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

基本信息

批准号：
8375159
负责人：
Alison Davis O'Brien
金额：
$ 30.69万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8375159
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

项目摘要

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.

1 型痢疾志贺氏菌的志贺毒素 (Stx)、大肠杆菌 O157:H7 的 Stx1 和 Stx2，以及其他产生志贺毒素的大肠杆菌 (STEC) 和来自蓖麻子植物的蓖麻毒素会导致 60S 核糖体 28S rRNA 中的关键残基，因此抑制蛋白质合成、细胞凋亡和细胞死亡。人类通常因摄入表达 Stx 的物质而面临 Stx 或蓖麻毒素中毒的风险分别是生物体或意外摄入蓖麻子。然而，这些毒素被 CDC 归类为选择特工是因为它们具有作为生物恐怖武器的效力和潜在用途。故意交付毒素可能是通过故意感染 STEC，或摄入、吸入或注射蓖麻毒素而产生的。当毒素通过口服引入时，它们必须首先通过以下方式与胃肠道的肠细胞相互作用：步骤尚未定义，并且在某些情况下会破坏粘膜。具体如何关联 Stxs 和结肠上皮细胞发生的情况为非侵入性大肠杆菌 O157:H7 和其他 STEC 尚不清楚因为人类的这些细胞不表达 Stx 受体三酰神经酰胺 (Gb3)。尽管如此，我们证明了 Stx1 和 Stx2 与人结肠上皮 HCT-8 细胞的剂量依赖性结合，尽管我们无法检测细胞表面的 Gb3。此外，索普等人。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，但无法针对 Stx2 显示），并且 Stxl、Stx2、蓖麻毒素对HCT-8细胞的蛋白质合成有不同程度的抑制作用。因此，这些核毒素似乎可以进入培养物中的人类肠道细胞并在某种程度上使人中毒，并且一种长范围的该项目的目标是确定 Stxs 和蓖麻毒素穿过粘膜屏障的机制。一秒钟最终目标是设计能够灭活中毒细胞中毒素的治疗化合物。这具体目标是： 1. 利用非 Gb3 表面表达的 HCT-8 细胞作为正常人类的替代细胞结肠细胞来评估 Stx 结合的性质，以及发现 Stx1 而不是 Stx2 可以在高剂量，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 3 维 (3-D) 类器官模型期间产生，以及评估添加到此类多层类器官组织表面的 Stxs 或蓖麻毒素是否可以从顶端表面到下面的细胞层和/或引起组织损伤； 3.开发重组Stx或蓖麻毒素细胞结合结构域作为选择性递送至毒素敏感细胞的缀合疗法的平台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证明其能够消除 Stx 或蓖麻毒素的致命作用。