Regulation of Bacteroides fragilis capsule expression

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DESCRIPTION (provided by the applicant): Bacteroides fragilis is the most frequently isolated anaerobic species from human infections such as intra-abdominal abscesses and bacteremia. The capsular polysaccharide complex of this organism has been identified as the primary virulence factor responsible for intra-abdominal abscesses. We identified two conserved genes (upxY and upxz) located upstream of eight distinct polysaccharide biosynthesis regions in a single B. fragilis strain. We hypothesize these products play an important role in the regulation of the capsular polysaccharides of B. fragilis, bestowing upon them a global function for polysaccharide regulation in this species. We have shown that deletion of upcY abrogates the ability of B. fragilis to produce one of its eight polysaccharides, and that this regulation occurs at the transcriptional level. We hypothesize that each tandem pair of genes specifically regulates the polysaccharide biosynthesis locus of which they are a part. An upaY mutant will be created and its phenotype will be analyzed to determine if it has a similar function in the specific regulation of the PS A biosynthesis locus as that of upcY in the regulation of the PS C biosynthesis operon. xylE reporter studies will determine if UpaY acts at the level of transcription to specifically regulate the PS A operon. Site-specific mutagenesis will be performed to determine which amino acids of UpaY are involved in conferring specific regulation of PS A expression. As UpcZ is not homologous to any sequences in the databases we will determine the role and mode of action of UpcZ in the regulation of PS C, by constructing a deletion mutant and performing transcriptional assays.