RESUMO
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.
Assuntos
Amidas/metabolismo , Cápsulas Bacterianas/metabolismo , Campylobacter jejuni/metabolismo , Ácidos Fosfóricos/metabolismo , Polissacarídeos Bacterianos/metabolismo , Animais , Cápsulas Bacterianas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Doenças das Aves/diagnóstico , Doenças das Aves/genética , Doenças das Aves/metabolismo , Doenças das Aves/microbiologia , Doenças das Aves/terapia , Infecções por Campylobacter/diagnóstico , Infecções por Campylobacter/genética , Infecções por Campylobacter/metabolismo , Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/terapia , Campylobacter jejuni/genética , Ceco/metabolismo , Ceco/microbiologia , Galinhas , Enterite/diagnóstico , Enterite/genética , Enterite/metabolismo , Enterite/microbiologia , Enterite/terapia , Teste de Complementação Genética , Humanos , Espectroscopia de Ressonância Magnética , Síndrome de Miller Fisher/diagnóstico , Síndrome de Miller Fisher/genética , Síndrome de Miller Fisher/metabolismo , Síndrome de Miller Fisher/microbiologia , Síndrome de Miller Fisher/terapia , Família Multigênica/genética , Mutação , Polissacarídeos Bacterianos/genética , Transferases/genética , Transferases/metabolismo , Tiflite/diagnóstico , Tiflite/genética , Tiflite/metabolismo , Tiflite/microbiologia , Tiflite/terapiaRESUMO
The indicator amino acid oxidation (IAAO) method allows the determination of amino acid requirements under conditions of low growth rate as found in pre-laying broiler breeder pullets. Cobb 500 breeder pullets (20 wk old; 2290 +/- 280 g, n = 4) were adapted (6 d) to a pelleted, purified control diet containing all nutrients at >or=110% of NRC recommendations. After recovery from surgery for implantation of a jugular catheter, each bird was fed, in random order, test diets containing one of nine levels of lysine (0.48, 0.96, 1.92, 2.88, 3.84, 4.80, 7.68, 9.60 and 14.40 g/kg of diet). Indicator oxidation was determined during 4-h primed (74 kBq/kg body), constant infusions (44 kBq x h(-1). kg body(-1)) of L-[1-(14)C]phenylalanine. Using the breakpoint of a one-slope broken-line model, the lysine requirement was determined to be 4.88 +/- 0.96 g/kg of diet or 366 +/- 72 mg x hen(-1) x d(-1) with an upper 95% CI of 6.40 g/kg of diet or 480 mg x hen(-1) x d(-1). IAAO allows determination of individual bird amino acid requirements for specific ages and types of birds over short periods of time and enables more accurate broiler breeder pullet diet formulation.