PolyGlcNAc-containing exopolymers enable surface penetration by non-motile Enterococcus faecalis

Bacterial pathogens have evolved strategies that enable them to invade tissues and spread within the host. Enterococcus faecalis is a leading cause of local and disseminated multidrug-resistant hospital infections, but the molecular mechanisms used by this non-motile bacterium to penetrate surfaces and translocate through tissues remain largely unexplored. Here we present experimental evidence indicating that E. faecalis generates exopolysaccharides containing -1,6-linked poly-N-acetylglucosamine (polyGlcNAc) as a mechanism to successfully penetrate semisolid surfaces and translocate through human epithelial cell monolayers. Genetic screening and molecular analyses of mutant strains identified glnA, rpiA and epaX as genes critically required for optimal E. faecalis penetration and translocation. Mechanistically, GlnA and RpiA cooperated to generate uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that was utilized by EpaX to synthesize polyGlcNAc-containing polymers. Notably, exogenous supplementation with polymeric N-acetylglucosamine (PNAG) restored surface penetration by E. faecalis mutants devoid of EpaX. Our study uncovers an unexpected mechanism whereby the RpiA-GlnA-EpaX metabolic axis enables production of polyGlcNAc-containing polysaccharides that endow E. faecalis with the ability to penetrate surfaces. Hence, targeting carbohydrate metabolism or inhibiting biosynthesis of polyGlcNAc-containing exopolymers may represent a new strategy to more effectively confront enterococcal infections in the clinic. Author summary Enterococcus faecalis is a microbial inhabitant of the human gastrointestinal tract that can cause lethal infections. Typically classified as a non-motile bacterium, E. faecalis can readily migrate and translocate across epithelial barriers to invade distant organs. Nevertheless, the molecular pathways driving enterococcal invasive attributes remain poorly understood. In this study, we uncover that E. faecalis produces a polyGlcNAc-containing extracellular glycopolymer to efficiently migrate into semisolid surfaces and translocate through human epithelial cell monolayers. Our work provides evidence that non-motile bacterial pathogens can exploit endogenous carbohydrate metabolic pathways to penetrate surfaces. Thus, targeting glycopolymer biosynthetic programs might be useful to control infections by Gram-positive cocci in the clinic.