Shiga toxin and ricin interaction with enterocytes and rescue of target cells

志贺菌1型的滋贺毒素（Stx），大肠杆菌O 157：H7的Stx 1和Stx 2，和 其它产滋贺毒素E.大肠杆菌（STEC）和蓖麻毒素从蓖麻子植物引起脱嘌呤的一个 60 S核糖体的28 S rRNA中的关键残基，因此，抑制蛋白质合成，细胞凋亡， 细胞死亡人类通常有通过摄入表达Stx的毒素而被Stx或蓖麻毒素中毒的风险。 有机体或意外摄入蓖麻子。然而，这些毒素被归类为CDC 选择代理人，因为他们的潜力和潜在的使用作为生物恐怖武器。故意交付 毒素可能通过故意感染STEC或通过摄入、吸入或注射蓖麻毒素而产生。 当毒素经口引入时，它们必须首先通过以下方式与胃肠道的肠上皮细胞相互作用： 尚未定义的步骤，并且在某些情况下，破坏粘膜。到底是什么让 Stxs和结肠上皮细胞发生的非侵袭性大肠杆菌O 157：H7等STEC尚不清楚 因为人体内的这些细胞不表达Stx受体神经酰胺三己糖苷（Gb 3）。然而，尽管如此， 我们证明了Stx 1和Stx 2与人结肠上皮HCT-8细胞的剂量依赖性结合， 我们无法在细胞表面检测到Gb 3。此外，Thorpe等人报道Stx 1具有细胞毒性， 对于HCT-8细胞，在高剂量下（我们证实了这一发现，但对于Stx 2不能显示），并且Stx 1，Stx 2， 和蓖麻毒素引起HCT-8细胞中蛋白质合成的不同程度的抑制。因此，这些核毒素 似乎进入并在一定程度上毒害培养中的人肠细胞， 本项目的目的是确定Stxs和蓖麻毒素穿过粘膜屏障的机制。第二 最终目标是设计治疗性化合物，其抑制中毒细胞中的毒素。的 具体目标是：1.利用不表达Gb 3表面的HCT-8细胞作为正常人的替代物 结肠细胞，以评估Stx结合的性质，以及发现Stx 1而不是Stx 2可以结合的基础。 高剂量，杀死HCT-8细胞; 2.通过免疫化学和生物活性监测Stx的易位 由E.大肠杆菌O 157：H7在感染HCT-8细胞的三维（3-D）类器官模型期间，和 评估添加到这种多层类器官组织表面的Stx或蓖麻毒素是否可以从细胞膜转运到细胞膜。 顶端表面到下面的细胞层和/或引起组织损伤;和，3.开发重组Stx或蓖麻毒素 作为选择性递送到毒素敏感细胞中的缀合治疗剂的平台的细胞结合结构域 毒素中和抗体或毒素抑制剂，然后在细胞系上测试这些嵌合分子， 在小鼠中消除Stx或蓖麻毒素的致死作用的能力。

Shiga toxin (Stx) of Shigella dysenteriae type 1, Stx1 and Stx2 of Escherichia coli O157:H7, and other Shiga toxin-producing E. coli (STEC), and ricin from the castor bean plant cause depurination of a critical residue in the 28S rRNA of 60S ribosomes and, hence, inhibition of protein synthesis, apoptosis, and cell death. Humans are usually at risk of intoxication by Stx or ricin through ingestion of Stx-expressing organisms or accidental intake of castor beans, respectively. Yet, these toxins are categorized as CDC Select Agents because of their potency and potential use as bioterrorist weapons. Deliberate delivery of the toxins may occur through intentional infection with STEC, or by ingestion, inhalation, or injection of ricin. When the toxins are introduced orally, they must first interact with enterocytes of the gastrointestinal tract by steps not yet defined, and, in some instances, breach the mucosa. Exactly how the association between Stxs and colonic epithelial cells occurs for the non-invasive E .coli O157:H7 and other STEC is unclear because these cells in humans do not express the Stx receptor globotriaosylceramide (Gb3). Nevertheless, we demonstrated dose-dependent binding of Stx1 and Stx2 to human colonic epithelial HCT-8 cells despite our inability to detect Gb3 on the surface of the cells. Moreover, Thorpe et al. reported that Stx1 is cytotoxic for HCT-8 cells at high doses (a finding that we confirmed but could not show for Stx2) and that Stxl, Stx2, and ricin cause varying degrees of inhibition of protein synthesis in HCT-8 cells. Thus, these ribotoxins appear to gain entry into and at some level intoxicate human intestinal cells in culture, and one long range goal of this project is to define the mechanisms by which Stxs and ricin cross the mucosal barrier. A second ultimate objective is to design therapeutic compounds that inactivate the toxins in intoxicated cells. The specific aims are to: 1. exploit non-Gb3-surface-expressing HCT-8 cells as a surrogate for normal human colonic cells to evaluate the nature of Stx binding and the basis for the finding that Stx1 but not Stx2 can, at high doses, kill HCT-8 cells; 2. monitor, by immunochemistry and biological activity, the translocation of Stxs produced by E. coli O157:H7 during infection of a 3-dimensional (3-D) organoid model of HCT-8 cells, and evaluate whether Stxs or ricin added to the surface of such multi-layered organoid tissue can transit from the apical surface to underlying cell layers and/or elicit tissue damage; and, 3.develop recombinant Stx or ricin cell-binding domains as platforms for conjugated therapeutics that selectively deliver into toxin-sensitive cells either toxin-neutralizing antibodies or toxin inhibitors and then test these chimeric molecules on cell lines and in mice for the capacity to ablate the lethal effects of Stx or ricin.