ABSTRACTBackground & AimClostridioides difficileinfection (CDI) is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis. Two protein toxins, TcdA and TcdB, produced byC. difficileare the major determinants of disease. However, the pathophysiological causes of diarrhea during CDI are not well understood. Here, we investigated the effects ofC. difficiletoxins on paracellular permeability and apical ion transporters in the context of an acute physiological infection.MethodsWe studied intestinal permeability and apical membrane transporters in female C57BL/6J mice. Üssing chambers were used to measure paracellular permeability and ion transporter function across the intestinal tract. Infected intestinal tissues were analyzed by immunofluorescence microscopy and RNA-sequencing to uncover mechanisms of transporter dysregulation.ResultsIntestinal permeability was increased through the size-selective leak pathwayin vivoduring acute CDI in a 2-day-post infection model. Chloride secretory activity was reduced in the cecum and distal colon during infection by decreased CaCC and CFTR function, respectively. SGLT1 activity was significantly reduced in the cecum and colon, accompanied by ablated SGLT1 expression in colonocytes and increased luminal glucose concentrations. SGLT1 and DRA expression was ablated by either TcdA or TcdB during acute infection, but NHE3 was decreased in a TcdB-dependent manner. The localization of key proteins that link filamentous actin to the ion transporters in the apical plasma membrane was unchanged. However,Sglt1, Nhe3, andDrawere drastically reduced at the transcript level, implicating downregulation of ion transporters in the mechanism of diarrhea during CDI.ConclusionsCDI increases intestinal permeability and decreases apical abundance of NHE3, SGLT1, and DRA. This combination likely leads to dysfunctional water and solute absorption in the large bowel, causing osmotic diarrhea. These findings provide insights into the pathophysiological mechanisms underlying diarrhea and may open novel avenues for attenuating CDI-associated diarrhea.