Abstract
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 β-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.
Highlights
Microbes use a variety of strategies to obtain nutrients and ensure survival
We uncover that E. faecalis produces a polyGlcNAc-containing extracellular glycopolymer to efficiently migrate into semisolid surfaces and translocate through human epithelial cell monolayers
An indelible bacterial “colony-print” developed inside modified medium optimal for lipopeptide production (MOLP) [24], when six-day-old colonies of the clinical isolate E. faecalis were extensively washed with water to remove adventitiously associated bacterial cells from the surface of the agar
Summary
While motility could be used as a means for accessing nutrient sources, non-motile bacterial species require unconventional mechanisms to accomplish this goal. Enterococcus faecalis is a non-motile, facultative anaerobic bacterium that inhabits the human gastrointestinal (GI) tract [1]. Hypervirulent E. faecalis strains resistant to multiple antibiotics often cause hospital-acquired urinary tract, wound and abdominal infections, as well as bacteremia and infective endocarditis [1]. E. faecalis translocation across the intestinal barrier enables bacterial spread and colonization of distal anatomical sites [2, 3]. E. faecalis extra-intestinal translocation appears to be promoted by association with epithelial cells in aggregates [3], a process that is partly mediated by the synthesis of adhesins [3,4,5] and additional unknown factors
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