PROJECT SUMMARY In recent decades there has been a rapid decline in effective antibiotic therapies and an increase in multidrug- resistant (MDR) bacterial infections. One common MDR pathogen is the Gram-positive intestinal bacterium Enterococcus faecalis which resists many antibiotics including “last-line-of-defense” drugs such as vancomycin. MDR enterococci can expand in the intestine in individuals undergoing broad-spectrum antibiotic therapy. This expansion can lead to E. faecalis translocation to the bloodstream, sepsis, and further shedding of the bacterium, thus perpetuating these hospital-acquired infections. One potential strategy to combat MDR enterococci is the use of bacteriophages (phages). Phages are viruses that infect and kill bacteria with high specificity. Phage infection relies on binding to the bacterial cell surface, ejection of phage DNA into the bacterial cell, replication of the phage genome, and viral particle release from the cell. The use of phages as therapeutics raises concern similar to antibiotic use; bacteria will become resistant to infection, diminishing therapeutic efficacy. Thus, before phages can become a standard clinical therapy, we must clearly understand the specific mechanisms used by phages to infect bacteria and how bacteria respond to phage infection. To begin to elucidate the mechanisms used by phages during infection, I challenged E. faecalis with lytic phages and found that the enterococcal polysaccharide antigen is used for initial phage adsorption to various E. faecalis strains, including strains that the phage cannot successfully infect. Along with this, I have shown that the presence of a mobile plasmid in E. faecalis restricts phage infection. The goals of this project are to fill key gaps in our basic knowledge of phage-bacteria interactions and to provide insights into the mechanisms that influence the outcomes of phage infection, which will be beneficial knowledge for applied phage therapies. This project encompasses the following two Specific Aims: Aim 1: Identify the receptor that promotes DNA entry of Epa-dependent phages. This will be addressed through complementary biochemical assays to identify the receptor that promotes phage DNA entry into E. faecalis. Aim 2: Define the genetic basis of endogenous plasmids in restricting enterococcal phage infection. Here, I will use genetic approaches to identify a novel, enterococcal anti-phage restriction mechanism encoded on a mobile plasmid. These experiments will reveal new insights into the mechanisms that dictate enterococcal phage recognition and infection that will ultimately aid in the development of informed phage therapies. In doing so, my results will expand our basic knowledge surrounding enterococcal cell biology during phage infection and potentially identify new targets for anti-enterococcal therapies.