Infectious diarrhea caused by food borne pathogens such as enteropathogenic E. coli (EPEC) or nosocomial pathogen Clostridium difficile result in significant morbidity and mortality and increased health care costs in the U.S. EPEC injects virulence factors into the host cells via a type 3-secretion system, whereas C. difficile produces two main toxins TcdA and TcdB as the virulence factors. To date, however, the molecular pathophysiology of infectious diarrhea caused by these two distinct pathogens is mostly unknown. Diarrhea results from decreased intestinal absorption and/or increased secretion of fluid and electrolytes. Intestinal luminal membrane proteins NHE3 (sodium hydrogen exchanger 3, SLC9A3) and DRA (Down Regulated in Adenoma, SLC26A3) play critical roles in electroneutral NaCl and fluid absorption in the human intestine. Indeed, both NHE3 and DRA knockout mice exhibit diarrheal phenotype. Recent studies have shown substantial decrease in DRA expression in diarrhea caused by infectious agents or in inflammation, thereby identifying DRA as a novel therapeutic target for diarrhea. Our preliminary data showed that EPEC infection of Caco-2 cells decreased DRA mRNA and promoter activity, had no effects on 3??-UTR activity, but substantially reduced DRA protein levels. In contrast, a complete loss of DRA protein was observed in response to TcdA and TcdB in Caco-2 cells and in biopsies from CDI patients with no effects on DRA mRNA, promoter and 3??- UTR activities. Thus, our novel data support both transcriptional and posttranslational downregulation of DRA by EPEC and involvement of only posttranslational mechanisms, such as via protein degradation, by C. difficile. Since DRA has emerged as a novel therapeutic target for diarrhea, detailed mechanisms underlying downregulation of DRA expression in infectious diarrhea caused by these two major but distinct pathogens warrant in-depth investigations. Therefore, we hypothesized that EPEC/C. difficile infection-induced inhibition of intestinal chloride absorption is secondary to downregulation of DRA expression involving distinct transcriptional and/or post-translational mechanisms orchestrated by specific pathogen/host cellular factors. The hypothesis will be tested utilizing in vitro models of human and mouse IECs, colonic organoid-derived monolayers, and in vivo models of infection. Studies in Aim 1 will determine the molecular mechanisms involved in EPEC/C. rodentium/C. difficile toxin-induced downregulation of DRA expression and function. Studies in Aim 2 will validate our in vitro mechanistic studies on modulation of DRA expression in mouse models of C. rodentium/C. difficile-induced diarrhea. The critical role of DRA in infectious diarrhea will be further evaluated in a novel transgenic mouse model generated by us with inducible intestine specific overexpression of DRA. Our proposed studies will not only highlight novel mechanisms underlying downregulation of chloride transporter DRA expression by two distinct diarrheal pathogens but will also substantiate the importance of DRA as a novel therapeutic target for diarrheal diseases.
Impaired intestinal absorption of electrolytes (NaCl) is a hallmark of diarrhea caused by pathogen infection or inflammation. Human intestinal luminal membrane protein DRA (Down Regulated in Adenoma, SLC26A3), a key mediator of chloride absorption in intestinal NaCl absorption, is emerging as a novel therapeutic target for diarrhea as its expression has been shown to be extensively reduced in infectious or inflammatory diarrhea. The studies proposed in the current application are designed to unravel detailed molecular mechanisms underlying inhibition of DRA expression and function in diarrhea caused by infection with food borne enteropathogenic E. coli or hospital acquired Clostridium difficile, two major but distinct diarrheal pathogens. The outcome of the proposed studies will highlight novel mechanisms of infectious diarrhea to help design better treatment modalities and also to establish the role of DRA as a novel therapeutic target for diarrhea.
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