Bacteroides fragilis is the anaerobic pathogen most frequently isolated from intraabdominal sepsis. The capsular polysaccharides of B. fragilis have been identified as the primary virulence determinants for this organism and purified capsule is able to induce abscesses in animal models. Elucidation of the structures of the capsular polysaccharides of one strain has demonstrated that the polysaccharides contain a charge motif that confers the ability to cause abscesses. This charge motif is not commonly found in bacterial polysaccharides. The long term goal of this proposal is to understand the genetic factors responsible for the ability of the B. fragilis capsular polysaccharides to induce abscesses. The experiments outlined in the specific aim will study the genetic factors underlying the formation and expression of these virulence determinants. Preliminary data suggest that the product of one of the genes of the capsular biosynthesis locus may be responsible for the free amino group on the capsular polysaccharide. Because this group is essential for the abscess-inducing capabilities of the polysaccharide, the product of this gene will be studied by creating a mutant and studying the effect on abscess formation, and by purifying the product and assaying for enzymatic activity. The structure of the capsular polysaccharides have been determined for only one strain of B. fragilis. Studies will be conducted to determine the genetic heterogeneity of the polysaccharide biosynthesis loci for both clinical and fecal isolates, with an emphasis on the presence of genes that will enable the bacteria to produce a polysaccharide that is able to induce abscesses. Additionally, experiments will be performed that will address the regulation of expression of these capsular polysaccharides both in vitro and in vivo using transcriptional fusion reporter strains with the promoters that regulate transcription of the polysaccharide biosynthesis loci. The involvement of regulatory genes demonstrated to be involved in the expression of one of the capsular polysaccharides will be studied by creating in-frame deletion mutants of each of the genes. These will be the first studies to address the regulation of expression of B. fragilis polysaccharides and may explain why these bacteria are able to reside in the intestine as normal flora but produce disease when released to extraintestinal sites.
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