Chloride Transporter Downregulation in Infectious Diarrhea

感染性腹泻中氯转运蛋白下调

• 批准号: 9177347
• 负责人: Pradeep K Dudeja
• 金额: $ 38.99万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9177347
• 关键词: 3' Untranslated Regions; Affect; Apical; Bicarbonates; Biopsy; Caco-2 Cells; Cell surface; Cells; Chloride Ion; Chlorides; Citrobacter rodentium; Clostridium difficile; Data; Degradation Pathway; Diarrhea; Disease; Down-Regulation; Electrolytes; Epithelial Cells; Escherichia coli Infections; Exhibits; Fluids and Secretions; Functional disorder; Funding; Genetic Transcription; Health Care Costs; Hospitals; Human; In Vitro; Infant; Infection; Infectious Agent; Inflammation; Inflammatory; Intestinal Absorption; Intestines; Investigation; Knockout Mice; Liquid substance; Long-Term Effects; Mediating; Mediator of activation protein; Membrane Proteins; Messenger RNA; Modality; Modeling; Molecular; Morbidity - disease rate; Mus; Organoids; Outcome; Patients; Phenotype; Play; Proteins; Proton Pump; Response Elements; Role; SLC26A3 gene; Secondary to; System; Testing; Therapeutic; Toxin; Transgenic Mice; Transgenic Organisms; Untranslated Regions; Virulence Factors; absorption; design; enteric pathogen; enteropathogenic Escherichia coli; foodborne; foodborne pathogen; in vitro Model; in vivo; in vivo Model; inhibitor/antagonist; luminal membrane; monolayer; mortality; mouse model; mutant; new therapeutic target; novel; overexpression; pathogen; promoter; protein degradation; response; sodium-hydrogen exchanger 3; transcription factor

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.

由食源性病原体引起的感染性腹泻，如肠致病性大肠杆菌。大肠杆菌（EPEC）或医院感染 病原体艰难梭菌导致显著的发病率和死亡率，并增加了健康护理成本， 美国EPEC通过3型分泌系统将毒力因子注入宿主细胞，而C.艰难 产生两种主要毒素TcdA和TcdB作为毒力因子。然而，到目前为止， 由这两种不同病原体引起的感染性腹泻的病理生理学大多是未知的。 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NHE 3（钠氢交换器3，SLC 9A 3）和NHE 3（Down SLC 26 A3在腺瘤中被调节，在人体电中性NaCl和液体吸收中起关键作用 肠子事实上，NHE 3和NHE 3基因敲除小鼠都表现出大肠杆菌表型。最近的研究表明 在由感染原引起的腹泻或炎症中， 确定了作为腹泻治疗新靶点的β-内酰胺酶。我们的初步数据显示， Caco-2细胞降低了p53 mRNA和启动子活性，对3 ′-UTR活性无影响，但显著降低了p53 mRNA和启动子活性。 降低了蛋白质水平。与此相反，TcdA处理后， 和TcdB在Caco-2细胞和CDI患者活检组织中的表达，对CDI mRNA、启动子和3 ′- UTR活性。因此，我们的新数据支持转录和翻译后的下调， 通过EPEC和只涉及翻译后机制，如通过蛋白质降解，通过C. 很难由于腹泻已成为一种新的治疗目标，详细的机制， 在由这两种主要但不同的病原体引起的感染性腹泻中， 值得深入调查因此，我们假设EPEC/C.艰难梭菌感染 抑制肠氯吸收是继发于下调RxB表达 涉及由特异性转录调控的不同转录和/或翻译后机制， 病原体/宿主细胞因子。将利用人和小鼠的体外模型来检验该假设。 IEC、结肠类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/C的分子机制。啮齿类C.艰难梭菌毒素诱导的β-内酰胺酶下调 表达和功能。目的2中的研究将验证我们在体外的机制研究， 在C.啮齿类C.艰难梭菌引起的腹泻在传染性疾病中的关键作用 腹泻将在一种新的转基因小鼠模型中进一步评估， 特异性过表达我们提出的研究不仅将突出潜在的新机制， 两种不同的病原体下调氯转运蛋白的表达，但也将 证实了作为一种新的治疗靶点的重要性，为乳腺疾病。