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: 8488964
• 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 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8488964
• 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（由适用提供）：来自肠毒素大肠杆菌（ETEC）的霍乱毒素（CT）和热剂量毒素（LT）是高度相关的AB5型蛋白毒素，由酶A1亚基组成，由A2链接和细胞结合B五角形。两种毒素都通过宿主蛋白GS¿的ADP-核糖基化产生腹泻反应。 CT和LT攻击在宿主细胞的细胞质内，但最初分泌到细胞外培养基中。 因此，毒素必须越过膜屏障才能发挥作用。这仅在完整的全毒素通过囊泡载体从细胞表面通过宿主内膜系统的多个隔室移动后才发生。 A1亚基与ER中的其余毒素分离，展开，并通过蛋白质传导通道进入胞质溶胶。然后，易位的A1亚基与宿主因子相互作用，使其保持了激活的功能构象。 据信，只有在进入肠道3-5天的进入囊细胞后才能从肠道上皮中恢复霍乱和ETEC介导的腹泻。许多观察结果表明事实并非如此。我们最近表明，胞质CTA1的阈值浓度需要引起细胞病变效应。我们还鉴定了两种治疗剂，它们将毒素转移到细胞质上，并记录了细胞质CTA1的泛素蛋白酶蛋白酶降解。其他报告表明，可以将GS的ADP-核糖基化形式取消激活。这些集体观察强烈表明霍乱和ETEC介导的腹泻是可逆的事件。如果CTA1/LTA1在毒素暴露后将其易位到细胞质，则毒素已经降解并且不会复制。因此，CTA1/LTA1的胞质池将降至必要的阈值浓度以下，从而使宿主细胞能够解散GS？并从中毒中恢复过来。为了测试该模型，我们将使用基于表面等离子体共振（SPR）技术的新型毒素检测系统来定量细胞质，恩膜膜系统和陶氏肠肠细胞寿命的细胞外培养基中的Holotoxin和A1亚基分布。这些参数尚未确定任何AB毒素。基于已发布和初步数据，我们预测，内膜系统是长期储层，可缓慢但连续地递送CTA1/LTA1向细胞质。我们进一步预测到达细胞质的CTA1/LTA1的低水平，结合细胞机制清除毒素的细胞质并逆转中毒的作用，将允许从CT/LT进行毒化的治疗。这将通过毒性测定和基于SPR的测定法对此进行测试，以监测用阻断毒素易位的药物治疗的细胞中胞质毒素的递送和持续性。对我们模型的验证将为易位事件的新暴露后热策略提供分子基础。 健康相关性：弧菌霍乱和肠毒素大肠杆菌产生高度相关的毒素，进入我们的肠细胞，诱导潜在的致命性腹泻病例。启动和维持腹泻反应所需的细胞内毒素量尚不清楚。在这里，我们将建立这些参数，并通过降低细胞内毒素水平的治疗方法证明可以从中毒中恢复。