HR-MAS NMR studies of 15N-labeled cells confirm the structure of the O-methyl phosphoramidate CPS modification in Campylobacter jejuni and provide insight into its biosynthesis

Abstract

Because Campylobacter jejuni is the leading cause of bacterial food-borne gastroenteritis throughout the world, there is intense effort to determine the mechanisms of infectivity associated with this bacterium. Capsular polysaccharide (CPS) has been shown to be an important virulence factor for C. jejuni and a recent study that examined the 11168V26 strain identified several phase-variable CPS modifications including an unusual O-methyl phosphoramidate (MeOPN) group on C-3 of the GalfNAc residue. In this study, we examined the MeOPN group using homo- and hetero-nuclear high-resolution magic angle spinning (HR-MAS) NMR experiments of whole bacterial cells grown on 15NH4Cl-enriched media. 1H–31P HSQC NMR experiments showed that the level of 15N labeling within the MeOPN reached 80%, and a large 1J(15N–31P) scalar coupling provided direct evidence that confirmed the structure of the MeOPN as CH3OP(O)(NH2)(OR). Because 15N was also detected within the major outer membrane protein as well as the NAc and NGro groups of CPS, ammonium was concluded to be an important building block used in the synthesis of amino acids and glycan structures in C. jejuni. HR-MAS NMR studies of 15N-labeled cells revealed an unanticipated level of complexity as multiple MeOPN signals were observed within 1H-31P HSQC spectra for the 11168V26 and 11168H strains. While some signals originated from the MeOPN at C-3 of GalfNAc, others were attributed to a novel MeOPN located on D-glycero-α-L-gluco-heptopyranose. Together, these HR-MAS NMR findings shed light on nitrogen metabolism in C. jejuni, confirm the chemical structure of the MeOPN, and demonstrate that the modification occurs on both furanose and pyranose CPS sugars for this bacterium.Key words: Campylobacter jejuni, capsular polysaccharide, HR-MAS NMR, isotope labeling, phosphoramidate.