Enterococcus faecium virulence: A molecular and therapeutic approach

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After acquiring expertise in molecular and translational research during the mentored (K99) phase of this award, I now seek to develop a career transition to independence in the ROO phase in the area of molecular pathogenesis of antibiotic-resistant bacteria. The long term goals of this work are to translate in vitro and in vivo observations relating to virulence and pathogenesis into new useful strategies to combat severe multidrug resistant enterococcal infections. Enterococcus faeclum (Efm) have now become the third most common organism isolated from hospital Infections, emerging as one of the most difficult bacteria to eradicate due to the development of resistance to virtually all antibiotics used In clinical practice. Research performed during the mentored phase of this award confirmed and further characterized a large plasmid carried by clinical isolates of Efm, which confers increased virulence in experimental peritonitis and indicated that a gene (designated hylEfm and encoding a putative glycosyl hydrolase), which was the focus of the research during the mentored phase, was not sufficient to explain the increased lethality conferred by the plasmid carrying it. The current hypotheses for this work are that i) other genes in the hylEfm region may be essential, in conjunction with hylEfm, for pathogenesis In Efm and 11) blocking the hylEfm gene product, as part of an important gene cluster involved in virulence, in addittlon to antibiotics, could be a novel therapeutic strategy to treat Efm infections. Thus, the specific aims of this part of the award will be to: I) Define the role and contribution of the hylEfm region to virulence In Efm and Ii) Evaluate an antibody-antibiotic approach for Efm infections using anti-HylEfm antibodies as part of the therapeutic combination. These aims will be addressed by using the tools developed during the mentored phase which included the application of the counterselection system PheS* for targeted mutagenesis to obtain non-polar mutations of genes in the hylEfm region, mRNA analysis to determine the patterns of In vitro and in vivo expression of this cluster and testing the mutants' phenotypes and response to therapies in animals models of Efm peritonitis, urinary tract infection and endocarditis.