Role of Enterococcal Membrane Proteins in Cell-Cell Communication and Stress Adaptation

Enterococcus faecalis has recently emerged as one of the leading causes of nosocomial infections. The ability to tolerate a variety of antibiotics and environmental stresses (biotic and abiotic) combined with a plethora of host adaptation strategies allows this bacterium to transition from its sessile state as gut commensal into an opportunistic pathogen that can colonize multiple niches in a host to cause persistent infections. Pheromone-mediated conjugation facilitates E. faecalis in acquiring a variety of antibiotic resistance genes and virulence factors which are often encoded by the pheromone-responsive plasmid that undergoes cell to cell transfer during conjugation. Although a lot is known about the mechanisms by which pheromones regulate gene expression in these systems, the transport of these pheromones (export and import) across the cell membrane is poorly understood. In the current study we have identified the ABC transporter (PptAB) that is required for the secretion of these peptide sex pheromones. Using 3 distinct pheromone-responsive plasmids systems we demonstrated that the deletion of this transporter (pptAB) resulted in a severe conjugation defect. We also identified a second oligopeptide permease (Opp) that is involved in the import of these secreted peptide sex pheromones into the cell. Deletion of the genes encoding these peptide transporters (pptAB, opp1 and opp2) resulted in a severe biofilm defect suggestive of a role played by these peptide pheromones in the development of these surface adherent structured communities. We also identified the additional roles played by the membrane protein Eep in the stress adaptation pathways of E. faecalis. Eep was originally described as a protein that was involved in the processing of lipoprotein derived peptide sex pheromones. We showed that Eep is the site-2 protease that is involved in the regulated intramembrane proteolysis of an anti-sigma factor called RsiV which in turn results in the activation of an extracytoplasmic function sigma factor called SigV that confers high level resistance to lysozyme. We also showed that the deletion of eep increases the susceptibility of E. faecalis towards high temperature, low pH and ethanol stresses indicative of a role played by Eep in multiple stress adaptation pathways