Characterization and biological role of the O-polysaccharide gene cluster of Yersinia enterocolitica serotype O:9

Abstract

Yersinia enterocolitica serotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) in Y. enterocolitica O:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer of N-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of the gnd gene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster, galF and galU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.

FIG. 1.

OPS gene cluster of Y. enterocolitica O:9. The gene cluster figures were generated from the sequence files under the accession numbers AJ605741 for Y. enterocolitica O:9 and AF047478 for B. melitensis using the ggnVIEW sequence file viewer (http://colibase.bham.ac.uk/cgi-bin/fileprepare.cgi). The identified and predicted promoter locations of the O:9 gene cluster are indicated with bent arrows. The orf1 gene (Table 3), present in the reverse direction upstream of the manC gene, is not shown. The regions of homology between the gene clusters are indicated with shadowed quadrangles, and the percentages of identity between the predicted amino acid sequences are given.

FIG. 2.

DOC-PAGE analysis of the LPS of wild-type and mutant strains grown overnight at RT. Strains are indicated at the top, and the different parts of LPS are indicated at the right. The peculiar situation of the LPS of Y. enterocolitica O:9 is demonstrated here; the OC likely represents a nonpolymerized O unit attached to typical (inner) core oligosaccharide.

FIG. 3.

Role of LPS phenotype in resistance of Y. enterocolitica O:9 to polymyxin B. Bacteria were grown either at RT (A) or at 37°C (B). *, P < 0.05, significant difference from wild-type strain; Δ, P < 0.05, significant difference from OC mutant.

FIG. 4.

Role of LPS phenotype in serum resistance of Y. enterocolitica O:9. Survival of bacteria in 66.7% NHS (CP/AP-killing bottom panel) and Mg-EGTA-treated serum (AP-killing, top panel) at 0.5- and 2-h time points. The columns indicate the mean survival percentage of the strain, and the bars indicate the ranges of standard errors. The filled columns show the results for the wild-type strain. In between the panels, the strains and their properties with respect to the presence of pYV (Yersinia virulence plasmid) and expression of OPS and OC are indicated. *, the AP killing results of YeO9-OC and YeO9-R1 at 2 h differed significantly (P < 0.001, Student's t test) from those of Ruokola/71.

FIG. 5.

Proposed biosynthesis pathway to GDP-N-formylperosamine in Y. enterocolitica O:9. The pathway up to GDP-4-keto-6-deoxy-d-mannose is identical to that of GDP-l-fucose biosynthesis; both Fcl (GDP-l-fucose synthetase) and Per (GDP-d-perosamine synthase) use this intermediate as a substrate.