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，但乳腺癌疾病每年仍导致全球约260万人死亡。因此，更好地了解牙周病的病理生理学对于设计新颖和上级干预策略至关重要。肠致病性E.大肠埃希菌（EPEC）是一种食源性致病菌，是世界范围内婴幼儿腹泻的主要致病菌，发病率和死亡率都很高。然而，迄今为止，EPEC诱导的腹泻的机制还不清楚。腹泻通过增加肠道分泌或减少吸收发生，或两者兼而有之。在这方面，电中性NaCl吸收是人体回肠和结肠中Na+和Cl-吸收的主要途径。肠腔膜NHE 3（钠氢交换器3）和NHE [在腺瘤中下调，SLC 26 A3 Cl-/HCO 3-（OH-）交换器]蛋白在人肠中的电中性NaCl吸收中起关键作用。我们小组早期发表的和初步的研究表明，EPEC诱导的腹泻后的早期事件涉及抑制NHE 3和NHE 3活性，这是由于它们通过不同的机制从肠上皮细胞（IEC）表面内化。NHE 3抑制需要E. coli分泌EspF蛋白和宿主IEC PDZ蛋白NHERF 2，而EPEC的内化需要EPEC分泌EspG（G1/G2）和IEC微管网络的破坏。基于这些数据，我们假设急性EPEC感染通过细胞表面NHE 3和NHE 4的细胞内化抑制肠NaCl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（埃兹蛋白、肌动蛋白）和PDZ结构域蛋白（NHERF）之间的复杂相互作用。因此，我们目前研究的目的是利用最先进的体外和体内方法详细探索EPEC诱导的肠上皮细胞中NHE 3和NHE 3内化的潜在运输机制。特定目标1旨在产生关于脂筏、细胞骨架和NHERF蛋白在EPEC和EspF诱导的NHE 3活性抑制中的作用的关键机制信息。具体目标2将研究潜在的机制，在体外，包括脂筏，微管和NHERF蛋白的作用，响应EPEC或EspGs的运输。具体目标3将利用体内小鼠模型，通过检查EPEC感染对NHE 3和NHE 3的影响以及利用最新技术、NHERF敲除小鼠和给予秋水仙碱的小鼠对偶联NaCl和水吸收的净影响来验证体外数据。我们提出的研究应提供新的见解EPEC诱导的腹泻的病理生理学和机制的基础上调节NaCl的吸收在哺乳动物肠道，因此，具有重要的生理和临床意义。这些研究可能会确定参与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感染性腹泻治疗管理策略。

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.