Norwood, J. S. | Search bioRxiv

bioRxiv2024

The Enterococcal Polysaccharide Antigen: from structure to biosynthesis and function

L-Rhamnose-containing polysaccharides are produced by Streptococci and Enterococci. They define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The Enterococcal Polysaccharide Antigen 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 for E. faecalis physiology and host-pathogen interactions, little information is available on its structure and biosynthesis. Here, we elucidate the structure of the intact EPA produced by E. faecalis OG1RF. We report the structure of the linkage unit, revealing an unprecedented complexity of the rhamnose backbone and decorations. 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 for the rational design of therapeutic strategies against a group of important pathogens.

microbiology↗

bioRxiv2024

Exploring the role of E. faecalis Enterococcal Polysaccharide Antigen (EPA) and lipoproteins in evasion of phagocytosis

Enterococcus faecalis is an opportunistic pathogen frequently causing nosocomial infections. The virulence of this organism is underpinned by its capacity to evade phagocytosis, allowing dissemination in the host. Immune evasion requires a surface polysaccharide produced by all enterococci, known as the Enterococcal Polysaccharide Antigen (EPA). EPA consists of a cell wall-anchored rhamnose backbone substituted by strain-specific polysaccharides called "decorations", essential for the biological activity of this polymer. However, the structural determinants required for innate immune evasion remain unknown, partly due to a lack of suitable validated assays. Here, we describe a quantitative, in vitro assay to investigate how EPA decorations alter phagocytosis. Using the E. faecalis model strain OG1RF, we demonstrate that a mutant with a deletion of the locus encoding EPA decorations can be used as a platform strain to express heterologous decorations, thereby providing an experimental system to investigate the inhibition of phagocytosis by strain-specific decorations. We show that the aggregation of cells lacking decorations is increasing phagocytosis and that this process does not involve the recognition of lipoproteins by macrophages. Collectively, our work provides novel insights into innate immune evasion by enterococci and paves the way for further studies to explore the structure/function relationship of EPA decorations.

microbiology↗