Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2

Abstract

e Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2 ؊/؊ ) mice were used to investigate the role of this receptor in E. faecalis-induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2 ؊/؊ mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2 ؊/؊ mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2 ؊/؊ mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2.A potential mechanism for the pathogenesis of inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS) involves disruption of the intestinal epithelial barrier and exposure of a genetically defective immune system to enteric microbial antigens. Indeed, IBD and IBS patients exhibit a higher level of intestinal permeability than healthy controls (1-7). Additionally, first-degree relatives of IBD patients display elevated levels of enteric permeability (8-10), suggesting that this abnormality is independent of inflammation. Consistent with this hypothesis are animal models of colitis that use chemical disruption of the epithelial barrier with trinitrobenzene sulfonic acid, dextran sodium sulfate, or nonsteroidal anti-inflammatory drugs (NSAIDs) to elicit inflammation (11). Further, disruption of the intestinal epithelial barrier by exposure of susceptible patients to NSAIDs (blockers of prostaglandin synthesis) is a risk factor for intestinal inflammation (12).Enteric proteases can act broadly as catalysts of protein degradation or specifically as selective agents that control physiological processes (13). Additionally, these enzymes can disrupt mucosal barriers and modulate the host immune resp...

Roles for Intestinal Bacteria, Viruses, and Fungi in Pathogenesis of Inflammatory Bowel Diseases and Therapeutic Approaches

“…The co-colonization of E faecalis and AIEC strain dramatically accelerate and potentiate experimental colitis, indicating inflammatory bacterial reciprocal interactions 119 . Mechanisms by which E faecalis induce injury include metalloprotease damage of epithelial barrier integrity through protease-activated receptor 2 120, 121 and activation of innate immune pathways via TLR2 ligation by membrane lipoproteins 122 . One caveat is that E faecalis has not been shown to be consistently increased in IBD patients.…”

“…For example, blocking AIEC epithelial adherence by glycopolymers or FimH antagonists, or inducing blocking antibodies to flagellin might inhibit AIEC epithelial invasion and translocation 116, 153 . Likewise, blocking the protease activity of E faecalis , or protease receptor binding, might inhibit the mucosal permeability mediated by these molecules 120, 121 . Similarly, specifically blocking expression of virulence gene products or their activity could diminish the pathogenic activity of aggressive bacterial populations that expand in the dysbiotic inflammatory environment.…”

TRPV4—A Missing Link Between Mechanosensation and Immunity

“…Conversely, activation of protease-activated receptor (PAR)-2 by thrombin suppresses TRPV4 activity in macrophages and resolves lung injury (74). Similarly, PARs are also activated by neutrophil elastase (NE), matrix metalloproteases (MMPs) or other microbial proteases (33,(75)(76)(77)(78)(79) which has been implicated to degrade ECM and thereby causing remodeling and matrix stiffening during infection (33,63,73,80).…”

Effects of artificially introduced Enterococcus faecalis strains in experimental necrotizing enterocolitis

“…In order to narrow down the list of E. faecalis strains for in vivo studies, we examined strains’ potential pathogenic properties. Of the phenotypes described above, antibiotic resistance [31], hemolysis, and proteolysis [32] may contribute to pathogenicity. Another pathogenic phenotype of relevance to NEC could be the ability of bacteria to trigger mucosal inflammatory response.…”