 and VC 167 are structurally and immunologically distinct and are synthesized by independent pathways in each organism. The data suggest that campylobacter flagellin needs to be glycosylated in order to be exported and/or assembled into a filament. A mutant in 81-176 that is unable to synthesize the acetamidino form of pseudaminic acid appears to be attenuated in virulence. The aim of this study is to further elucidate the pathways by which the different forms of pseudaminic acid are synthesized and to study unique aspects of the regulation of these glycosylation genes. Site-specific mutagenesis will be done on flagellin to eliminate modification sites sequentially in order to determine sites that are critical for flagella function and the rules of site occupancy. The biological role of flagella glycosylation will be studied by examining a series of mutants in in vitro and in vivo assays of virulence.