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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
The Journal of Biological Chemistry (2006)
  • Somalinga Vijayakumar , The University of Western Ontario
  • Alexandra Merkx-Jacques , The University of Western Ontario
  • Dinath B. Ratnayake , The University of Western Ontario
  • Irene Gryski , The University of Western Ontario
  • Ravinder Kaur Obhi , The University of Western Ontario
  • Sébastien Houle
  • Charles M Dozois
  • Carole Creuzenet , The University of Western Ontario
Abstract

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-beta-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.

Keywords
  • Bacterial Proteins,
  • Campylobacter jejuni,
  • Catalysis,
  • Flagella,
  • Glutamic Acid,
  • Glycosylation,
  • Hydrogen-Ion Concentration,
  • Intestinal Mucosa,
  • Magnetic Resonance Spectroscopy,
  • Temperature,
  • Transaminases,
  • Uridine Diphosphate N-Acetylglucosamine,
  • Virulence
Publication Date
September 22, 2006
Citation Information
Somalinga Vijayakumar, Alexandra Merkx-Jacques, Dinath B. Ratnayake, Irene Gryski, et al.. "Cj1121c, a Novel UDP-4-keto-6-deoxy-GlcNAc C-4 Aminotransferase Essential for Protein Glycosylation and Virulence in Campylobacter Jejuni" The Journal of Biological Chemistry Vol. 281 Iss. 38 (2006)
Available at: http://works.bepress.com/alexandra_merkx-jacques/2/