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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 One 8, e73204 (2013); S. Kozar et al., Cell Stem Cell 13, 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 pathogen Clostridioides 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.

SUBMITTER: Mileto SJ

PROVIDER: S-EPMC7149309 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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

Mileto Steven J SJ Jardé Thierry T Childress Kevin O KO Jensen Jaime L JL Rogers Ashleigh P AP Kerr Genevieve G Hutton Melanie L ML Sheedlo Michael J MJ Bloch Sarah C SC Shupe John A JA Horvay Katja K Flores Tracey T Engel Rebekah R Wilkins Simon S McMurrick Paul J PJ Lacy D Borden DB Abud Helen E HE Lyras Dena D

Proceedings of the National Academy of Sciences of the United States of America 20200320 14

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 One 8, e73204 (2013); S. Kozar et al., Cell Stem Cell 13, 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 ...[more]

PMID: 32198200

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Project description:Clostridioides difficile is a Gram-positive bacterium that can cause mild to severe diarrhea, inflammation, and colonic tissue damage in animal hosts. Symptoms of the disease can be attributed to the activity of toxin B (TcdB) secreted by C. difficile during infection. TcdB can engage multiple host cell surface receptors in vitro; however, little is known about where these receptors localize on colonic tissue and how these interactions promote disease. Here, we used immunofluorescence microscopy to first visualize TcdB interactions with two of the reported receptors, CSPG4 and Nectin-3, on cells in vitro. In cells expressing both receptors, we found that TcdB preferentially interacts with CSPG4. In moving to host colonic tissue, we observed novel localization of Nectin-3 within the brush border of epithelial cells and CSPG4 localization at epithelial cell junctions. The unexpected junctional CSPG4 signal led us to the hypothesis that the signal could represent a soluble form of the CSPG4 extracellular domain (ECD) being shed from fibroblasts in the underlying stromal layer of the tissue. To test, we set up a co-culture of epithelial cells and fibroblasts separated by transwell inserts. We observed CSPG4-ECD shed into the media of cultured fibroblasts and an accumulation in epithelial cells following co-culture. We also found that soluble CSPG4-ECD present in the conditioned media from fibroblasts can potentiate TcdB-mediated cytopathic responses in epithelial cells lacking CSPG4 expression. Based on these observations, we propose that Nectin-3 can facilitate the binding of TcdB at the epithelial surface and that a soluble form of CSPG4 derived from stromal cells can contribute to TcdB intoxication of epithelial cells in vivo. IMPORTANCE Toxin B (TcdB) is a major virulence factor of Clostridioides difficile, a Gram-positive pathogen that is a leading cause of hospital-acquired diarrhea. While previous studies have established that TcdB can engage multiple cell surface receptors in vitro, little is known about how these interactions promote disease and where these receptors localize on colonic tissue. Here, we used immunofluorescence microscopy to visualize Nectin-3 and CSPG4 on tissue, revealing unexpected localization of both receptors on colonic epithelial cells. We show that Nectin-3, which was previously characterized as an adherens junction protein, is also localized to the brush border of colonocytes. Staining for CSPG4 revealed that it is present along epithelial cell junctions, suggesting that it is shed by fibroblasts along the crypt-surface axis. Collectively, our study provides new insights into how TcdB can gain access to the receptors Nectin-3 and CSPG4 to intoxicate colonic epithelial cells.

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

Publications

<i>Clostridioides difficile</i> infection damages colonic stem cells via TcdB, impairing epithelial repair and recovery from disease.

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