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

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.

Exopolysaccharide-mediated surface penetration as new virulence trait in Enterococcus faecalis

“…Chatterjee and collaborators recently demonstrated that mutations in genes involved in decorating EPA reduced E. faecalis resistance to antibiotics, but conferred elevated resistance to phage infections in both cultures and in an intestinal colonization mouse model [25]. Our work demonstrated that the absence of either a polyGlcNAc decoration of EPA or a new polyGlcNAc-containing polysaccharide impaired the ability of E. faecalis to move into surfaces, suggesting a new role for these exopolysaccharides [8].…”

“…Indeed, we demonstrated that the RpiA-GlnA-EpaX metabolic axis was also necessary for movement through human intestinal barriers, and this process involved the formation of bacterial aggregates. Thus, strains devoid of either GlnA, RpiA or EpaX had impaired ability to translocate through epithelial cell monolayers [8]. Altogether these results revealed a new role for polyGlcNAc-containing extracellular polysaccharides as key mediators of E. faecalis migration traits.…”

“…Moreover, the use of broad-spectrum antimicrobials has been suggested to be a predisposing factor that promotes the translocation of vancomycinresistant enterococci [5]. While the mechanisms of enterococcal migration remain largely unexplored, recent evidence indicates that this process is mediated by the formation of bacterial aggregates through cellsurface appendages, adhesins, and other unknown factors [4][5][6][7][8][9][10]. A deeper understanding of the molecular processes that enable E. faecalis movement and translocation across epithelial barriers is required to more effectively control systemic infections by this opportunistic pathogen.…”

“…Our group recently uncovered a new mechanism used by E. faecalis to penetrate semisolid surfaces and translocate through human epithelial cell monolayers, where synthesis and secretion of enterococcal extracellular polysaccharides is requiered [8]. When grown on semisolid agar, E. faecalis cells penetrated and invaded into the medium, thereby creating a "colony-print" (penetrating cells;).…”

“…Further, we found that the absence of these polysaccharides rendered E. faecalis incapable of efficiently penetrating the semisolid surface. We determined that E. faecalis aggregates were formed by metabolically active cells [8] and exhibited an organized spatial distribution. When two E. faecalis strains constitutively expressing m-Cherry or GFP reporters were co-cultured, spatially-differentiated red or green regions, respectively, were observed within the penetrating colony print (Figure 1(c)), suggesting that an unknown process might dictate the cellular localization within the aggregates.…”