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

描述(申请人提供)：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AB5型蛋白毒素，由酶A1亚基、A2接头和细胞结合B五聚体组成。这两种毒素都通过宿主蛋白Gs？的ADP核糖化作用产生腹泻反应。CT和LT攻击宿主细胞胞浆内的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。只有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进入内质网(ER)后，才会发生这种情况。A1亚基与内质网中的其余毒素解离，展开，并通过蛋白质传导通道进入胞浆。然后，转位的A1亚基与宿主因子相互作用，重新获得激活Gs？的功能构象。 据信，霍乱和ETEC介导的腹泻只有在中毒的肠道细胞从肠道上皮细胞上脱落后才能恢复，这些细胞的寿命为3-5天。许多观察表明，情况并非如此。我们最近已经证明，胞浆CTA1需要一个阈值浓度才能引起细胞病变效应。我们还确定了两种治疗药物，可以阻止毒素转位到胞浆，我们还记录了胞浆CTA1的泛素非依赖性蛋白酶体降解。其他报告表明，有可能使ADP核糖化形式的Gs？失活。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转位在毒素暴露后被阻止到细胞质，细胞质中已经存在的毒素池将被降解，不会被补充。因此，CTA1/LTA1的胞液池将降至必要的阈值浓度以下，从而允许宿主细胞去激活Gs？从醉酒中恢复过来。为了测试这一模型，我们将使用一种基于表面等离子共振(SPR)技术的新型毒素检测系统，以量化全毒素和A1亚基在中毒肠细胞的胞浆、内膜系统和细胞外介质中的分布。尚未确定任何AB毒素的这些参数。基于已发表的和初步的数据，我们预测内膜系统是CTA1/LTA1缓慢但持续输送到细胞质的长期储存库。我们进一步预测，到达胞浆的低水平CTA1/LTA1，结合清除胞浆毒素和逆转中毒影响的细胞机制，将使CT/LT中毒的治疗和康复成为可能。这将通过毒性分析和基于SPR的分析进行测试，以监测胞质毒素在用阻止毒素转位的药物处理的细胞中的传递和持久性。我们模型的验证将为关注易位事件的暴露后新的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，进入我们的肠道细胞，导致潜在的致命腹泻病例。启动和维持腹泻反应所需的细胞内毒素的数量尚不清楚。在这里，我们将建立这些参数，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过来。

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.