Nectin-3 and shed forms of CSPG4 can serve as epithelial cell receptors for Clostridioides difficile TcdB

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.