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

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-␤-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.

Biochemical Characterization of dTDP- d -Qui4N and dTDP- d -Qui4NAc Biosynthetic Pathways in Shigella dysenteriae Type 7 and Escherichia coli O7

“…Sequence analysis reveals that the two SATs share 53% identity (67% similarity), which is much higher than the levels they share with other reported SATs (Table S1 in the supplemental material). Based on the position of the amino receptor, SATs can be divided into three subgroups acting on scylloinosose (1, 13, 14), NDP-3-keto sugars (7,23,30,35), or NDP-4-keto sugars (2,5,12,27,34,36). This is also supported by the phylogenetic analysis of SATs (Fig.…”

“…Many O-antigen gene clusters have been sequenced, with gene functions proposed (http://www.mmb.usyd.edu.au/BPGD/default.htm). Biosynthetic pathways for several O-antigen sugar precursors, such as UDP-L-FucNAc, have been characterized biochemically (15,16,22,27,30,36).…”

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…Impaired glycosylation of the VirB10 glycoprotein, a type IV secretion system glycosylated at two sites, for instance, resulted in reduced natural transformation (329). Mutations affecting the pgl locus resulted in altered phenotypes, such as decreased adhesion and invasion efficiency in vitro and abolishment of mouse and chicken colonization in vivo (5,315,330,331). The glycans were also hypothesized to play a role in immune evasion by masking primary amino acid sequences, based on studies with human antisera (304).…”

“…PglF (CJ1120c) is a C-6 dehydratase, which, together with the UDP-4-keto-6-deoxy-GlcNAc C-4-aminotransferase PglE (CJ1121c) (315), is involved in the biosynthesis of the Bac moiety (316). PglD, an acetyltransferase modifying the UDP-4-aminosugar, is coupled to the reactions catalyzed by PglF and PglE (317).…”

Mass Spectrometry in the Elucidation of the Glycoproteome of Bacterial Pathogens

“…In tropical developing countries Campylobacter infection is hyper-endemic among young people causing high mortality rates in children and infants. Several studies estimate infection rates in the under fives to be as much as 60,000 per 100,000 population [100,[164][165][166][167]. This bacterium is commensal in both poultry and cattle, with birds acting as its natural reservoir.…”