Complete 6-deoxy-D-altro-heptose biosynthesis pathway from Campylobacter jejuni: more complex than anticipated

The Campylobacter jejuni capsule is important for colonization and virulence in various infection models. In most strains, the capsule includes a modified heptose whose biological role and biosynthetic pathway are unknown. To decipher the biosynthesis pathway for the 6-deoxy-D-altro-heptose of strain 81-176, we previously showed that the 4,6-dehydratase WcbK and the reductase WcaG generated GDP-6-deoxy-D-manno-heptose, but the C3 epimerase necessary to form GDP-6-deoxy-D-altro-heptose was not identified. Herein, we characterized the putative C3/C5 epimerase Cjj1430 and C3/C5 epimerase/C4 reductase Cjj1427 from the capsular cluster. We demonstrate that GDP-6-deoxy-D-altro-heptose biosynthesis is more complex than anticipated and requires the sequential action of WcbK, Cjj1430, and Cjj1427. We show that Cjj1430 serves as C3 epimerase devoid of C5 epimerization activity and that Cjj1427 has no epimerization activity and only serves as a reductase to produce GDP-6-deoxy-D-altro-heptose. Cjj1430 and Cjj1427 are the only members of the C3/C5 epimerases and C3/C5 epimerase/C4 reductase families shown to have activity on a heptose substrate and to exhibit only one of their two to three potential activities, respectively. Furthermore, we show that although the reductase WcaG is not part of the main pathway, its presence and its product affect the outcome of the pathway in a complex regulatory loop involving Cjj1427. This work provides the grounds for the elucidation of similar pathways found in other C. jejuni strains and other pathogens. It provides new molecular tools for the synthesis of carbohydrate antigens useful for vaccination and for the screening of enzymatic inhibitors that may have antibacterial effects.