Regulation of Enteric Virus Secretory Diarrhea

肠道病毒分泌性腹泻的调节

• 批准号: 9316119
• 负责人: Joseph M. Hyser
• 金额: $ 7.93万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-15 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9316119
• 关键词: 5 year old; Address; Antidiarrheals; Astroviridae; Astrovirus; Attenuated; Automobile Driving; Biological Assay; Biological Models; Biosensor; Calcium; Cause of Death; Cell Line; Cell Surface Receptors; Cells; Cessation of life; Child; Chlorides; Cholera Toxin; Cognitive; Critical Pathways; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic Nucleotides; Cystic Fibrosis Transmembrane Conductance Regulator; Dehydration; Developing Countries; Development; Diarrhea; Disease; Drug Targeting; Drug usage; Endoplasmic Reticulum; Enteral; Enterocytes; Enterotoxins; Family; Fluids and Secretions; Fluorescence; Foundations; Future; Gene Expression; Genes; Goals; Growth; Homeostasis; Human; In Vitro; Inactivated Vaccines; Individual; Infant; Infection; Intestines; Knowledge; Life; Liquid substance; Measures; Mediating; Minor; Morbidity - disease rate; Mus; Neurotransmitters; Nonstructural Protein; Norovirus; Oral Rehydration Therapy; Pathway interactions; Phospholipase C; Phospholipases A; Play; Production; Regulation; Role; Rotavirus; Rotavirus Infections; Rotavirus Vaccines; Signal Pathway; Signal Transduction; Swelling; System; Testing; Vaccines; Viral; Viral Gastroenteritis; Virulence Factors; Virus; channel blockers; design; drug development; enteric pathogen; extracellular; genetic regulatory protein; global health; in vivo; inhibitor/antagonist; insight; live cell imaging; mortality; mouse model; novel therapeutics; pathogen; research study; response; sensor; small molecule

Diarrheal is a leading cause of death and illness throughout the world and causes 560 million deaths of children under 5 years old. Rotavirus (RV) causes about 70% of viral diarrheas and even with two vaccines kills 215,000 children yearly. RV diarrhea is mainly due to excess Cl- secretion that drives fluid loss, but the cellular pathways responsible for RV-induced fluid secretion are not well defined, which limits efforts to develop and testing of potential anti-diarrheal drugs that can be used to treat children when protection by vaccines is insufficient. Activation of chloride (Cl-) secretory channels is regulated by intracellular calcium (Ca2+) and cyclic nucleotide (cAMP) signals These signals in turn activate Ca2+-activated Cl- channels (CaCC), such as anoctamin (Ano) family, and cAMP-activated Cl- channels (cAMP-CC), such as the cystic fibrosis transmembrane regulator (CFTR). RV diarrhea is the classic example of Cl- secretion due to elevated Ca2+ because a hallmark of RV infection is increased Ca2+ signaling, which is induced by the RV nonstructural protein 4 (NSP4). Ano channels are the prominent CaCCs in the gut; however, the Cl- channels responsible for RV diarrhea have yet to be identified. Additionally, NSP4 is considered the primary initiator of RV-induced Ca2+ signals; however, the mechanisms by which NSP4-derived signals cause Cl- channel activation and fluid secretion have not been adequately explored. In Aim 1, we will identify the Cl- channels responsible for RV diarrhea using both in vitro and in vivo approaches. Little is known about the expression of different Cl- channels throughout the human intestine, particularly children, we will use human intestinal enteroids (HIEs) to determine gene expression of the key Cl- channels and their regulatory proteins. Next, we will measure the ability of RV to activate individual Cl- channels using a fluorescence quench assay and determine whether those channels contribute to fluid secretion using the enteroid swelling assay (ESA) in the presence of specific Cl- channel blockers. Lastly, we will determine whether inhibitors of Ano1 or CFTR will attenuate diarrhea and if so whether intestinal specific deletion of those channels also reduces diarrhea. In Aim 2, we will determine whether RV NSP4 activates both Ca2+ and cAMP through activation of the sensor stromal interacting molecule 1 (STIM1). STIM1 activation can generate Ca2+ signals through the store-operated Ca2+ entry (SOCE) pathway, as well as cAMP signals through the store-operated cAMP signaling (SOcAMPs) pathway. We will use live cell imaging of intestinal cell lines and HIEs expressing fluorescent Ca2+ and cAMP biosensors to determine whether RV and NSP4 activate both SOCE and SOcAMPs and use the ESA to determine whether the Ca2+/cAMP signals generated by NSP4 cause fluid secretion. Lastly, we will determine whether blockers of SOCE or SOcAMPs will attenuate RV diarrhea in vivo. Our results will generate critical insights into the mechanisms of viral diarrhea and new biosensor HIE model systems that can be applied to future studies enteric virus, such as noroviruses and astroviruses.

