GENETICS OF CAPSULAR POLYSACCHARIDE PRODUCTION IN ENTEROCOCCUS FAECALIS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Entercocci are leading causes of hospital-acquired infections, accounting for nearly 10% of all nosocomial infections. Infections caused by enterococci constitute a significant treatment challenge due to the presence of multi-drug resistance. The proposed studies will determine how capsule diversity is achieved at the genetic level. Recent evidence suggests that E. faecalis has a limited number of capsular serotypes and that the majority of isolates fall into 4 predominant serotypes (A-D). Recent clinical evidence suggests that E. faecalis strains possessing capsule types C and D maybe more pathogenic than strains expressing other capsule types. Serotypes C and D appear to be structurally related based on extensive immunologic cross-reactivity. The genetic basis for serotype C capsule biosynthesis is encoded by an operon of 9 genes, designated cpsC-K. Genetic comparison between serogroup C and D strains revealed extensive sequence conservation for each gene in the operon with the exception of cpsF, which was only present in serotype C strains. We aim to address this difference by constructing an in-frame deletion mutant of cpsF in E. faecalis V583, a serotype C strain. Complementation of type D strains with cpsF expressed from the native capsule promoter on a multi-copy plasmid will also be used to confirm the hypothesis that the presence of cpsF confers type C specificity. These derived strains will be examined by ELISA using type-specific antisera. In addition, carbohydrate compositional analysis on purified capsular material using GC/MS will be performed to determine the compositional basis for the serotype differences between types C and D.