Decoration of the enterococcal polysaccharide antigen EPA is essential for virulence, cell surface charge and resistance to innate immunity

Abstract

1Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to 3 2 lysozyme, a key effector of the innate immune system. This high level of resistance requires 3 3 several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic 3 4 and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes 3 5 modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in 3 6 the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent 3 7insertions clustered on the chromosome. These mutations were located in a locus referred to as 3 8 the enterococcal polysaccharide antigen (EPA) variable region located downstream of the 3 9 highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa 4 0 variable region was previously proposed to be responsible for EPA decorations, but the role of 4 1 this locus remains largely unknown. Here, we show that EPA decoration contributes to 4 2 resistance towards charged antimicrobials and underpins virulence in the zebrafish model of 4 3 infection by conferring resistance to phagocytosis. Collectively, our results indicate that the 4 4

Carbohydrate Metabolism Affects Macrophage-Mediated Killing of Enterococcus faecalis

“…The other genes with disruptions that caused fitness reductions included those encoding an acetyltransferase (log 2 FC, −1.66), aspartate aminotransferase (log 2 FC, −1.59), and a LysR family transcriptional regulator (log 2 FC, −1.73) ( Table S1 ), of which all will be further investigated in the future. In addition, mutants in OG1RF_11714, which encodes a polysaccharide antigen EPA-related protein, glycosyltransferase, displayed an abundance reduction after 48 h (log 2 FC, −0.713) or 72 h (log 2 FC, −0.829) of survival in RAW264.7 cells ( Table S1 ), which was consistent with the results reported by Smith ( 30 ).…”

“… E. faecalis resists immune clearance by the host and causes severe infectious diseases worldwide. Although several factors have been reported to contribute to the resistance mechanisms of E. faecalis against host immunity, such as the enterococcal polysaccharide antigen (EPA) ( 30 ), the impact of metabolites or metabolic pathways on the ability of E. faecalis to resist macrophage-mediated killing has rarely been reported. According to the results of transposon insertion sequencing, a total of 15 and 19 mutants with decreased fitness in RAW264.7 cells were identified from the 48- and 72-h groups, respectively, of which 9 overlapped ( Fig.…”

“…The in-frame deletion of E. faecalis OG1RF was constructed by allele exchange using the procedure previously described ( 30 ). pGhost9 derivatives, which were replicated in a temperature-dependent manner in host cells, were electroporated, and transformants were selected at 28°C on BHI agar plates with erythromycin (30 μg/ml).…”