Transporter Trafficking Mechanisms in Infectious Diarrhea

传染性腹泻中的转运蛋白贩运机制

• 批准号: 8332247
• 负责人: Pradeep K Dudeja
• 金额: $ 25.73万
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8332247
• 关键词: Actins; Acute; Age; Bacteria; Bacterial Infections; Bicarbonates; Biochemical; Biotinylation; Caco-2 Cells; Caveolins; Cell Communication; Cell surface; Cells; Cessation of life; Child; Clathrin; Colchicine; Colon; Complex; Coupled; Cytoskeletal Proteins; Data; Development; Diarrhea; Disease; Down-Regulation; Endocytosis; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Event; Exocytosis; Functional disorder; Future; Goals; Human; Image; In Vitro; Infantile Diarrhea; Infection; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Intervention; Intestinal Secretions; Intestines; Knockout Mice; Mediating; Medical; Membrane; Membrane Microdomains; Microtubules; Modality; Molecular; Molecular Target; Morbidity - disease rate; Mus; PDZ protein; Pathway interactions; Physiological; Play; Proteins; Publishing; Regulation; Role; Route; SLC26A3 gene; Small Interfering RNA; Sodium Chloride; Surface; System; Techniques; Tertiary Protein Structure; Therapeutic; Therapeutic Intervention; Treatment Protocols; United States National Institutes of Health; Vesicle; Water; Widespread Disease; Work; absorption; apical membrane; base; clinically significant; design; enteric pathogen; enteropathogenic Escherichia coli; ezrin; foodborne; foodborne pathogen; grasp; ileum; in vivo; in vivo Model; innovation; insight; luminal membrane; mortality; mouse model; mutant; novel; programs; response; sodium-hydrogen exchanger 3; sodium-hydrogen exchanger regulatory factor; trafficking; treatment strategy

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.

描述（由申请人提供）：尽管医学取得了重大进展，腹泻病每年仍导致全世界约 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且优越的干预策略至关重要。肠病性大肠杆菌 (EPEC) 是一种食源性病原体，是全世界婴儿腹泻的主要原因，导致高发病率和死亡率。然而，迄今为止，EPEC 引起的腹泻的机制尚不清楚。腹泻是通过肠道分泌增加或吸收减少或两者兼而有之而发生的。在这方面，电中性的 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 NHE3（钠氢交换器 3）和 DRA [腺瘤中下调，SLC26A3 Cl-/HCO3-(OH-) 交换器] 蛋白在人体肠道电中性 NaCl 吸收中发挥关键作用。我们小组早期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，因为它们通过不同的机制从肠上皮细胞 (IEC) 表面内化。 NHE3 抑制需要大肠杆菌分泌蛋白 EspF 和宿主 IEC PDZ 蛋白 NHERF2，而 DRA 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 NaCl 吸收。这种内化需要 EPEC 分泌蛋白与宿主 IEC 脂筏、细胞骨架（埃兹蛋白、肌动蛋白）和 PDZ 结构域蛋白 (NHERF) 之间复杂的相互作用。因此，我们当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 诱导肠上皮细胞中 NHE3 和 DRA 内化的运输机制。具体目标 1 旨在产生有关脂筏、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 体外运输的机制，包括脂筏、微管和 NHERF 蛋白响应 EPEC 或 EspG 的作用。 Specific Aim 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、NHERF 敲除小鼠和施用的小鼠检查 EPEC 感染对 NHE3 和 DRA 的影响以及对 NaCl 和水吸收耦合的净影响秋水仙碱。我们提出的研究应该为 EPEC 引起的腹泻的病理生理学和哺乳动物肠道中 NaCl 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。这些研究可能会识别出参与 EPEC-IEC 相互作用的新分子靶标，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。