Enterococcus faecalis plays a major role in the current antibiotic resistance crisis. It is one of the leading causes of antibiotic-resistant nosocomial infections and, as part of the normal flora of the intestinal tract, also serves as a reservoir of antibiotic resistance genes. Peptide pheromone-induced conjugative plasmid transfer contributes to the spread of these antibiotic resistance genes and of other enterococcal virulence factors. The focus of this research is on the role of peptide signaling in plasmid maintenance, specifically of the tetracycline-resistance plasmid pCF10 in Enterococcus faecalis. The original observations of pheromone signaling in conjugative plasmid biology found that the peptide signal cCF10 produced by plasmid-free recipient cells is used for the induction of conjugative pCF10 transfer. cCF10 is produced at the cell surface and internalized by pCF10-containing donor cells by interaction of the cCF10 receptor with the oligopeptide permease. Once inside the cell cCF10 interacts with intracellular effector molecules to induce conjugation. Recently, we have found a second role, for donor-cell produced cCF10, in maintaining pCF10 in the Enterococcus faecalis cell population. In a donor cell population, cCF10 is also produced and its internalization is required to maintain pCF10 in the apparent absence of conjugation. The maintenance functions occur with a minimum pCF10 replicon construct that is unable to be conjugatively transferred, suggesting the role of cCF10 in pCF10 maintenance is distinct from its role in conjugative transfer. The main goal of the research proposed in this application is to gain a basic understanding of how cCF10 is affecting pCF10 maintenance. (1) Does cCF10 act as a pheromone in maintenance of pCF10? (2) Are the oligopeptide permease and the cCF10 receptor PrgZ required for efficient transport of cCF10 (produced extracellularly)? (3) What are the basic features of the pCF1 0 replicon? (4) What are the intracellular effector molecules responsible for cCF10-dependent pCF10 maintenance? This will lay the groundwork for future experiments to determine how the same peptide can be used for plasmid maintenance without inducing detectable levels of conjugation.
