Enterococcal Polysaccharide Antigen (EPA) rhamnan backbone contributes to cell wall architecture and is essential for antimicrobial resistance, innate immune evasion and phage infection

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ABSTRACT Enterococci are opportunistic pathogens classified by the World Health Organization as high-priority microorganisms. They cause a broad spectrum of infections, and their intrinsic and acquired resistance to antimicrobials makes these infections particularly difficult to treat and eradicate. In Enterococcus faecalis , the most frequently isolated enterococcal pathogen in humans, antimicrobial resistance and innate immune evasion are largely driven by the Enterococcal Polysaccharide Antigen (EPA). This surface polymer underpins key virulence traits, including resistance to host defence mechanisms, reduced susceptibility to multiple classes of antimicrobials, and susceptibility to bacteriophage infection. EPA consists of a rhamnan backbone decorated with strain-specific substituents that are essential for its biological activity. Here, we show that epaB encodes the enzyme responsible for the first committed step in assembling the EPA rhamnan chain. Using NMR spectroscopy, we demonstrate that E. faecalis lacking epaB produces an EPA polymer composed solely of decorations directly anchored to the peptidoglycan, with no detectable rhamnan backbone. The absence of this rhamnan moiety profoundly alters cell wall architecture, as revealed by atomic force microscopy of the mutant cell walls. The epaB mutation also abolishes innate immune evasion and virulence in the zebrafish infection model, while conferring resistance to bacteriophages. Collectively, these findings demonstrate that both the rhamnan backbone and its decorations are required for EPA’s full biological activity, establishing the structural and functional interdependence of these two components. IMPORTANCE The Enterococcal Polysaccharide Antigen (EPA) is essential for normal growth and division, virulence, antimicrobial resistance, and phage infection in enterococci. This surface polymer comprises a structurally conserved rhamnan backbone substituted with strain-specific decorations. These variable decorations have been directly linked to the biological functions of EPA, whereas the rhamnan backbone has been proposed to serve primarily as a structural scaffold. Here, we use NMR spectroscopy to show that mutation of epaB , the gene responsible for the first biosynthetic step in rhamnan backbone formation, results in an EPA polymer composed exclusively of decorations anchored to the peptidoglycan. We reveal that the lack of rhamnan backbone is associated with a change in the cell wall architecture and abolishes virulence and infection by bacteriophages. Together, these findings demonstrate that assembly of the conserved rhamnan backbone is indispensable for EPA function and highlight this biosynthetic step as a promising target to combat enterococcal infections.