Retrograde trafficking of Shiga toxin 2

摘要 在美国，产生志贺毒素的大肠杆菌是食源性疾病的主要原因，并通过 每年有10万人。这些细菌产生两种志贺毒素(STX)：STX1和STX2。这个 毒素是由A亚单位与A亚基结合形成的，A亚单位通过阻止蛋白质合成来杀死宿主细胞 五聚体B亚基，它调节从细胞表面到细胞质的运输。没有任何治疗方法 用于由产生志贺毒素的大肠杆菌引起的疾病，包括危及生命的肾脏并发症。 由于STX1和STX2必须运输到胞质才能引起毒性，所以在设计中有相当大的兴趣 用于治疗的毒素转运的抑制剂。2012年，我们确定金属锰是一种有效的抑制 STX1交易保护小鼠免受STX1诱导的死亡。然而，疾病的严重程度与 STX2生产。然而，STx2的转运机制还没有被阐明，也没有被批准为STx2的抑制剂 STX2交易不可用。因此，我们最近的努力集中在STx2传输上。 我们发现，在宿主细胞中，stx1和stx2都使用一个保守的结构域(β4-β5环，其B- 亚基)，以避免溶酶体的降解，并从早期的内体直接运输到高尔基体。β4-β5 STX1B亚基的环与宿主蛋白GPP130相互作用，GPP130起着内体受体的作用 STX1，并将该毒素从早期内体中伴随到高尔基体上。锰通过以下方式阻止STX1贩运 诱导GPP130降解。相反，STx2的转运是不依赖于GPP130的(并且对MN不敏感) 因为GPP130结合所需的残基在其B亚基中不保守。尽管分子上 不同的是，STX1和STX2使用共同的结构基序来经历早期内体到高尔基体的过程 运输增加了潜在机制可能在概念上有相似之处的可能性。 我们假设，与GPP130-STX1范例类似，STx2B亚基的β4-β5环相互作用 带有一种宿主蛋白，它充当STx2的内体受体。这种交互允许STx2将流量传输到 将高尔基体从早期内体中分离出来，从而避免溶酶体的降解。在这个试探性的提案中，我们将 通过确定早期内胚体向高尔基体运输所需的寄主因素来检验这一假说 STx2，并随后确定这些因子中是否有任何一个作为内体STx2受体发挥作用。至 为了实现我们的实验目标，我们将在Aim 1中将高通量蛋白质组筛选与基因组- 目的2.由于锰诱导STX1内体受体GPP130的降解， 在保护STX1毒性方面非常有效，STX2内体受体的鉴定是 有望为STx2转运抑制剂的设计提供理想的分子靶点。因此，这种探索性的 这项提议有可能对开发一种治疗癌症的方法做出深远的贡献 由产志贺毒素的大肠杆菌引起的疾病，基于阻止STx2的运输。

Abstract Shiga toxin-producing E. coli are a major cause of foodborne illness in the United States and infect over 100,000 individuals annually. These bacteria produce two types of Shiga toxin (STx): STx1 and STx2. The toxins are formed by the association of an A subunit, which kills host cells by blocking protein synthesis, with a pentameric B-subunit, which mediates trafficking from the cell surface to the cytosol. There are no treatments for disease caused by Shiga toxin-producing E. coli, including for life-threatening renal complications. As STx1 and STx2 must traffic to the cytosol to induce toxicity, there is considerable interest in designing inhibitors of toxin trafficking for therapy. In 2012, we identified the metal manganese to be a potent inhibitor of STx1 trafficking that protected mice against STx1-induced lethality. However, disease severity correlates with STx2 production. Yet, mechanisms of STx2 trafficking have not been elucidated and approved inhibitors of STx2 trafficking are not available. Therefore, our recent efforts focused on STx2 transport. We discovered that, in host cells, both STx1 and STx2 used a conserved domain (β4-β5 loop of their B- subunits) to avoid lysosomal degradation and directly traffic to the Golgi from early endosomes. The β4-β5 loop of STx1 B-subunit interacted with a host protein, GPP130, which functioned as the endosomal receptor for STx1 and chaperoned the toxin to the Golgi from early endosomes. Manganese blocked STx1 trafficking by inducing GPP130 degradation. In contrast, STx2 trafficking was GPP130 independent (and Mn insensitive) because residues required for GPP130 binding were not conserved in its B-subunit. Despite the molecular difference, the fact that STx1 and STx2 use a common structural motif to undergo early endosome-to-Golgi transport raises the possibility that the underlying mechanisms may have conceptual similarities. We hypothesize that, similar to the GPP130-STx1 paradigm, the β4-β5 loop of STx2 B-subunit interacts with a host protein, which acts as the endosomal receptor for STx2. This interaction allows STx2 to traffic to the Golgi from early endosomes, and thereby avoid lysosomal degradation. In this exploratory proposal, we will test this hypothesis by identifying host factors that are required for the early endosome-to-Golgi transport of STx2 and subsequently determining whether any of these factors function as the endosomal STx2 receptor. To achieve our experimental goals, we will combine a high-throughput proteomic screen in Aim 1 with a genome- wide siRNA screen in Aim 2. As Mn-induced degradation of GPP130, the STx1 endosomal receptor, is remarkably effective in protecting against STx1 toxicity, identification of the endosomal receptor for STx2 is expected to provide an ideal molecular target for design of STx2 transport inhibitors. Thus, this exploratory proposal has the potential to make far-reaching contributions towards the development of a treatment for disease caused by Shiga toxin-producing E. coli, based on blocking STx2 trafficking.