Characterization of the ADP--d-manno-heptose biosynthetic enzymes from two pathogenic Vibrio strains

ADP-activated beta-d-manno-heptoses (ADP-beta-d-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-d-glycero-beta-d-manno-heptose (ADP-d,d-manno-heptose) and its C-6 '' epimer, ADP-l-glycero-beta-d-manno-heptose (ADP-l,d-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-d,d-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-beta-d-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-d,d-manno-heptose and the epimerase that converts it to ADP-l,d-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that beta-d-mannose 1-phosphate, but not alpha-d-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldE(VC) (from V. cholerae) and HldE(VP) (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldE(VC) and HldE(VP) together with the E. coli-derived HldE(EC) were thus carried out using beta-d-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-beta-d-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains.