Cj1121c, a novel UDP-4-keto-6-deoxy-GlcNAc C-4 aminotransferase essential for protein glycosylation and virulence in Campylobacter jejuni.
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.
Vijayakumar
Somalinga
S
Department of Microbiology and Immunology, Infectious Diseases Research Group, University of Western Ontario, London, Ontario N6A 5C1.
Merkx-Jacques
Alexandra
A
Ratnayake
Dinath B
DB
Gryski
Irene
I
Obhi
Ravinder Kaur
RK
Houle
Sébastien
S
Dozois
Charles M
CM
Creuzenet
Carole
C
D001426:Bacterial Proteins / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000201:enzymology* / Q000472:pathogenicity; D002384:Catalysis; D005407:Flagella / Q000502:physiology; D018698:Glutamic Acid / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000382:microbiology; D009682:Magnetic Resonance Spectroscopy; D013696:Temperature; D000637:Transaminases / Q000502:physiology*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism; D014774:Virulence
D001426:Bacterial Proteins; D018698:Glutamic Acid; D014537:Uridine Diphosphate N-Acetylglucosamine; D000637:Transaminases