Transporter Trafficking Mechanisms in Infectious Diarrhea

描述（由申请人提供）：尽管有重大的医学进步，腹泻疾病每年仍在全球造成约260万人死亡。因此，更好地了解腹泻疾病的病理生理学对于设计新颖和优越的干预策略至关重要。肠致病性大肠杆菌（EPEC）是一种食源性病原体，是世界范围内引起婴儿腹泻的主要原因，造成高发病率和死亡率。然而，迄今为止，epec诱发腹泻的机制尚不清楚。腹泻的发生可能是由于肠道分泌增加或吸收减少，或两者兼而有之。在这方面，电中性NaCl吸收是人类回肠和结肠吸收Na+和Cl-的主要途径。肠腔膜NHE3（钠氢交换剂3）和DRA[在腺瘤中下调，SLC26A3 Cl-/HCO3-(OH-)交换剂]蛋白在人肠内的电中性NaCl吸收中起关键作用。本小组早期发表的研究和初步研究表明，epec诱导腹泻的早期事件涉及NHE3和DRA活性的抑制，这是由于它们通过不同的机制从肠上皮细胞表面内化。NHE3抑制需要大肠杆菌分泌EspF蛋白和宿主IEC pdz蛋白NHERF2，而DRA内化需要EPEC分泌espg （G1/G2）并破坏IEC微管网络。基于这些数据，我们假设急性EPEC感染通过细胞表面NHE3和DRA的细胞内化抑制肠道NaCl吸收。这种内化需要epec分泌蛋白与宿主IEC脂筏、细胞骨架（ezrin、actin）和PDZ结构域蛋白（NHERFs）之间复杂的相互作用。因此，我们目前的研究目的是利用最先进的体外和体内方法，详细探讨epec诱导的NHE3和DRA内化在肠上皮细胞中的运输机制。特异性Aim 1被设计用于提供关于脂筏、细胞骨架和NHERF蛋白在EPEC和EspF诱导的NHE3活性抑制中的作用的关键机制信息。特异性目标2将研究DRA在体外运输的机制，包括脂筏、微管和NHERF蛋白在EPEC或espg反应中的作用。Specific Aim 3将利用体内小鼠模型验证体外数据，利用最先进的技术，研究EPEC感染对NHE3和DRA的影响，以及对耦合NaCl和水吸收的净影响，NHERF敲除小鼠和秋水仙碱小鼠。我们提出的研究将为epec诱导腹泻的病理生理学和哺乳动物肠道NaCl吸收调节机制提供新的见解，因此具有重要的生理和临床意义。这些研究可能会发现EPEC-IEC相互作用的新分子靶点，最终导致NaCl吸收受损，并可能有助于未来开发更好的治疗感染性腹泻的管理策略。

DESCRIPTION (provided by applicant): Despite major medical advances, diarrheal diseases still cause ~2.6 million deaths per year worldwide. Therefore, a better understanding of the pathophysiology of diarrheal diseases is critical for designing novel and superior strategies for interventions. Enteropathogenic E. coli (EPEC), a food-borne pathogen, is a major cause of infantile diarrhea worldwide causing high rate of morbidity and mortality. To date, however, the mechanisms underlying EPEC-induced diarrhea are not well understood. Diarrhea occurs either via increased intestinal secretion or decreased absorption, or both. In this regard, electroneutral NaCl absorption is the predominant route for Na+ and Cl- absorption in the human ileum and colon. Intestinal luminal membrane NHE3 (sodium hydrogen exchanger 3) and DRA [Down Regulated in Adenoma, SLC26A3 Cl-/HCO3-(OH-) exchanger] proteins play critical roles in electroneutral NaCl absorption in the human intestine. Earlier published and preliminary studies from our group demonstrated that the early events following EPEC-induced diarrhea involved inhibition of NHE3 and DRA activity due to their internalization from surface of intestinal epithelial cells (IECs) via distinct mechanisms. NHE3 inhibition required E. coli secreted protein EspF and host IEC PDZ-protein NHERF2, whereas internalization of DRA required EPEC secreted EspGs (G1/G2) and disruption of IEC microtubular network. Based on these data, we hypothesize that acute EPEC infection inhibits intestinal NaCl absorption via cellular internalization of cell surface NHE3 and DRA. This internalization requires complex interactions between EPEC-secreted proteins and host IEC lipid-rafts, cytoskeletal (ezrin, actin) and PDZ domain proteins (NHERFs). Therefore, the objective of our current studies is to explore in detail the trafficking mechanisms underlying EPEC-induced internalization of NHE3 and DRA in intestinal epithelial cells utilizing state-of-the art in vitro and in vivo approaches. Specific Aim 1 has been designed to yield critical mechanistic information on contribution of lipid rafts, cytoskeletal and NHERF proteins in EPEC and EspF- induced inhibition of NHE3 activity. Specific Aim 2 will investigate mechanisms underlying DRA trafficking in vitro, including the role of lipid rafts, microtubules and NHERF proteins in response to EPEC or EspGs. Specific Aim 3 will utilize in vivo mouse models to validate in vitro data by examining the effects of EPEC infection on NHE3 and DRA and net impact on coupled NaCl and water absorption utilizing state-of-the-art techniques, NHERF knockout mice and mice administered colchicine. Our proposed studies should provide new insights into the pathophysiology of EPEC-induced diarrhea and mechanisms underlying regulation of NaCl absorption in the mammalian intestine and are, therefore, of great physiological and clinical significance. It is likely that these studies may identify novel molecular targets involved in EPEC-IEC interactions culminating in impaired NaCl absorption and may aid in the future development of better management strategies for the treatment of infectious diarrhea. PUBLIC HEALTH RELEVANCE: Despite major advances in medical advancements, diarrheal diseases cause ~2.6 million deaths per year worldwide, out of which about 1.5 million deaths are seen in children below the age of 5. Therefore, a better understanding of the basis of diarrheal diseases is critical for designing new and superior strategies for the treatment. Diarrhea occurs via increased intestinal secretion, decreased absorption, or both. In this regard, enteropathogenic E. coli (EPEC), an important food borne bacteria is known to cause widespread disease and death in children worldwide. In spite of knowing about this disease for many decades, the mechanisms involved in diarrhea in response to infection by these bacteria had not been well understood. Recent studies from our group suggested that one of the important mechanisms for early events of EPEC mediated diarrhea involved a decrease in salt (NaCl) and water absorption. The proposed studies in this application are designed to understand the detailed mechanisms of this reduction in salt and water by studying the molecular and cellular events underlying this reduction in salt and water absorption in response to bacterial infection. It is expected that these detailed molecular and cellular studies will identify new targets for the development of better treatment protocols for diarrheal diseases in future.