Table 1.
Virulence factors identified to be relevant for colitogenic activity of E. faecalis in the IL-10-/- mouse model and their proposed cellular mechanisms.
Fig 1.
E. faecalis colitogenic activity is mediated by epaB and lgt.
(A) Expression profile of selected virulence-related genes of E. faecalis wild type OG1RF isolated from colon content of monoassociated IL-10-/- mice vs. monoassociated wild type (WT) mice: differential expression of genes in relation to a chronically inflamed environment is shown for virulence-related genes including the epa cluster (green-labeled locus tags) and lgt-dependent (predicted) lipoproteins putatively involved in E. faecalis virulence (blue-labeled locus tags); epaB (OG1RF_11737) and lgt (OG1RF_11459) are highlighted by red letters. Samples and genes are hierarchically clustered according to Ward-Spearman correlation and log2 ratio of mean abundance (mIL10/mWT) of normalized expression levels is shown (up-regulation is indicated by red bar color, down-regulation is indicated by blue bar color). Please see Table 2 for annotation of locus tags. (B) Histological scores of distal colon from wild type (Wt) and IL-10-/- mice monoassociated with E. faecalis OG1RF, ΔepaB or Δlgt strain. (C) Representative hematoxylin/eosin-stained sections of distal colon from wild type and IL-10-/- mice monoassociated with E. faecalis OG1RF, ΔepaB or Δlgt strain. (D) E. faecalis presence in luminal contents from colon of wild type and IL-10-/- mice monoassociated with E. faecalis OG1RF, ΔepaB or Δlgt mutant strain according to the CFU counts/mL. Differences were considered significant for *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Table 2.
Virulence-related genes of E. faecalis OG1RF selected for RNA-sequencing analysis.
Fig 2.
Colitogenic activity of E. faecalis is associated with infiltration of different immune cell subsets in monoassociated IL-10-/- mice.
(A) Representative images of distal colon sections from IL-10-/- mice monoassociated with E. faecalis OG1RF stained by immunofluorescence for F4/80+ (red), Ly6G+ (red), CD11c+ (red) or CD3+ (red) cells, E-cadherin (intracellular domain, green), nuclei (blue) and magnifications of respective images as indicated by white frames (scale bar = 100μm). (B) Relationship between F4/80+, Ly6G+, CD11c+ or CD3+ cells infiltrating the distal colon of monoassociated IL-10-/- mice with respective histological scoring for distal colon was assessed by Pearson correlation coefficient test (F4/80+ cells: Pearson r = 0.6460, ***p<0.001; Ly6G+ cells: Pearson r = 0.7382, ****p<0.0001; CD11c+ cells: Pearson r = -0.05360, p>0.05; CD3+ cells: Pearson r = 0.6661, ***p<0.001).
Fig 3.
Bacterial structures responsible for colitogenic activity also direct virulence of E. faecalis.
(A) Survival of G mellonella larvae after injection of E. faecalis OG1RF, ΔepaB or Δlgt strain and (B) E. faecalis OG1RF, ΔepaB or reconstituted ΔepaB mutant or (C) E. faecalis OG1RF, Δlgt or reconstituted Δlgt mutant. (D) Survival of C. elegans nematodes after oral administration of E. faecalis OG1RF, ΔepaB or Δlgt strains. Differences were considered significant for *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 4.
EpaB mediates E. faecalis adhesion to intestinal mucus and mucosal surfaces in vivo.
(A) Representative images of distal colon sections from wild type (Wt) and IL-10-/- mice monoassociated with E. faecalis OG1RF, ΔepaB or Δlgt mutant strain, stained by immunofluorescence for MUC2 (green), nuclei (blue) and FISH for E. faecalis (red) (scale bar = 120μm). The epithelial cell surface is indicated by interrupted white line, white arrows indicate representative FISH-labeled E. faecalis. In the representative pictures for E. faecalis ΔepaB the inner-to-outer mucus interface is indicated by dotted white line with white long arrows indicating the distance from this interface layer to the epithelial cell surface. (B, C) Histograms showing the depth of penetration of the mucus layer by E. faecalis cells in the corresponding representative distal colon sections from (B) wild type and (C) IL-10-/- mice monoassociated with E. faecalis OG1RF, ΔepaB or Δlgt mutant strain (0 to 400μm distance from epithelial cell surface as indicated by interrupted white line in the representative pictures; grey areas indicate the average thickness of the inner mucus layer). (D) Adhesion of E. faecalis OG1RF, ΔepaB or Δlgt strains to the intestinal midgut epithelium of monoassociated M. sexta larvae as shown by representative bright-field images from sections stained for E. faecalis (red) by immunofluorescence (scale bar = 100μm). Epithelial cell surface is indicated by interrupted white line.
