Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384 - Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-MHz H-1 and C-13 NMR analysis

We have applied two strategies for the cloning of four genes responsible fo r the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barre syndrome. We first cloned a gene encoding a n alpha-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed s equence from C.jejuni NCTC 11168 to amplify a region involved in LOS biosyn thesis from C, jejuni OH4384, The LOS biosynthesis locus from C, jejuni OH4 384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C,jejuni NCTC 11168 spans 13.49 k ilobase pairs and contains 15 open reading frames, indicating a different o rganization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coil, and assayed using synthetic fluorescent oligosaccharides as accepters. We identified genes en coding a beta-1,4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1,3-gal actosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), w hich transfers sialic acid to 0-3 of galactose and to 0-8 of a sialic acid that is linked alpha-2,3 to a galactose, The linkage specificity of each id entified glycosyltransferase was confirmed by NMR analysis at 600 MHz on na nomole amounts of model compounds synthesized in vitro. Using a gradient in verse broadband nano-NMR probe, sequence information could be obtained by d etection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and csf-II in the synthesis of the GT1a mimic in C,jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homol ogues in two related C,jejuni strains that express shorter ganglioside mimi cs in their LOS.