Enterococcal Pathogenesis:Role of Cytolysin

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DESCRIPTION (provided by applicant): Enterococci have emerged as leading causes of multidrug resistant hospital infection. Yet they have existed as commensal microbes of everything from insects to man for over 400 million years. Our previous work, as well as the work of others, has shown that one of the factors that makes hospital isolate of Enterococcus faecalis more virulent is a toxin termed "cytolysin." The E. faecalis cytolysin has antibacterial properties, as well as being a cytolytic toxin for human cells. We previously showed that this toxin contributed a 5-fold increased risk of death from infection, that it was conveyed on a pathogenicity island along with several additional virulence traits, and that its expression is regulated by a novel process termed "telesensing" that allows the bacterium to detect the presence of target cells in the vicinity, and in respose produce high levels of the toxin. Although we have shown that the E. faecalis cytolysin toxi is novel in structure and regulation, we do not yet know how it contributes to the severity of infection in man, or in models of infection where it renders E. faecalis infection as much as 100 times more acutely toxic. We also do not know exactly how "telesensing" works at the level of transcription. Therefore, we propose to determine whether the antibacterial property of the toxin allows E. faecalis entry into the GI tract microbiota, whether it there causes changes in the bacterial composition and overgrows within the colon because of the antibacterial activity, whether it compromises the barrier function of the colon through its cytolytic activity for mammalian cells, or whether it exacerbates infection by impeding the innate immune system from clearing the organism from a site of infection. To understand how its expression is regulated, we also propose to dissect the novel two component regulatory system which has been shown to be necessary for its repression and depression. This will be done by mapping the precise points where transcription of the cytolysin operon is initiated, and determining the relationship between those start sites and structural features of the regulatory region of the operon. We will determine whether the inducing signal that results in high level toxin expression is communicated from the outside of the cell, or whether t is internalized and directly causes operon depression. Finally, we will determine exactl how each of the regulatory proteins relate to each other, and relate to structural features within the control region of the cytolysin operon. These results will provide needed information for knowing how the cytolysin exacerbates E. faecalis infection, as well as the information needed for controlling its expression.