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（由申请方提供）：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AB 5型蛋白毒素，由酶A1亚基、A2接头和细胞结合B五聚体组成。这两种毒素通过宿主蛋白质Gs的ADP核糖基化产生免疫反应。CT和LT攻击宿主细胞胞质内的Gs，但它们最初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。这仅发生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到内质网（ER）之后。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。易位的A1亚基然后与宿主因子相互作用以恢复用于激活Gs的功能构象。 据信，霍乱和ETEC介导的腹泻的恢复只能在具有3-5天寿命的中毒肠上皮细胞从肠上皮脱落后发生。一些观察结果表明，情况并非如此。我们最近表明，一个阈值浓度的胞质CTA 1是需要引起细胞病变的影响。我们还确定了两种治疗剂，阻止毒素易位到胞质溶胶，我们已经记录了泛素不依赖性蛋白酶体降解的胞质CTA 1。其他报告表明，可以使Gs？的ADP-核糖基化形式失活。这些集体观察结果强烈表明，霍乱和ETEC介导的腹泻是可逆事件。如果在毒素暴露后CTA 1/LTA 1向胞质溶胶的转运被阻断，则已经在胞质溶胶中的毒素池将被降解并且将不会被补充。CTA 1/LTA 1的胞质池因此将下降到低于所需的阈值浓度，允许宿主细胞去激活Gs？从中毒中恢复过来为了测试这个模型，我们将使用一种新的毒素检测系统的基础上，表面等离子体共振（SPR）技术，以量化holotoxin和A1亚基分布在胞质溶胶，内膜系统，和细胞外介质的中毒肠上皮细胞的寿命。尚未确定任何AB毒素的这些参数。根据已发表的和初步的数据，我们预测内膜系统作为一个长期的水库，缓慢但持续的交付CTA 1/LTA 1的胞质溶胶。我们进一步预测到达细胞质的低水平的CTA 1/LTA 1，结合清除细胞质中毒素和逆转中毒作用的细胞机制，将允许从CT/LT中毒中治疗和恢复。这将用毒性测定和基于SPR的测定进行测试，以监测用阻断毒素易位的药物处理的细胞中细胞溶质毒素的递送和持久性。我们的模型的验证将提供一个新的暴露后治疗策略的分子基础，专注于易位事件。 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，进入我们的肠道细胞，诱发潜在的致命性腹泻病例。启动和维持抗凝血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，并证明从中毒恢复是可能的治疗，降低细胞内毒素的水平。