Enterococci represent one of the leading causes of infections among hospitalized patients and residents of long-term care facilities. In the USA, there were over 50,000 cases of enterococcal infections in 2017, with ~10% fatality rate. The majority of infections are caused by Enterococcus faecalis and E. faecium, which include bacteremia, urinary tract and surgical sites infections, and endocarditis. E. faecalis and E. faecium display high levels of natural and acquired antibiotic resistance that severely limits the treatment options. The major component of streptococcal cell wall is the Enterococcal Polysaccharide Antigen (EPA). The recently solved structure of EPA consists of a polyrhamnose backbone decorated with a polyribitol phosphate- containing teichoic acid. While a hypothetical mechanism of EPA biosynthesis has been proposed, studies are required to test it. The goal of this application is to decipher the critical steps of EPA polyrhamnose and teichoic acid assembly. In addition, we will examine the role of EPA in binding to the enterococcal autolysins and the resistance mechanism to host antimicrobial protein expressed in the intestinal epithelial cells, ReGIIIγ. We will utilize a combination of genetic, analytical and biochemical methods to answer these questions. The enzymes of EPA biosynthetic pathway constitute promising targets for the development of novel antimicrobials to combat enterococcal infections. Thus, the proposed experiments will provide a better understanding of cell wall biogenesis in enterococci to enable the development of alternative approaches to treat drug-resistant infections. Furthermore, the proposed studies will enhance our understanding of the functions of EPA in antimicrobial resistance and persistence of enterococci in gut microbiota.