ABSTRACT
BACKGROUND: In V. cholerae, the biogenesis of capsule polysaccharide is poorly understood. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. V. cholerae serogroup O31 NRT36S, a human pathogen that produces a heat-stable enterotoxin (NAG-ST), is encapsulated. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S. RESULTS: The structure of the capsular (CPS) polysaccharide was determined by high resolution NMR spectroscopy and shown to be a complex structure with four residues in the repeating subunit. The gene cluster of capsule biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data (GenBank accession DQ915177). The capsule gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. Other than V. cholerae O139, this is the first V. cholerae CPS for which a structure has been fully elucidated and the genetic locus responsible for biosynthesis identified. CONCLUSION: The co-location of CPS and LPS biosynthesis genes was unexpected, and would provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. This, in turn, may be a key element for V. cholerae to evolve new strains that can escape immunologic detection by host populations.
Subject(s)
Bacterial Capsules/biosynthesis , Lipopolysaccharides/biosynthesis , Vibrio cholerae non-O1/genetics , Vibrio cholerae non-O1/metabolism , Amino Acid Sequence , Bacterial Capsules/chemistry , Base Sequence , Chromatography, Gas/methods , Chromatography, Gel , DNA Transposable Elements/genetics , Genome, Bacterial , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Immunoblotting , Lipopolysaccharides/chemistry , Microscopy, Electron/methods , Molecular Sequence Data , Mutagenesis , Nuclear Magnetic Resonance, Biomolecular , O Antigens/biosynthesis , O Antigens/geneticsABSTRACT
In the past decade, several outbreaks of cholera have been reported to be caused by Vibrio cholerae O139, a strain which differs from the more common O1 strain in that the former is encapsulated. The hexasaccharide repeating subunit has been isolated from the V. cholerae O139 capsular polysaccharide by digestion with a recently discovered polysaccharide lyase derived from a bacteriophage specific for this serogroup. It specifically cleaves at a single position of the 4-linked galacturonic acid producing an unsaturated sugar product in quantities for conformational studies by (1)H and (13)C NMR spectroscopy. We report conformational studies on this oligosaccharide by molecular modeling and NMR spectroscopy including nuclear Overhauser effects and residual dipolar coupling of a sample weakly oriented in liquid crystalline solution. The structure contains a tetrasaccharide epitope homologous to the human Lewis(b) blood group antigen, which adopts a relatively well-defined single conformation. Comparison of these results with those of a previously published study of the intact capsular polysaccharide indicates that the conformations of the epitope in the two cases are identical or at least closely similar. Thus, this epitope, which may be essential for the pathogenicity of this V. cholerae strain, is not a "conformational epitope" requiring a certain critical size for antigenicity as has been reported for several other bacterial capsular antigens.
