Clostridioides difficile infection damages colonic stem cells via TcdB, impairing epithelial repair and recovery from disease

Abstract

Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith,PLoS One8, e73204 (2013); S. Kozaret al.,Cell Stem Cell13, 626–633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogenClostridioides difficile,we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.

Research in a time of enteroids and organoids: how the human gut model has transformed the study of enteric bacterial pathogens

“…Mileto et al demonstrated that TcdB induced pathogenic effects, particularly stem cell death, through both FZD7-dependent and -independent mechanisms in 3D colonoids, supporting evidence that receptors in addition to FZD7 are essential for toxin-induced pathology. 42 The interaction of C. difficile with intestinal mucus and subsequent colonization in humans has not been thoroughly investigated. Engevik et al reported that patients with C. difficile infection have disrupted intestinal mucus, and biopsies from infected patients exhibited almost complete loss of the loose outer MUC2 layer as assessed by confocal microscopy, exposing the firmly attached inner MUC1 layer.…”

“…demonstrated that TcdB induced pathogenic effects, particularly stem cell death, through both FZD7-dependent and – independent mechanisms in 3D colonoids, supporting evidence that receptors in addition to FZD7 are essential for toxin-induced pathology. 42 …”

Murine intrarectal instillation of purified recombinant C. difficile toxins enables mechanistic studies of pathogenesis

“…Previously, we showed E-cadherin and β-catenin mislocalization in the spore gavage mouse model occurs after 24 hours (40). Surprisingly, pseudomembrane formation can occur 4 hours post-instillation without dramatically affecting adherens junctions in more basally located cells.…”

“…Histopathological damage occurred near the lumen without crypt base changes. In a CDI mouse model using oral gavage of infectious spores, adherens junction instability was described throughout the crypt axis from base to lumen (40). We used immunofluorescent staining to examine adherens junctions and the apical brush border after intrarectal intoxication.…”

“…The most significant aspect of this recombinant toxin approach is the ability to interrogate easily how changes in toxin structure affect its function. As an example, we have previously shown how a TcdB mutation ( 1595 VNFLQS -> 1596 GFE) abolishing Frizzled binding induces the same histopathological response as wild-type (40). Others have used purified recombinant toxin via systemic injection or on explants to show the L543A mutation renders TcdB unable to undergo cysteine protease cleavage while retaining the capacity to induce histological damage (45,46).…”