腹泻是全世界死亡和疾病的主要原因，导致 5.6 亿人死亡 5 岁以下儿童。约 70% 的病毒性腹泻由轮状病毒 (RV) 引起，即使使用两种疫苗 每年杀死 215,000 名儿童。 RV 腹泻主要是由于 Cl- 分泌过多导致体液流失，但 负责 RV 诱导液体分泌的细胞途径尚未明确定义，这限制了开发的努力 并测试可用于在疫苗保护作用下治疗儿童的潜在抗腹泻药物 不足的。氯 (Cl-) 分泌通道的激活受细胞内钙 (Ca2+) 和循环调节 核苷酸 (cAMP) 信号 这些信号依次激活 Ca2+ 激活的 Cl- 通道 (CaCC)，例如 anoctamin (Ano) 家族和 cAMP 激活的 Cl-通道 (cAMP-CC)，例如囊性纤维化 跨膜调节因子（CFTR）。 RV 腹泻是由于 Ca2+ 升高导致 Cl- 分泌的典型例子 因为 RV 感染的一个标志是 Ca2+ 信号传导增加，这是由 RV 非结构性诱导的 蛋白质 4 (NSP4)。 Ano 通道是肠道中最重要的 CaCC；然而，Cl-通道负责 RV 腹泻尚未确定。此外，NSP4 被认为是 RV 诱导的 Ca2+ 的主要引发剂 信号；然而，NSP4 衍生信号引起 Cl 通道激活和液体的机制 分泌尚未得到充分研究。在目标 1 中，我们将确定负责 RV 的 Cl- 通道 使用体外和体内方法进行腹泻。对于不同 Cl- 的表达知之甚少 整个人类肠道的通道，特别是儿童，我们将使用人类肠道肠类（HIE）来 确定关键 Cl- 通道及其调节蛋白的基因表达。接下来，我们将测量 RV 使用荧光淬灭测定激活单个 Cl-通道的能力，并确定是否 在特定的存在下，使用肠样肿胀测定 (ESA)，这些通道有助于液体分泌 Cl-通道阻滞剂。最后，我们将确定 Ano1 或 CFTR 抑制剂是否会减轻腹泻，以及是否会减轻腹泻。 那么肠道特异性删除这些通道是否也会减少腹泻。在目标 2 中，我们将确定 RV NSP4 是否通过激活传感器基质相互作用分子来激活 Ca2+ 和 cAMP 1（刺激1）。 STIM1激活可以通过存储操作的Ca2+入口（SOCE）产生Ca2+信号 途径，以及通过库操作的 cAMP 信号传导 (SOcAMPs) 途径的 cAMP 信号。我们将 使用表达荧光 Ca2+ 和 cAMP 生物传感器的肠道细胞系和 HIE 的活细胞成像 确定 RV 和 NSP4 是否同时激活 SOCE 和 SOcAMP，并使用 ESA 确定是否 NSP4 产生的 Ca2+/cAMP 信号导致液体分泌。最后，我们将确定是否有阻止者 SOCE 或 SOcAMP 将减轻体内 RV 腹泻。我们的结果将产生对以下问题的重要见解： 病毒性腹泻的机制和可应用于未来研究的新型生物传感器 HIE 模型系统 肠道病毒，例如诺如病毒和星状病毒。