The Ion Transport Basis of Clostridium difficile Associated Diarrhea

Clostridium difficile infection is the primary cause of nosocomial diarrhea in the United States. While C. difficile toxins A and B are the primary mediators of CDI, the overall pathophysiology underlying C. difficile associated diarrhea remains poorly understood. In inflammatory and infectious diarrhea, symptoms are caused by increased secretion and/or decreased absorption of water and solutes in the intestine. The functional coupling of intestinal luminal membrane transporters NHE3 (Na+/H+ exchanger) and DRA (Cl-/HCO3- exchanger) is the predominant route of colonic electroneutral NaCl absorption. Studies have shown that a decrease in both NHE3 and DRA are implicated in infectious and inflammatory diarrhea. Furthermore, studies have shown that NHE3 is depleted at the apical surface of intestinal epithelial cells and down-regulated in patients with CDI. Our data demonstrated that DRA protein levels were significantly reduced in the presence of TcdA and TcdB in intestinal epithelial cells. This effect was also specific to DRA, as NHE3 and PAT-1 (Putative Anion Transporter 1) protein levels were unaffected by TcdA and TcdB. The toxin-mediated downregulation of DRA likely occurred at the post-transcriptional level as DRA mRNA levels were unchanged in the presence of either toxin. Additionally, co-treatment of toxin-treated Caco2 monolayers with bafilomycin A1, a known inhibitor of lysosomal degradation, abrogated the effects of C. difficile toxins on DRA protein. Thus, C. difficile toxins may decrease total DRA protein levels via increased degradation in lysosomes. In a toxigenic mouse model of CDI, purified TcdA and TcdA + TcdB, but not TcdB alone, resulted in a decrease in colonic DRA and NHE3 protein levels. Colonic inflammatory markers (MPO release and inflammatory cytokine mRNA levels) were only increased in mice administered purified TcdA alone. Finally, patients with recurrent CDI also exhibited significantly reduced expression of colonic DRA protein. Together these findings indicate that C. difficile toxins markedly downregulate intestinal expression of DRA which may contribute to the diarrheal phenotype of CDI.