Inhibition of Toxin Translocation Can Reverse Cholera and ETEC-Mediated Diarrhea

抑制毒素易位可以逆转霍乱和 ETEC 介导的腹泻

• 批准号: 8604141
• 负责人: KENNETH R TETER
• 金额: $ 18.13万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-15 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8604141
• 关键词: 26S proteasome; ADP Ribose Transferases; ADP ribosylation; Acids; Adenylate Cyclase; Antineoplastic Agents; Apical; Back; Binding; Biological Assay; Cell surface; Cells; Chloride Channels; Chloride Ion; Chlorides; Cholera; Cholera Toxin; Clinical Trials; Cultured Cells; Cyclic AMP; Cytosol; Data; Detection; Diarrhea; Disulfides; Drops; Endoplasmic Reticulum; Endosomes; Enterocytes; Event; FDA approved; Funding Mechanisms; Geldanamycin; Golgi Apparatus; Health; Heating; Integration Host Factors; Intestines; Intoxication; Lead; Life; Link; Longevity; Lysine; Mediating; Membrane; Modeling; Molecular; Molecular Conformation; Monitor; Nature; Organelles; Pharmaceutical Preparations; Process; Proteins; Publishing; Quality Control; Recovery; Reporting; Rest; Route; Signal Transduction; Surface Plasmon Resonance; System; Technology; Testing; Therapeutic; Therapeutic Agents; Toxic effect; Toxin; Travel; Ubiquitin; Ubiquitination; Validation; Vesicle; Vibrio cholerae; Water Movements; Work; base; enterotoxigenic Escherichia coli; extracellular; high reward; high risk; holotoxins; intestinal epithelium; new therapeutic target; novel; prevent; response

DESCRIPTION (provided by applicant): Cholera toxin (CT) and heat-labile toxin (LT) from enterotoxigenic Escherichia coli (ETEC) are highly related AB5-type protein toxins composed of an enzymatic A1 subunit, an A2 linker, and a cell-binding B pentamer. Both toxins generate a diarrheatic response through the ADP-ribosylation of a host protein, Gs¿. CT and LT attack Gs¿ within the cytosol of the host cell, but they are initially secreted into the extracellular medium. The toxins must therefore cross a membrane barrier in order to function. This only occurs after the intact holotoxin moves by vesicle carriers from the cell surface through multiple compartments of the host endomembrane system en route to the endoplasmic reticulum (ER). The A1 subunit dissociates from the rest of the toxin in the ER, unfolds, and passes through a protein-conducting channel to enter the cytosol. The translocated A1 subunit then interacts with host factors to regain a functional conformation for the activation of Gs¿. It is believed that recovery from cholera and ETEC-mediated diarrhea can only occur after intoxicated enterocytes, which have a 3-5 day lifespan, are sloughed from the intestinal epithelium. A number of observations indicate this is not the case. We have recently shown that a threshold concentration of cytosolic CTA1 is required to elicit a cytopathic effect. We have also identified two therapeutic agents that block toxin translocation to the cytosol, and we have documented the ubiquitin-independent proteasomal degradation of cytosolic CTA1. Other reports indicate it is possible to de-activate the ADP-ribosylated form of Gs?. These collective observations strongly suggest that cholera and ETEC-mediated diarrhea are reversible events. If CTA1/LTA1 translocation to the cytosol is blocked after toxin exposure, the pool of toxin already in the cytosol will be degraded and will not be replenished. The cytosolic pool of CTA1/LTA1 will therefore drop below the requisite threshold concentration, allowing the host cell to de-activate Gs? and recover from intoxication. To test this model, we will use a novel toxin detection system based on surface plasmon resonance (SPR) technology to quantify holotoxin and A1 subunit distributions in the cytosol, endomembrane system, and extracellular medium over the lifespan of an intoxicated enterocyte. These parameters have not been determined for any AB toxin. Based on published and preliminary data, we predict the endomembrane system serves as a long-term reservoir for the slow but continual delivery of CTA1/LTA1 to the cytosol. We further predict the low levels of CTA1/LTA1 which reach the cytosol, combined with cellular mechanisms to clear the cytosol of toxin and reverse the effects of intoxication, will permit treatment and recovery from CT/LT intoxication. This will be tested with toxicity assays and SPR-based assays to monitor the delivery and persistence of cytosolic toxin in cells treated with drugs that block toxin translocation. Validation of our model will provide a molecular basis for new post-exposure therapeutic strategies focused on the translocation event. HEALTH RELEVANCE: Vibrio cholerae and enterotoxigenic Escherichia coli produce highly related toxins that enter our intestinal cells to induce a potentially fatal case of diarrhea. The amounts of intracellular toxin needed to initiate and sustain a diarrheatic response are unknown. Here, we will establish these parameters and demonstrate recovery from intoxication is possible with treatments that lower the levels of intracellular toxin.

描述（由申请人提供）：来自产肠毒素大肠杆菌（ETEC）的霍乱毒素（CT）和不耐热毒素（LT）是高度相关的AB5型蛋白质毒素，由酶促A1亚基、A2接头和细胞-两种毒素通过宿主蛋白 Gs¿ 的 ADP 核糖基化产生腹泻反应。 .CT和LT攻击Gs¿在宿主细胞的细胞质内，但它们最初被分泌到细胞外介质中。 因此，毒素必须穿过膜屏障才能发挥作用，这只有在完整的全毒素通过囊泡载体从细胞表面穿过宿主内膜系统的多个区室到达内质网 (ER) 后才会发生。内质网中毒素的其余部分展开，并通过蛋白质传导通道进入细胞质，然后与宿主相互作用。重新获得激活 Gs 的功能构象的因子¿ 。 人们认为，只有在中毒的肠细胞从肠上皮脱落后，霍乱和 ETEC 介导的腹泻才会恢复。我们最近的研究表明，情况并非如此。需要细胞质 CTA1 的阈值浓度才能引起细胞病变效应。我们还鉴定了两种阻止毒素易位至细胞质的治疗剂，并且我们已经记录了这一点。其他报告表明，如果 CTA1/LTA1 易位，则有可能使 ADP 核糖基化形式失活。毒素暴露后，细胞质被堵塞，细胞质中已有的毒素池将被降解，并且不会得到补充。因此，CTA1/LTA1 将降至所需的阈值浓度以下，从而使宿主细胞失活 Gs? 并从中毒中恢复。为了测试该模型，我们将使用基于表面等离子共振 (SPR) 技术的新型毒素检测系统。量化中毒肠细胞生命周期内的全毒素和 A1 亚基分布。这些参数尚未根据任何 AB 毒素确定。根据已发表的初步数据，我们预测内膜系统可作为缓慢但连续地将 CTA1/LTA1 递送至细胞质的长期储存库，并结合细胞机制进一步预测到达细胞质的 CTA1/LTA1 水平较低。清除细胞质中的毒素并逆转中毒的影响，将允许 CT/LT 中毒的治疗和恢复。这将通过毒性测定和基于 SPR 的测定进行测试，以监测毒素的传递和持久性。用阻断毒素易位的药物处理的细胞中的胞质毒素对我们模型的验证将为专注于易位事件的新暴露后治疗策略提供分子基础。 健康相关性：霍乱弧菌和产肠毒素大肠杆菌产生高度相关的毒素，这些毒素进入我们的肠道细胞，诱发潜在致命的腹泻反应。在此，我们将确定这些毒素的量。参数并证明通过降低细胞内毒素水平的治疗可以从中毒中恢复。