Metabolic Pathways for the Biosynthesis of Heptoses Used in the Construction of Capsular Polysaccharides in the Human Pathogen Campylobacter jejuni

Abstract

Campylobacter jejuni is the leading cause of food poisoning in North America. The exterior surface of this bacterium is coated with a capsular polysaccharide (CPS) that consists of a repeating sequence of 2−5 different carbohydrates that is anchored to the outer membrane. Heptoses of various configurations are among the most common monosaccharides that have been identified within the CPS. It is currently thought that all heptose variations derive from the modification of GDP-D-glycero-α-D-manno-heptose (GMH). From the associated gene clusters for CPS biosynthesis, we have identified 20 unique enzymes with different substrate profiles that are used by the various strains and serotypes of C. jejuni to make six different stereoisomers of GDP-6-deoxy-heptose, four stereoisomers of GDP-D-glycero-heptoses, and two stereoisomers of GDP-3,6-dideoxyheptoses starting from D-sedoheptulose-7-phosphate. The modification enzymes include a C4-dehydrogenase, a C4,6-dehydratase, three C3-and/or C5-epimerases, a C3dehydratase, eight C4-reductases, two pyranose/furanose mutases, and four enzymes for the formation of GMH from D-sedoheptulose-7-phosphate. We have mixed these enzymes in different combinations to make novel GDP-heptose modifications, including GDP-6-hydroxy-heptoses, GDP-3-deoxy-heptoses, and GDP-3,6-dideoxy-heptoses.

Biosynthesis of UDP-α-N-Acetyl-d-mannosaminuronic Acid and CMP-β-N-Acetyl-d-neuraminic Acid for the Capsular Polysaccharides of Campylobacter jejuni

“…The nonhydrolyzing UDP-GlcNAc 2-epimerase and the UDP-ManNAc 6-dehydrogenase from the HS:11 serotype of C. jejuni were purified according to a slight modification of previously reported procedures. − Similarly, the hydrolyzing UDP-GlcNAc 2-epimerase, Neu5Ac synthase, and CMP-Neu5Ac synthase from C . jejuni strain 81116 were expressed and purified according to a modification of previously reported procedures. − Escherichia coli BL21­(DE3) competent cells were transformed with the appropriate plasmids. Single colonies were inoculated in 50 mL of LB medium [20 g/L yeast extract, 35 g/L tryptone, and 5 g/L sodium chloride (pH 7.0)] supplemented with 50 μg/mL kanamycin and grown at 37 °C overnight while being shaken.…”

“…The repeating chemical structures for capsular polysaccharides have been determined from at least 12 different strains and serotypes of C. jejuni . A total of 24 different sugar moieties have thus far been identified, and these include glycerol, three pentoses ( d -ribose, l -arabinose, and d -xylulose), 11 hexoses ( d -glucose, d -galactose, d -mannose, d -glucitol, d -fructose, l -sorbose, d -fucose, 6-deoxy- l -altrose, d -glucuronate, N -acetyl- d -glucosamine, and N -acetyl- d -galactosamine), and nine heptoses ( d - glycero - d - manno -heptose, d - glycero - l - gluco -heptose, l - glycero - d - ido -heptose, 6-deoxy- l - galacto -heptose, 6-deoxy- d - ido -heptose, 6-deoxy- d - altro -heptose, 6-deoxy- d - manno -heptose, 6-deoxy- l - gulo -heptose, and 3,6-dideoxy- l - ribo -heptose). ,− ,− Our efforts have been directed at improving our understanding of how individual NDP-activated monosaccharides are synthesized using the enzymes identified from the various gene clusters and the biochemical pathways for how these moieties are functionally decorated. The ultimate goal, however, is to ascertain the specific glycosyl transferases needed for the mating of the appropriate sugar donor with the proper sugar acceptor during polysaccharide formation.…”

Biosynthesis of Cytidine Diphosphate-6-d-Glucitol for the Capsular Polysaccharides of Campylobacter jejuni

“…The putative nucleotidyltransferase and the NAD­(P)-oxidoreductase from the HS:5 serotype of C. jejuni were purified according to the procedures reported previously. − Escherichia coli BL21­(DE3) competent cells were transformed with the appropriate plasmids. Single colonies were inoculated in 50 mL of LB medium (5 g/L yeast extract, 10 g/L tryptone, 5 g/L sodium chloride) supplemented with 50 μg/mL kanamycin and grown at 37 °C overnight with shaking.…”

“…The capsular polysaccharides from C. jejuni are composed of a repeating series of monosaccharide units attached to one another via glycosidic bonds. The carbohydrates are further decorated by methylations, methyl phosphoramidylations, and amidations. , At least 12 unique chemically determined CPS structures from more than 33 different C. jejuni serotypes have been identified thus far. , Among the most common monosaccharide units that have been identified and investigated in the CPS of C. jejuni are the relatively rare seven-carbon heptoses. − Twelve different heptoses have been chemically and structurally identified …”

“…The genes required for the biosynthesis of GDP- d - glycero - d - manno -heptose include hddC (HS5.8) for d - glycero - d - manno -heptose 1-phosphate guanosyltransferase; gmhA (HS5.9) for d -sedoheptulose 7-phosphate isomerase; and hddA (HS5.10) for d - glycero - d - manno -heptose 7-phosphate kinase. Similarly, the genes needed for the biosynthesis of 3,6-dideoxy- ribo -heptose include those for the expression of a 4,6-dehydratase (HS5.11), a C3-dehydratase (HS5.12), a C5-epimerase (HS5.14), and a C4-reductase (HS5.13). ,− A cursory examination of the gene cluster for the biosynthesis of the capsular polysaccharide of C. jejuni serotype HS:5 indicates the presence of a pair of genes currently annotated as a sugar nucleotidyltransferase (UniProt entry: A0A0Q3NN41; HS5.18) and a nucleotide sugar dehydratase or NAD­(P)-dependent oxidoreductase (UniProt entry: A0A0U3AP28; HS5.17), which we suggest are potential candidates for the biosynthesis of the nucleotide activated d -glucitol.…”