Dissecting the enterococcal polysaccharide antigen (EPA) structure to explore innate immune evasion and phage specificity

Streptococci, Lactococci and Enterococci all produce L-rhamnose-containing cell wall polysaccharides which define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The L-rhamnose containing Enterococcal Polysaccharide Antigen (EPA), produced by the opportunistic pathogen Enterococcus faecalis, plays a critical role in normal growth, division, biofilm formation, antimicrobial resistance, phage susceptibility, and innate immune evasion. Despite the critical role of this polymer in E. faecalis physiology and host-pathogen interactions, little information is available on its structure and biosynthesis. Here, using an NMR approach, we elucidate the structure of EPA and propose a model for biosynthesis. We report the structure of the EPA-peptidoglycan linkage unit and reveal an unprecedented complexity of the EPA rhamnose backbone and decoration subunits. Finally, we explore the impact of several EPA structural modifications on innate immune evasion and recognition by bacteriophages. This work represents a first step towards the functional characterisation of EPA and the rational design of therapeutic strategies against a group of important pathogens.