The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae NRT36S: genes are embedded in the LPS region

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.

Figure 1

Panel A shows the HSQC spectrum of the de-O-acetylated capsular polysaccharide from NRT36S. The strong signal at 3.78, 60 ppm is a low molecular weight impurity. Panel B shows the signal from the anomeric region. The methyl group region is not shown.

Figure 2

Proposed structure of NRT36S CPS repeating unit. Parentheses indicate partial O-acetylation of residue B at the 3-position.

Figure 3

Size exclusion chromatography of the capsule prep. A). Capsule prep from control (NRT36S and A5) compared to purified CPS of NRT36S. The size was estimated by thyroglobulin. The arrow indicates the peak of the capsule at about 13.2 minutes retention time, which corresponds to about 670 k Dalton molecular weight. B). Capsule prep from mutants and control.

Figure 4

Immuno blot of NRT36S antiserum against external polysaccharide preparations from various strains.

Figure 5

Thin sections of V. cholerae NRT36S and its translucent mutants stained with polycationic ferritin. (A) NRT36S; (B) TR3; (C) TR296; (D) TR17. Bar, 200 nm.

Figure 6

Map of the CPS/O-antigen region of V. cholerae NRT 36s. JUMPstart site is indicated by a diamond. Transposon insertion sites are indicated by black arrows. Design patterns of open reading frames indicate different classes of genes: vertical lines, pathway genes; diagonal lines, processing and transportation genes; grey box, glycosyltransferase; white box, functions not clear.

Figure 7

Pictorial representation of the genes in the LPS/CPS regions of O1, O37, O22, O139 and O31 Vibrio cholerae. The galE and wbeW genes found in common between all five sequences are marked in blue. The housekeeping genes gmhD and rjg that delineate the region are marked in black. The genes in common between O22 aand O139 are marked in grey. The transport genes and the IS elements are labeled. The unlabeled genes represented by white boxes are not found in common across the regions.