title: Lactate enhancement of sialylation of gonococcal lipopolysaccharide and of induction of serum resistance by CMP-NANA is not due to direct activation of the sialyltransferase: metabolic events are involved.
abstract: Lactate enhances lipopolysaccharide (LPS) sialylation and induction of serum resistance in gonococci by CMP-NANA. To investigate whether the enhancement is due to a direct effect on the sialyltransferase, an improved extraction of the enzyme and a reliable quantitative assay were devised. Gonococci (strain F62) were disrupted in a French pressure cell and the bacterial membranes were extracted for 1 h at 37 degrees C with a detergent, NONIDET (1% v/v). The assay involved sialylation of LPS by CMP-14CNANA and scintillation counting of the labelled LPS after fixing it on filter paper strips by trichloracetic acid (TCA) and washing away unincorporated CMP-14CNANA. It was rapid, reproducible and, although the enzyme preparations contained endogenous LPS, was dependent upon added LPS for maximum activity. At 37 degrees C the rate was constant for up to 5 min and proportional to the concentration of extract in the assay. A wide range of concentrations of lithium-L-lactate did not enhance the activity of the extracted sialyltransferase. At concentrations above 22 microM, it was inhibitory. Pre-incubation of gonococci with lactate enhanced subsequent LPS sialylation and induction of serum resistance by CMP-NANA. Hence, the process whereby lactate enhances the effect of CMP-NANA is separate from the action of CMP-NANA itself. Both processes were inhibited by a sublethal concentration of chloramphenicol, indicating that metabolic events are required. Evidently, the enhancement process does not involve a direct activation of the sialytransferase.
authors.affiliation: School of Biochemistry, University of Birmingham, UK.
authors.forename: N J
authors.initials: NJ
authors.lastname: Parsons
authors.forename: J P
authors.initials: JP
authors.lastname: Emond
authors.forename: M
authors.initials: M
authors.lastname: Goldner
authors.forename: J
authors.initials: J
authors.lastname: Bramley
authors.forename: H
authors.initials: H
authors.lastname: Crooke
authors.forename: J A
authors.initials: JA
authors.lastname: Cole
authors.forename: H
authors.initials: H
authors.lastname: Smith
mesh_terms: D001770:Blood Bactericidal Activity; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000494:pharmacology*; D004789:Enzyme Activation; D019344:Lactic Acid / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008954:Models, Biological; D009344:Neisseria gonorrhoeae / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D019344:Lactic Acid; D012799:Sialyltransferases