Conformation of the hexasaccharide repeating subunit from the Vibrio cholerae O139 capsular polysaccharide.

ABSTRACT : In the past decade, several outbreaks of cholera have been reported to be caused by Vibriocholerae 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 polysaccharideby digestion with a recently discovered polysaccharide lyase derived from a bacteriophage specific forthis serogroup. It specifically cleaves at a single position of the 4-linked galacturonic acid producing anunsaturated sugar product in quantities for conformational studies by 1 H and 13 C NMR spectroscopy. Wereport conformational studies on this oligosaccharide by molecular modeling and NMR spectroscopyincluding nuclear Overhauser effects and residual dipolar coupling of a sample weakly oriented in liquidcrystalline 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 theseresults with those of a previously published study of the intact capsular polysaccharide indicates that theconformations 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 capsularantigens.Although improved sanitation in the 20th century haseliminated major outbreaks of cholera in North America, itremains a debilitating disease throughout much of thedeveloping world with epidemics resulting in hundreds ofthousands of cases and thousands of deaths in many partsof Africa, South America, and Asia. The disease is causedby Vibrio cholerae, a Gram-negative bacterium that isassociated with brackish water. While before 1992 it wasbelieved that cholera was caused only by serogroup O1strains, an outbreak of disease caused by a new serogroup,O139, has spread from India and Bangladesh throughoutmuch of Asia (1).V. cholerae O139 Bengal is genetically similar to O1 ElTor strains, including the presence of cholera toxin genes,but differing in that the former has a capsular polysaccharidewhile the latter is not encapsulated (2). There is considerableevidence that the capsule is an essential virulence factor ofthe strain O139. Although the immunological specificity ofO-serotypes in Gram-negative bacterial strains is generallyassumed to arise from the polysaccharide chain of thelipopolysaccharide (LPS), strain O139 is unusual in that thereis cross-reactivity between the capsule and the LPS (3). Thisphenomenon is explained by the observation that the capsuleand LPS O-chain share an identical chemical structure withthe LPS containing a single repeat of the subunit found inthe capsule (4).The structure of the capsule is unusual in that it containsa cyclic phosphate substituent on a galactopyranoside residueand its hexasaccharide repeating unit features a tetrasaccha-ride epitope which is homologous to the human Lewis