Clostridioides difficile Reduces Chemosensory Tuft Cells in the Colonic Epithelium

Background: Clostridioides difficile is a significant human pathogen responsible for severe diarrhea and life-threatening colitis. Each year, C. difficile infection (CDI) affects thousands of patients in the United States, incurring an estimated annual cost exceeding $1 billion. C. difficile colonizes the colonic mucus layer, producing toxins that cause inflammation, diarrhea, epithelial damage, and pseudomembranous colitis. CDI has been shown to alter the composition of gut epithelial cells. The normal colonic epithelium comprises primarily absorptive enterocytes, mucus-producing goblet cells, and proliferative stem cells, alongside hormone-producing enteroendocrine cells and chemosensory tuft cells, which are present in lower abundance. While C. difficile is known to reduce goblet cell numbers, its impact on other epithelial cell types remains poorly understood. Tuft cells, a chemosensory cell type involved in sensing the luminal environment and relaying information to the immune system, have gained increasing attention in recent years. Tuft cells play a critical role in infectious diseases, but their response to many pathogens, including C. difficile remains largely unknown. Hypothesis: We hypothesized that C. difficile infection decreases tuft cell numbers in the colon. Methods & Results: Analysis of human single cell RNA sequencing data revealed that tuft cells harbor toxin A, toxin B and binary toxin receptors, suggesting that these cells could respond to C. difficile toxins. To model CDI, adult C57BL/6 mice were treated with an antibiotic cocktail in their drinking water for four days, followed by a single intraperitoneal injection of clindamycin. Mice were then orally gavaged with C. difficile strain R20291 or PBS vehicle control and monitored over time. RNA sequencing analysis of colonic tissue from mice sampled from day 0 to day 10 post-infection demonstrated a significant decrease in the mouse tuft cell marker Dclk1 expression on days 3, 4, and 5, with resolution by days 8 and 10. Additionally, we observed reduced levels of IL-25, a cytokine secreted by tuft cells, as well as other tuft cell markers ( Pou2F3, Sucnr1, Ckb, Aldh1a1, Trpm5, and Bmx ). Immunostaining at the peak of infection, day 3 post-gavage, we observed decreased numbers of tuft using tuft cell markers including: DCLK1, cytokeratin 18, Pou2f3 and P-EGFR in the colons of infected mice compared to PBS vehicle controls. To confirm that C. difficile alone could elicit these effects, murine colonic organoids were treated with live C. difficile. RNA sequencing analysis of these organoids revealed decreased Dclk1 expression following treatment. Conclusions: These findings demonstrate that C. difficile infection significantly influences colonic tuft cells, reducing their abundance and associated markers. This work provides new insights into the interaction between C. difficile and the gut epithelium, particularly chemosensory tuft cells. MUSC start up funds. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.