Retrograde trafficking of Shiga toxin 2

摘要 产滋贺毒素E.大肠杆菌是美国食源性疾病的主要原因， 每年10万人。这些细菌产生两种类型的滋贺毒素（STx）：STx 1和STx 2。的 毒素是由A亚基与A亚基结合形成的，A亚基通过阻断蛋白质合成杀死宿主细胞， 五聚体B亚基，其介导从细胞表面到胞质溶胶的运输。没有治疗方法 对产滋贺毒素E.大肠杆菌，包括危及生命的肾脏并发症。 由于STx 1和STx 2必须运输到胞质溶胶以诱导毒性，因此设计STx 1和STx 2具有相当大的兴趣。 用于治疗的毒素运输抑制剂。2012年，我们发现金属锰是一种有效的抑制剂， STx 1运输，保护小鼠免受STx 1诱导的致死性。然而，疾病的严重程度与 STx 2生产。然而，STx 2运输的机制尚未阐明，并且尚未批准STx 2的抑制剂。 STx 2交易不可用。因此，我们最近的努力集中在STx 2运输上。 我们发现，在宿主细胞中，STx 1和STx 2都使用一个保守的结构域（B-β4-β5环）， 亚基），以避免溶酶体降解，并直接运输到高尔基体从早期内涵体。β4-β5 STx 1 B亚基环与宿主蛋白GPP 130相互作用，GPP 130作为内体受体， STx 1和陪伴毒素从早期内体到高尔基体。锰通过以下途径阻断STx 1运输： 诱导GPP 130降解。相比之下，STx 2运输是GPP 130独立的（并且Mn不敏感）。 因为GPP 130结合所需的残基在其B亚基中不保守。尽管分子 不同之处在于，STx 1和STx 2使用共同的结构基序进行早期内体到高尔基体的过程， 运输提出了一种可能性，即基本机制可能具有概念上的相似性。 我们假设，类似于GPP 130-STx 1范式，STx 2 B亚基的β4-β5环相互作用 与作为STx 2的内体受体的宿主蛋白质结合。这种交互允许STx 2将流量传输到 高尔基体从早期的内体，从而避免溶酶体降解。在这个探索性的建议中，我们将 通过鉴定早期内体到高尔基体转运所需的宿主因子来验证这一假设。 STx 2，并随后确定这些因子中的任一个是否作为内体STx 2受体起作用。到 为了实现我们的实验目标，我们将联合收割机结合Aim 1中的高通量蛋白质组学筛选和基因组- Aim 2中的宽siRNA筛选。由于锰诱导的GPP 130（STx 1内体受体）降解， STx 2的内体受体的鉴定在抵抗STx 1毒性方面是非常有效的， 有望为STx 2转运抑制剂的设计提供理想的分子靶点。因此，这种探索性 该提案有可能为开发治疗方法做出深远的贡献。 由产滋贺毒素E.大肠杆菌，基于阻断STx 2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.