EpaX is necessary for <i>E</i>. <i>faecalis</i> semisolid surface penetration and synthesis of polyGlcNAc-containing polysaccharides.

(A—H) Penetration analysis for E. faecalis VE14089 (closely-related to MMH594) and the EpaX-deficient mutant (ΔepaX) (A) Invasion was tested for mutant and its parental strain with (right panel) or without exogenous GlcNAc, glucosamine or fructose. Complementation in-trans of epaX WT allele restored invasion (p-epaX). Scale bar: 6,000 μm. (B) SEM images of external cells at (left) low magnification (scale bar: 5 μm). Yellow squared areas were imaged at higher magnifications (right; scale bar: 1 μm), where extracellular matrix is indicated (yellow arrow head). (C) Fractions of cells and extracellular matrix found on the visible surface of SEM images of non-penetrating WT and mutant strains. (mean±SE; n = 3; *Pt–test) (D) Glycosyl composition of polysaccharides extracted from liquid cultures of WT and ΔepaX grown to exponential phase in MOLP broth (mean±SE; n = 3). (E) Fluorescence microscopy images of external cells from MOLP-grown colonies incubated with mAb F598 to visualize GlcNAc-containing exopolysaccharides (green fluorescence). DAPI was used to stain bacterial DNA (blue fluorescence). Scale bar: 20 μm. (F) Immunoblot (left panel) of 24-hour-grown colonies. The relative intensity obtained upon incubation with mAb F598 was calculated for each colony using Image J, right panel; (mean±SE; n = 8; ****PG) Semisolid media penetration of WT and ΔepaX grown with and without 200 μM PNAG purified from S. aureus MN8. Scale bar: 5,000 μm. (H) Quantification of invasion was expressed as CFUs/mL (mean±SE; n = 4; **P<0.01 for both the one-way ANOVA and Tukey’s multiple comparison test).