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

志贺毒素和蓖麻毒素与肠上皮细胞相互作用并拯救靶细胞

• 批准号: 8233379
• 负责人: Alison Davis O'Brien
• 金额: $ 31.46万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8233379
• 关键词: 3-Dimensional; Aerosols; Apical; Apoptosis; Binding; Biological; Bioterrorism; Breathing; Cell Death; Cell Line; Cell surface; Cells; Centers for Disease Control and Prevention (U.S.); Cleaved cell; Depurination; Distal; Dose; Enterocytes; Epithelial; Epithelial Cells; Escherichia coli EHEC; Escherichia coli O157; Event; Family; Food; Galactose; Gastrointestinal tract structure; Genetic; Goals; Hemolytic-Uremic Syndrome; Human; Immune Sera; Immunochemistry; Infection; Ingestion; Injection of therapeutic agent; Intake; Intestines; Intoxication; Link; Modeling; Monitor; Movement; Mucous Membrane; Mus; N glycosidase; Nature; Organism; Organoids; Plants; Poison; Protein Synthesis Inhibition; Recombinants; Reporting; Ribosomal RNA; Ribosomes; Ricin; Ricinus communis; Risk; Shiga Toxin; Shigella dysenteriae; Site; Structure; Subgroup; Surface; Testing; Therapeutic; Tissues; Toxin; Water Pollution; base; biodefense; cytotoxic; design; globotriaosylceramide; glycolipid receptor; holotoxins; inhibitor/antagonist; killings; member; neutralizing antibody; polypeptide; therapy design; weapons

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.

志贺氏菌1型志贺毒素(STX)、O157：H7志贺氏菌STX1和STX2，以及 其他产生志贺毒素的大肠杆菌(STEC)和来自蓖麻的蓖麻毒素会导致一种 60S核糖体28S rRNA中的关键残基，从而抑制蛋白质合成、细胞凋亡和 细胞死亡。人类通常有被STX或通过摄入表达STX的蓖麻毒素中毒的风险 生物体或意外摄入蓖麻豆。然而，这些毒素被归类为疾控中心 选择毒剂是因为它们的效力和作为生物恐怖武器的潜在用途。故意交付的 毒素可通过故意感染STEC，或通过摄入、吸入或注射蓖麻毒素而发生。 当口服毒素时，它们必须首先通过以下方式与胃肠道的肠道细胞相互作用 步骤尚未确定，在某些情况下，会破坏粘膜。究竟两者之间的关联是如何 STX和结肠上皮细胞发生于非侵袭性大肠杆菌O157：H7和其他STEC尚不清楚 因为人类体内的这些细胞不表达STX受体球状三糖神经酰胺(Gb3)。不过， 我们证明了STX1和STX2与人结肠上皮细胞HCT-8的结合具有剂量依赖性，尽管 我们无法在细胞表面检测到Gb3。此外，Thorpe et al.据报道，STX1具有细胞毒性 对于高剂量的HCT-8细胞(我们证实了这一发现，但不能显示Stx2)，以及Stx1，Stx2， 蓖麻毒素对HCT-8细胞蛋白质合成有不同程度的抑制作用。因此，这些核毒素 似乎进入并在某种程度上使培养的人肠道细胞中毒，并在很长一段时间内 该项目的目标是确定Stxs和蓖麻毒素通过粘膜屏障的机制。一秒钟 最终目标是设计治疗化合物，使中毒细胞中的毒素失活。这个 具体的目的是：1.利用非gb3表面表达的hct-8细胞作为正常人的替代品 结肠细胞评估STX结合的性质以及发现STX1而不是StX2可以的基础，在 高剂量，杀死hct-8细胞；2.通过免疫化学和生物活性监测stxs的易位 由大肠杆菌O157：H7在感染HCT-8细胞的三维(3-D)有机物模型时产生，以及 评估添加到这种多层类器官组织表面的Stxs或蓖麻毒素是否可以从 根尖表面到下层细胞层和/或引起组织损伤；以及3.开发重组STX或蓖麻毒素 细胞结合域作为结合疗法的平台选择性地输送到毒素敏感细胞 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 在小鼠身上进行实验，以获得消除STX或蓖麻毒素致死效应的能力。