Transporter Trafficking Mechanisms in Infectious Diarrhea

描述(由申请人提供)：尽管医学取得了重大进展，但全世界每年仍有约260万人死于腹泻疾病。因此，更好地了解腹泻病的病理生理学对于设计新的和更好的干预策略至关重要。肠致病性大肠杆菌(EPEC)是一种食源性致病菌，是全球婴幼儿腹泻的主要病原菌，发病率和死亡率均较高。然而，迄今为止，EPEC引起腹泻的机制还不是很清楚。腹泻要么通过肠道分泌物增加，要么通过吸收减少，或者两者兼而有之。在这一点上，电中性盐吸收是人类回肠和结肠吸收Na+和Cl-的主要途径。肠腔膜蛋白NHE3和DRA[在腺瘤中下调，SLC26A3Cl-/HCO3-(OH-)交换器]在人体肠道对电中和盐的吸收中起着关键作用。我们课题组早先发表的初步研究表明，EPEC诱导的腹泻后的早期事件涉及NHE3和DRA活性的抑制，这是因为它们通过不同的机制从肠上皮细胞(IECS)表面内化。NHE3的抑制需要大肠杆菌分泌ESPF蛋白和宿主IEC PDZ蛋白NHERF2，而DRA的内化需要EPEC分泌EspGs(G1/G2)和破坏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对盐的吸收。这种内化需要EPEC分泌的蛋白质与宿主IEC脂筏、细胞骨架(Ezrin、肌动蛋白)和PDZ结构域蛋白(NHERF)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NHE3和DRA内化在肠上皮细胞中的转运机制。特定目的1旨在提供关于脂筏、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。具体目标2将研究DRA在体外转运的机制，包括脂筏、微管和NHERF蛋白在EPEC或EspGs反应中的作用。特定目标3将利用体内小鼠模型来验证体外数据，方法是利用最先进的技术，检测EPEC感染对NHE3和DRA的影响以及对耦合的氯化钠和水吸收的净影响，NHERF基因敲除小鼠和注射秋水仙素的小鼠。我们提出的研究将对EPEC诱导的腹泻的病理生理学和哺乳动物肠道中盐分吸收的调节机制提供新的见解，因此具有重要的生理和临床意义。这些研究可能会确定EPEC-IEC相互作用中涉及的新分子靶点，最终导致盐吸收受损，并可能有助于未来制定更好的感染性腹泻治疗策略。

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.