The genetic bases for the variation in the liposaccharide of the mucosal pathogen, Campylobacter jejuni : biosynthesis of sialylated ganglioside mimics in the core oligosaccharide

We have compared the lipo-oligosaccharide (LOS) biosynthesis loci from 11 Campylobacter jejunistrains expressing a total of 8 different ganglioside mimics in their LOS outer cores. Based on the organization of the genes, the 11 corresponding loci could be classified into three classes, with one of them being clearly an intermediate evolutionary step between the other two. Comparative genomics and expression of specific glycosyltransferases combined with in vitro activity assays allowed us to identify at least five distinct mechanisms that allowC. jejuni to vary the structure of the LOS outer core as follows: 1) different gene complements; 2) phase variation because of homopolymeric tracts; 3) gene inactivation by the deletion or insertion of a single base (without phase variation); 4) single mutation leading to the inactivation of a glycosyltransferase; and 5) single or multiple mutations leading to \u201callelic\u201d glycosyltransferases with different acceptor specificities. The differences in the LOS outer core structures expressed by the 11 C. jejuni strains examined can be explained by one or more of the five mechanisms described in this work.Peer reviewed: YesNRC publication: Ye

A single bifunctional UDP-GlcNAc/Glc 4-epimerase supports the synthesis of three cell surface glycoconjugates in campylobacter jejuni

The major cell-surface carbohydrates (lipooligosaccharide, capsule and glycoprotein N-linked heptasaccharide) of Campylobacter jejuni NCTC 11168 contain Gal and/or GalNAc residues. GalE is the sole annotated UDP-glucose 4-epimerase in this bacterium. The presence of GalNAc residues in these carbohydrates suggested that GalE may be an UDP-GlcNAc 4-epimerase. GalE was shown to epimerize UDP-Glc and UDP-GlcNAc in coupled assays with C. jejuni glycosyltransferases and in sugar nucleotide epimerization equilibria studies. Thus, GalE possesses UDP-GlcNAc 4-epimerase activity and was renamed Gne. The Km(app) of a purified MalE-Gne fusion protein for UDP-GlcNAc and UDP-GalNAc are 1087M and 1070 M while those for UDP-Glc and UDP-Gal are 780 M and 784 M. The kcat and kcat/ Km(app) were three to four times higher for UDP-GalNAc and UDP-Gal than for UDP-GlcNAc and UDP-Glc. The comparison of the kinetic parameters of MalE-Gne to those of other characterized bacterial UDP-GlcNAc 4-epimerases indicated that Gne is a bifunctional UDP-GlcNAc/Glc 4-epimerase. The UDP-sugar binding site of Gne was modeled using the structure of the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa. Small differences were noted and these may explain the bifunctional character of the C. jejuni Gne. In a gne mutant of C. jejuni, the LOS was shown by CE-MS to be truncated by at least five sugars. Furthermore, both the glycoprotein N-linked heptasaccharide and capsule were no longer detectable by high resolution magic angle spinning NMR. These data indicate that Gne is the enzyme providing Gal and GalNAc residues for the synthesis of all three cell-surface carbohydrates in C. jejuni NCTC 11168NRC publication: Ye