Fig 5.
E. faecalis biofilm and associated microcolony formation are dependent on epaB.
(A) Microcolonies formed by E. faecalis OG1RF, ΔepaB, Δlgt or reconstituted ΔepaB strain in vitro after incubation for 20 hours on a fixed monolayer of murine Ptk6 intestinal epithelial cells. Representative images stained by immunofluorescence for E. faecalis (red), E-cadherin (intracellular domain, green) and nuclei (blue) showing 3D-reassembling of single stacks and (B) quantitation of total microcolony biomass. (C) Biofilm indices representing total biofilm formation of E. faecalis OG1RF, ΔepaB or Δlgt or reconstituted ΔepaB strain on polystyrene surface after 20 hours incubation stained for biofilm matrix with Hucker’s crystal violet. Differences were considered significant for *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 6.
Gelatinase E activity of E. faecalis is not dependent on epaB or lgt.
(A) Presence of E-cadherin in a fixed monolayer of murine Ptk6 intestinal epithelial cells incubated for 20 hours with E. faecalis OG1RF, ΔepaB or Δlgt strain or an E. faecalis mutant lacking gelatinase E (ΔgelE) as reference. Representative images stained by immunofluorescence for E-cadherin (intracellular domain, green) and E. faecalis (red) (scale bar = 100μm) showing E-cadherin of the intestinal epithelial cell monolayer and (B) according quantification of E-cadherin (normalized to value for E. faecalis ΔgelE) as indicator for gelatinase E presence and/or activity secreted by E. faecalis. (C) Representative images of distal colon sections from wild type and IL-10-/- mice monoassociated with E. faecalis OG1RF, ΔepaB or Δlgt strain stained by immunofluorescence for E-cadherin (extracellular domain, green) and nuclei (blue) to visualize degradation of E-cadherin by gelatinase E in vivo (scale bar = 100μm). Differences were considered significant for *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 7.
Activation of dendritic cells but not reactivation of T cells is dependent on E. faecalis lipoproteins.
(A) TNF secretion by bone marrow-derived dendritic cells (BMDC) from wild type (Wt) mice and TLR2-/- mice after stimulation with lysates of E. faecalis OG1RF, ΔepaB, Δlgt or reconstituted Δlgt strain or LPS as control for 24 hours in vitro. (B) Time-dependent secretion of IL-12p40 by BMDCs isolated from IL-10-/- mice after stimulation with lysates of E. faecalis OG1RF, ΔepaB, Δlgt or reconstituted Δlgt strain or LPS as control for 3, 6, 12 or 24 hours in vitro. (C) IFN-γ and (D) IL-12p40 secretion in an DC-T cell co-culture system, where DCs isolated from bone-marrow of IL-10-/- mice were pulsed with lysates from E. faecalis OG1RF, ΔepaB or Δlgt strains and afterwards co-cultured for 72 hours with CD4+ T cells isolated and pooled from mesenteric lymph nodes (MLN) of IL-10-/- mice monoassociated with E. faecalis OG1RF. (E) IFN-γ secretion by MLN cells isolated from wild type (Wt) and IL-10-/- mice monoassociated with E. faecalis OG1RF, ΔepaB or Δlgt strains that were reactivated with the corresponding lysate for 72 hours. (F) IFN-γ cytokine levels in plasma of wild type (Wt) and IL-10-/- mice monoassociated with E. faecalis OG1RF, ΔepaB or Δlgt strain. Differences were considered significant for *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 8.
Proposed mechanisms of E. faecalis virulence factors responsible for colitogenic activity in the disease susceptible host.
(A) Dynamic contribution of virulence factors to colitogenic activity of E. faecalis: (1) Gelatinase E secreted by E. faecalis triggers degradation of E-cadherin in intestinal epithelial cells (IEC) impairing the intestinal barrier. (2) The enterococcal polysaccharide antigen mediates adhesion of E. faecalis to mucosal surfaces and facilitates resistance against lysozyme secreted by neutrophil granulocytes (NG) infiltrating the lamina propria. (3) Cell surface-associated lipoproteins are essential for colitogenic activity of E. faecalis promoting activation of innate immune cells through TLR2, such as dendritic cells (DC) for example, and infiltration of immune cells.
Table 3.
E. faecalis strains used in this study.
Table 4.
Primers used for generation of E. faecalis deletion or reconstitution mutants.