INTRACELLULAR TRANSPORT OF SHIGA TOXIN

溶血性尿毒症综合征是导致儿童急性肾功能衰竭的主要原因，通常与志贺和志贺类毒素的肠道感染有关。对这些毒素的致病机制进行了深入的研究，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式系统。志贺毒素要到达目标宿主蛋白，必须内化并通过宿主细胞内的一系列细胞器转运，最终转运到宿主胞浆空间。尽管扰乱细胞内的这种行程可以消除毒素的作用，但毒素作用所需的确切途径仍然没有明确的定义。在这方面，我们最近阐明了在哺乳动物细胞内细胞内运输的两个关键节点的运输调节机制。由ADP核糖化因子6(ARF6)编码的小GTP酶调节质膜和早期内体之间的运输，而细胞内的KDEL受体调节从高尔基复合体到内质网(ER)的逆行运输。因此，我们将通过确定过表达阻断内吞作用的突变ARF6是否取消毒素作用来测试志贺毒素的作用是否需要通过早期内吞作用进行内吞作用。我们还将通过确定突变的KDEL受体的过度表达是否阻止从高尔基复合体到内质网的运输来测试毒素作用是否需要转运到内质网。这些结果将提供有关志贺菌在宿主细胞内的运输路线的更准确的信息。由于许多细菌毒素都有类似的细胞内行程，我们的发现可能适用于了解各种细菌毒素如何与宿主相互作用而致病。此外，由于不同的细胞内间隔可能具有独特的特征，我们所建议的研究成果可能被应用于针对志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.