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J. Biol. Chem., Vol. 281, Issue 38, 27733-27743, September 22, 2006

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Cj1121c, a Novel UDP-4-keto-6-deoxy-GlcNAc C-4 Aminotransferase Essential for Protein Glycosylation and Virulence in Campylobacter jejuni^*

Somalinga Vijayakumar¹, Alexandra Merkx-Jacques¹², Dinath B. Ratnayake, Irene Gryski, Ravinder Kaur Obhi, Sébastien Houle, Charles M. Dozois, and Carole Creuzenet³

From the Department of Microbiology and Immunology, Infectious Diseases Research Group, University of Western Ontario, London, Ontario N6A 5C1 and INRS-Institut Armand Frappier, Laval, Québec H7V 1B7, Canada

Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins carry diacetamidobacillosamine (DAB), a sugar that is not found in humans. Hence, the enzymes responsible for DAB synthesis represent potential therapeutic targets. We describe the biochemical characterization of Cj1121c, a putative aminotransferase encoded by the general protein glycosylation locus, to assess its role in DAB biosynthesis. By using overexpressed and affinity-purified enzyme, we demonstrate that Cj1121c has pyridoxal phosphate- and glutamate-dependent UDP-4-keto-6-deoxy-GlcNAc C-4 transaminase activity and produces UDP-4-amino-4,6-dideoxy-GlcNAc. This is consistent with a role in DAB biosynthesis and distinguishes Cj1121c from Cj1294, a homologous UDP-2-acetamido-2,6-dideoxy--L-arabino-4-hexulose C-4 aminotransferase that we characterized previously. We show that Cj1121c can also use this 4-keto-arabino sugar indirectly as a substrate, that Cj1121c and Cj1294 are active simultaneously in C. jejuni, and that the activity of Cj1121c is preponderant under standard growth conditions. Kinetic data indicate that Cj1121c has a slightly higher catalytic efficiency than Cj1294 with regard to the 4-keto-arabino substrate. By site-directed mutagenesis, we show that residues Glu-158 and Leu-131 are not essential for catalysis or for substrate specificity contrary to expectations. We further demonstrate that a cj1121c knock-out mutant is impaired for flagella-mediated motility, for invasion of intestinal epithelial cells, and for persistence in the chicken intestine, clearly demonstrating that Cj1121c is essential for host colonization and virulence. Finally, we show that cj1121c is necessary for protein glycosylation by lectin Western blotting. Collectively, these results validate Cj1121c as a promising drug target and provide the means to assay for inhibitors.

Received for publication, October 31, 2005 , and in revised form, May 10, 2006.

^* This work was supported in part by Operating Grant MOP-62775 from the Canadian Institutes of Health Research (to C. C.) and by funding from a Canada Research Chair and a Fonds de la Recherche en Santé du Québec research award (to C. M. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Recipient of a Canadian Digestive Health Foundation/Canadian Institutes of Health Research doctoral research award.

³ Recipient of a university faculty award from the Natural Sciences and Engineering Research Council of Canada and a Premier's research excellence award (Ontario, Canada). To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Infectious Diseases Research Group, University of Western Ontario, DSB 3031, London, Ontario N6A 5C1, Canada. Tel.: 519-661-3204; Fax: 519-661-3499; E-mail: ccreuzen{at}uwo.ca.

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