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094

GENETICS AND BIOCHEMISTRY OF PROTEIN GLYCOSYLATION IN CAMPYLOBACTER JEJUNI

A Merkx-Jacques1, S Vijayakumar1, M Demendi1, D Ratnayake1, C Dozois2, C Creuzenet1
1Microbiology and Immunology, University of Western Ontario, London, Ontario; 2INRS-Institut Armand Frappier, Laval, Quebec

BACKGROUND: C. jejuni produces numerous glycoproteins, including flagellins, which are important for virulence. The flagellins harbour pseudaminic acid (PA) whereas other glycoproteins harbour diacetamidobacillosamine (DAB). We are investigating the genetics and biochemistry of protein glycosylation in C. jejuni to identify the enzymes involved, and determine their activity and roles in virulence. We focused on two homologous pathways comprising each a putative dehydratase, aminotransferase and acetyltransferase, namely {Cj1293, Cj1294 and Cj1298} and {Cj1120c, Cj1121c and Cj1123c}.
METHODS: All enzymes were overexpressed and purified before monitoring their activity by capillary electrophoresis. The cj1121c and cj1294 genes were disrupted by a chloramphenicol resistance cassette and virulence-related phenotypes were investigated.
RESULTS: We determined that Cj1293 is a UDP-GlcNAc C6 dehydratase. It leads to the formation of 4-keto-arabino and 4-keto-gluco intermediates. We showed that Cj1294 and Cj1121c are aminotransferases that use the arabino and gluco intermediates generated by Cj1293 as substrates, respectively. Both activities are present in C. jejuni extracts, with preponderance of the Cj1121c activity. We showed that Cj1123c is a N-acetyltransferase that uses the Cj1121c reaction product as a substrate. The cj1294 and cj1121c mutants are non-motile but the cj1121c mutant produces normal flagellins and flagella, whereas the cj1294 mutant does not. cj1121c is important for interactions with Caco2 cells whereas cj1294 is not. Finally, cj1121c is essential for colonization of chicken intestine and for the glycosylation of proteins other than flagellins.
DISCUSSION: Our data indicate that Cj1293 and Cj1294 are involved in flagellin glycosylation via PA biosynthesis, and that Cj1121c and Cj1123c are involved in general protein glycosylation via DAB biosynthesis. The data demonstrate a cross-talk between both pathways, with a preponderant role of Cj1121c on virulence, hence identifying Cj1121c as a target for inhibitor development.

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