| With the goal of identifying new targets for the rational design of novel antimicrobial agents and immunotherapeutics for use in the treatment of infections caused by multi-drug resistant strains of Enterococcus faecalis, the biology and chemistry of prominent cell surface components were investigated. It was found that E. faecalis produces three anionic polysaccharides of varying molecular mass, ranging from 15 kDa to 130 kDa. The 130 kDa polysaccharide was identified as a serotype capsular antigen. This polysaccharide was purified and found to consist of glycerol phosphate, glucose, and galactose. The synthesis of the capsular polysaccharide was shown to be encoded by a cluster of genes arranged in an operon. Inactivation of several genes in this operon resulted in mutant strains that failed to produce capsular polysaccharide, which compromised the ability of these mutants to persist at sites of infection in the host. In addition, the capsular polysaccharide was opsonized with antibodies, which led to phagocytic killing by neutrophils, establishing its value as a basis for the design of immunotherapeutics for treatment of antibiotic resistant enterococcal infections. We also identified an invariant cell wall polysaccharide, the conservation of which suggests that it could be a target for a broadly applicable antimicrobial therapy. Purification of this polysaccharide revealed it to be a rhamnopolysaccharide similar to those known to constitute a structural element of the cell walls of other streptococci. In streptococci, these rhamnopolysaccharides do not elicit a protective immune response. Despite its conservation, the rhamnopolysaccharide of E. faecalis is unlikely to be an effective immunotherapeutic target. The genetic regulation of the capsular polysaccharide encoding operon was explored using promoter fusions to the β-galactosidase reporter, and by real time RT-PCR. Data obtained from both reporter fusion and real time RT-PCR indicate that this operon is maximally expressed under logarithmic growth conditions. Further understanding of how capsular polysaccharide expression relates to pathogenesis will provide important insights into targeting this process in the development of antimicrobial strategies. |
