Bacteroides fragilis NCTC9343 produces at least three distinct capsular polysaccharides: cloning, characterization, and reassignment of polysaccharide B and C biosynthesis loci

Abstract

Bacteroides fragilis produces a capsular polysaccharide complex (CPC) that is directly involved in its ability to induce abscesses. Two distinct capsular polysaccharides, polysaccharide A (PS A) and PS B, have been shown to be synthesized by the prototype strain for the study of abscesses, NCTC9343. Both of these polysaccharides in purified form induce abscesses in animal models. In this study, we demonstrate that the CPC of NCTC9343 is composed of at least three distinct capsular polysaccharides: PS A, PS B, and PS C. A previously described locus contains genes whose products are involved in the biosynthesis of PS C rather than PS B as was originally suggested. The actual PS B biosynthesis locus was cloned, sequenced, and found to contain 22 genes in an operon-type structure. A mutant with a large chromosomal deletion of the PS B biosynthesis locus was created so that the contribution of PS B to the formation of abscesses could be assessed in a rodent model. Although purified PS B can induce abscesses, removal of this polysaccharide does not attenuate the organism's ability to induce abscesses.

FIG. 1

Western immunoblot analysis of B. fragilis wild-type and mutant strains. Bacterial cultures were grown to an optical density at 600 nm of 0.8, and the bacteria were pelleted by centrifugation and lysed with 1× loading buffer. Bacteria (equivalent to 40 μl of culture) were added to each well of the 4 to 20% gradient SDS-polyacrylamide gel, transferred to PVDF, and probed with MAb G9 (PS B specific) (A) or MAb QUBF7 (PS C specific) (B).

FIG. 2

ORF map of the B. fragilis NCTC9343 PS B biosynthesis locus. The direction of transcription of each ORF is designated with an arrow. The region deleted in mutant 9343ΔPS B is indicated above the ORFs. Cosmid clones, subclones, and PCR products used as sequencing templates are shown. The average G+C content of each ORF is presented below.