INTRACELLULAR TRANSPORT OF SHIGA TOXIN

溶血性尿毒综合征是儿童急性肾功能衰竭的主要原因，通常与滋贺和志贺样毒素引起的肠道感染有关。 对这些毒素的致病机制的深入研究使它们成为阐明许多具有相似作用模式的细菌毒素的作用的模型系统。 滋贺毒素要到达其靶宿主蛋白，必须通过宿主细胞内的一系列细胞器内化和转运，并最终易位到宿主细胞溶质空间。虽然扰乱这种细胞内行程废除毒素的行动，所需的毒素行动的精确途径仍然不明确。在这方面，我们最近阐明了在哺乳动物细胞内的细胞内运输的两个关键路口的运输调节机制。 小的GTmanned ADP-核糖基化因子6（ARF 6）调节质膜和早期内体之间的转运，而细胞内KDEL受体调节从高尔基复合体到内质网（ER）的逆行转运。 因此，我们将测试滋贺毒素的作用是否需要通过早期内体的内吞作用，通过确定是否过表达突变ARF 6，阻断内吞作用废除毒素的作用。 我们还将测试毒素的作用是否需要运输到ER，通过确定是否过表达的突变KDEL受体，从高尔基体复合体的转运阻断ER废除毒素的作用。 这些结果将为滋贺在宿主细胞内的运输路线提供更精确的信息。 由于许多细菌毒素具有相似的细胞内行程，我们的研究结果可能适用于了解各种细菌毒素如何与宿主相互作用引起疾病。 此外，由于不同的细胞内隔室具有独特的特性，从我们提出的研究中获得的知识可以应用于针对滋贺毒素的治疗干预的合理设计。

Hemolytic uremic syndrome is a leading cause of acute renal failure in children, and is typically associated with enteric infections by shiga and shiga-like toxins. Intensive research into the pathogenic mechanisms of these toxins has resulted in them being a model system of elucidating the actions of many bacterial toxins that share a similar mode of action. To reach its target host protein, shiga toxin must be internalized and transported through a series of organelles within the host cell, and ultimately be translocated into the host cytosolic space. Although perturbing this intracellular itinerary abrogates toxin action, the precise pathways that are required for toxin action remains ill-defined. In this regard, we have recently elucidated mechanisms of transport regulation at two critical junctures of intracellular transport within mammalian cells. The small GTPase maned ADP- Ribosylation Factor 6 (ARF6) regulates transport between the plasma membrane and the early endosome, while the intracellular KDEL receptor regulates retrograde transport from the Golgi complex to the endoplasmic reticulum (ER). Thus, we will test whether the action of shiga toxin requires endocytosis through the early endosome by determining whether overexpression of a mutant ARF6 that blocks endocytosis abrogates toxin action. We will also test whether toxin action requires transport to the ER by determining whether overexpression of a mutant KDEL receptor that blocks transport from the Golgi complex to the ER abrogates toxin action. These results will provide more precise information regarding the transport itinerary of shiga within the host cell. As many bacterial toxins share a similar intracellular itinerary, our findings will likely be applicable to understanding how a variety of bacterial toxins interface with their host to cause disease. Moreover, as different intracellular compartments possess characteristics that may be unique, knowledge derived from our proposed studies may be applied to a rational design of therapeutic intervention against shiga toxin.