Chloride Transporter Downregulation in Infectious Diarrhea

食源性致病性大肠杆菌(EPEC)或医院内致病菌引起的感染性腹泻 病原体艰难梭菌导致严重的发病率和死亡率，并增加了卫生保健费用 美国EPEC通过3型分泌系统将毒力因子注入宿主细胞，而艰难梭菌 产生两种主要毒素TcdA和TcdB作为毒力因子。然而，到目前为止，分子 由这两种不同的病原体引起的感染性腹泻的病理生理学大多不清楚。 腹泻是由于肠道吸收减少和/或液体和电解质分泌增加所致。 肠腔膜蛋白NHE3(钠氢交换蛋白3，SLC9A3)和DRA(向下 在腺瘤中调节，SLC26A3)在人体的电子中性盐和液体吸收中起关键作用 肠子。事实上，NHE3和DRA基因敲除小鼠都表现出腹泻表型。最近的研究表明 在感染性或炎症引起的腹泻中，DRA的表达显著减少，从而 确定DRA为治疗腹泻的新靶点。我们的初步数据显示，EPEC感染 Caco-2细胞DRA mRNA和启动子活性降低，对3ʹ‘-UtR活性无影响，但显著 降低DRA蛋白水平。相反，观察到对TcdA的反应完全丧失了DRA蛋白 对DRA mRNA、启动子和3-ʹ‘-1无影响。 UTR活动。因此，我们的新数据支持DRA的转录和翻译后下调 只参与翻译后机制，如通过蛋白质降解，由C. 艰难抉择。由于DRA已成为治疗腹泻的新靶点，详细的机制可能 这两种主要但不同的病原体引起的感染性腹泻中DRA表达的下调 有必要进行深入调查。因此，我们假设EPEC/艰难梭菌感染诱导 抑制肠道氯吸收是DRA表达下调的次要因素 涉及不同的转录和/或翻译后机制，由特定的 病原体/宿主细胞因素。这一假说将利用人类和小鼠的体外模型进行验证。 肠内皮细胞、结肠器官衍生的单层和体内感染模型。目标1中的研究将确定 EPEC/罗齿梭菌/艰难梭菌毒素诱导DRA下调的分子机制 表情和功能。AIM 2的研究将验证我们对DRA调节的体外机制研究 罗齿梭菌/艰难梭菌致小鼠腹泻模型的表达。DRA在传染性疾病中的关键作用 腹泻将在我们建立的具有可诱导肠道的新型转基因小鼠模型中进一步评估 DRA的特异性过表达。我们提议的研究不仅将突出新的机制 两种不同的腹泻病原体下调氯转运蛋白DRA的表达，但也将 证实DRA作为治疗腹泻疾病的新靶点的重要性。

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 3ʹ′-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 3ʹ′- 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.