Transporter Trafficking Mechanisms in Infectious Diarrhea

传染性腹泻中的转运蛋白贩运机制

• 批准号: 8716742
• 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 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8716742
• 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）：尽管有重大医学进展，但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出色的干预策略至关重要。肠病大肠杆菌（EPEC）是一种食物传播的病原体，是全世界婴儿腹泻的主要原因，导致高发病率和死亡率高。然而，迄今为止，尚不清楚EPEC诱导的腹泻的基础机制。腹泻是通过增加的肠道分泌或吸收减少而发生的，或两者兼而有之。在这方面，电反性NaCl吸收是人回肠和结肠中Na+和Cl-吸收的主要途径。肠腔膜膜NHE3（氢氢交换器3）和DRA（在腺瘤中调节，SLC26A3 Cl-/hco3-（OH-）交换剂]蛋白质在人类肠中的电自动性NaCl吸收中起关键作用。早期发表和初步研究的研究表明，EPEC诱导的腹泻后的早期事件涉及抑制NHE3和DRA活性，这是由于它们通过不同的机制从肠上皮细胞（IEC）表面进行了内在化而导致的。 NHE3抑制需要大肠杆菌分泌的ESPF和宿主IEC PDZ蛋白NHERF2，而DRA的内在化需要EPEC分泌的ESPGS（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NHE3和DRA的细胞内在化抑制了肠NaCl的吸收。这种内部化需要EPEC分泌的蛋白质与宿主IEC脂质抗体，细胞骨架（Ezrin，肌动蛋白）和PDZ结构蛋白（NHERFS）之间的复杂相互作用。因此，我们目前的研究的目的是详细探讨使用EPEC诱导的NHE3和DRA内部化的运输机制，利用肠道上皮细胞利用了最新的体外和体内方法。特定的目标1旨在产生有关脂质筏，细胞骨架和NHERF蛋白在EPEC中的贡献以及ESPF诱导的NHE3活性抑制的关键机理信息。具体目标2将研究体外DRA运输的机制，包括脂质筏，微管和NHERF蛋白在响应EPEC或ESPG的作用。特定的目标3将利用体内小鼠模型来验证EPEC感染对NHE3和DRA的影响以及净影响NHERF敲除小鼠和给药的小鼠，对耦合的NaCl和吸水吸收。我们提出的研究应提供对EPEC诱导的腹泻的病理生理学的新见解，以及哺乳动物肠中NaCl吸收的基础机制，因此具有很高的生理和临床意义。这些研究可能会发现与EPEC-EEC相互作用有关的新型分子靶标，最终导致NaCl吸收受损，并可能有助于未来发展更好的管理策略来治疗感染性腹泻。