Abstract
In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.
Highlights
Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis, a causative agent of child morbidity in underdeveloped countries and an antecedent to the MillerFisher and Guillain-Barreneuropathies [1,2,3,4,5]
The study uncovered the presence of a highly conserved gene cluster within those strains that produce the methyl phosphoramidate CH3OP(O)(NH2)(OR) (MeOPN) capsular polysaccharides (CPS) modification (NCTC11168, NCTC12517, G1, 81-176). Because these genes have no apparent role in sugar biosynthesis and because their presence coincides with the presence of MeOPN on CPS sugars, it was hypothesized that they might be involved in MeOPN biosynthesis
Two anomeric signals were observed for residue C as a ificity of MeOPN expression among Campylobacter isolates is result of structural heterogeneity generated by the phase variable important to determine the abundance of this CPS modifica- MeOPN group at C-3 of this sugar [13]
Summary
Bacterial Strains and Growth Conditions—C. jejuni NCTC11168 (HS:2) and its motile variant, 11168H (HS:2) were routinely grown on Mueller Hinton (MH) agar (Difco) at 37 °C under microaerobic conditions (85% N2, 10% CO2, 5% O2). 1H NMR spectra of bacterial cells were acquired using the Carr-Purcell-Meiboom-Gill pulse sequence (90-(-180-)n acquisition) to remove broad signals originating from lipids and solid-like materials [22] and were typically obtained using 256 transients (11 min). Bacterial Colonization of Specific Pathogen-free Leghorn Chicks—The inoculum for each chick colonization experiment was prepared by harvesting C. jejuni 11168H cells, grown for 18 h into a 0.1 M (pH 7.4) phosphate-buffered saline solution (supplemented with 0.14 M NaCl and 0.002 M KCl). Detection of MeOPN from the Cecal Contents of Colonized Chickens—To assess the expression of the MeOPN within the natural avian host, a chicken model was develgies, Phoenix, AZ) using 15 mM ammonium acetate/ammo- oped using 1-day-old specific pathogen-free chicks that were inocnium hydroxide in deionized water, pH 9.0, containing 5% ulated with a high dose of C. jejuni 11168H cells.
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