Effect of Shiga toxin, OMVs, and innate immune cells on epithelial integrity of human colonoids during EHEC infection

志贺毒素、OMV 和先天免疫细胞对肠出血性大肠杆菌感染期间人结肠上皮完整性的影响

基本信息

批准号：
9978339
负责人：
JOHN M LEONG
金额：
$ 21.24万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-21 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9978339
关键词：
Actins Acute Animals Antibiotic Therapy Antibiotics Antibodies Apical Apoptosis Apoptotic Architecture Bacteria Bacterial Infections Bacterial Toxins Bacteriophages Binding Biological Models Blood Circulation Brush Border Cell Death Cells Cessation of life Childhood Coculture Techniques Colon Complex Cultured Cells Cytolysis Development Disease Drug Targeting Enterocytes Environment Epithelial Epithelial Cells Epithelium Escherichia coli EHEC Escherichia coli Infections Escherichia coli O157:H7 Evaluation Event Evolution Experimental Models Future Glycolipids Goals Goblet Cells Hemolytic-Uremic Syndrome Human Immune Individual Infant Infection Inflammation Inflammatory Integration Host Factors Intestines Intoxication Kidney Diseases Kidney Failure Knowledge Label Laboratories Lead Lesion Life MAP Kinase Kinase Kinase Measures Mediating Membrane Methods Modeling Movement Mucous Membrane Mucous body substance Mus Neuroendocrine Cell Organoids Oryctolagus cuniculus Pathway interactions Physiology Production Protein Synthesis Inhibitors Risk Role Septic Toxemia Serotyping Shiga Toxin Shiga-Like Toxin II Signal Transduction Stress Supportive care Surface System Systemic disease Testing Tight Junctions Toxin Tube Type III Secretion System Pathway Tyrosine Kinase Inhibitor Validation Variant Vesicle Virulence Factors Work absorption alpha Toxin base beef cell type cellular targeting diarrheal disease foodborne globotriaosylceramide gut colonization human model in vivo inflammatory disease of the intestine insight intestinal barrier intestinal epithelium macrophage microbiota migration monolayer pathogen prevent receptor expression response stem cells tissue culture uptake

项目摘要

Enterohemorrhagic E. coli (EHEC) serotype O157:H7 is a foodborne diarrheal illness typically transmitted by contaminated beef or produce. EHEC produce Shiga toxin (Stx), a protein synthesis inhibitor that can cause severe disease, and upon systemic absorption, can lead to the devastating illness, hemolytic uremic syndrome (HUS). Because Stx is encoded by a lysogenic bacteriophage, antibiotic therapy is precluded, as antibiotics may induce the phage and increase toxin production, exacerbating disease. EHEC-mediated damage to the intestinal epithelium is critical for HUS by facilitating movement of Stx from the intestine into the bloodstream. Upon colonizing the intestinal epithelium, EHEC penetrates the mucus layer and injects effectors into host cells via a type 3 secretion system, triggering formation of “attaching and effacing” (AE) lesions characterized by microvillar effacement and actin pedestal formation beneath bound bacteria. Stxs can induce proinflammatory responses in intoxicated cells and cause cell death. It has been postulated that EHEC disrupts intestinal barrier function when cytoskeletal rearrangements are induced by injected type 3- secreted effectors, by Stx-mediated enterocyte death, and/or by the influx of inflammatory cells. Unfortunately, we lack an understanding of how epithelial damage and subsequent toxin uptake occurs, in part because we lack models that mimic the complex disease-promoting interactions that occur in vivo between EHEC, Stx and the multiple host cell types. A recently developed 2-D colonoid model permits an evaluation of the contributions of bacteria, host cells, and the toxin itself in uptake of Stx into intestinal cells and transfer across the intestinal epithelium. Colonoid monolayers, derived from stem cells, polarize and differentiate into the four cell types that comprise human colon, so cell-specific virulence factor target(s) can be distinguished, and their apical and basolateral surfaces are accessible. Like in human colon, an apical adherent mucus layer is present. Inflammatory cells such as macrophages can be introduced, adding to the ability to study EHEC- colonic epithelial interactions in the presence of immune cells. The 2-D colonoid model allows for assessment of how individual virulence factors may work in concert to cause maximal intestinal damage. In this proposal, we identify intestinal epithelial targets of Stx and key events that promote toxin binding and uptake. We assess how Stx2 and outer membrane vesicles (OMV’s) containing Stx2 and other EHEC virulence factors interact with colonoid cells in the presence and absence of macrophages and EHEC. . We test whether intoxication by Stx itself influences these interactions.. Finally, we assess the effect of Stx, OMV’s, EHEC and/or macrophage co-culture on the apical junction complex, epithelial cell apoptosis, and proinflammatory molecule expression. By understanding the sequential proinflammatory, intestinal epithelium- damaging events that occur during EHEC infection that lead to systemic uptake of Stx, we hope to identify which of these events may be critical and drug-targetable, thus preventing HUS.

Enterohehemorrhagic E. coli（EHEC）血清型O157：H7是一种通常通过被污染的牛肉或农产品。 EHEC产生Shiga毒素（STX），这是一种可能引起的蛋白质合成抑制剂严重的疾病以及全身虐待，可能导致造成毁灭性疾病，溶血性尿毒症综合征（HUS）。由于STX是由溶酶因细菌噬细胞编码的，因此抗生素治疗被排除为抗生素可能诱导噬菌体并增加毒素产生，加剧疾病。 EHEC介导的对肠上皮的损害对HUS对HUS至关重要肠道进入血液。在定居肠上皮时，EHEC穿透了粘液层，通过3型分泌系统注入宿主细胞，触发“连接和eSf缩”的形成（AE）以微绒毛能和肌动蛋白基座形成为特征的病变。 STX可以诱导陶醉细胞中促炎反应并导致细胞死亡。它已经假设当细胞骨架重排被注入3型 - STX介导的肠细胞死亡和/或受炎症细胞的影响，分泌作用。不幸的是，我们缺乏对上皮损害和随后的毒素摄取的理解，部分部分是因为我们缺乏模仿体内发生的复杂促进疾病相互作用的模型 EHEC，STX和多种主机单元类型。最近开发的二维结构化模型允许评估细菌，宿主细胞和毒素本身在摄入STX进入肠细胞的贡献并转移跨肠上皮。源自干细胞的结肠单层，极化并分化为包括人类结肠的四种细胞类型，因此可以区分细胞特异性病毒因子靶标，并且可以访问它们的顶端和基底外侧表面。就像人类结肠一样，顶端粘附层在场。可以引入炎性细胞，例如巨噬细胞，从而增加了研究EHEC-的能力在免疫细胞存在下结肠上皮相互作用。 2-D结肠模型允许评估关于单个病毒因素如何协同起作用，从而导致最大的肠道损伤。在此提案中，我们确定了STX的肠上皮靶标和促进毒素结合和的关键事件吸收。我们评估含有STX2和其他EHEC的STX2和外膜蔬菜（OMV）在存在和不存在巨噬细胞和EHEC的情况下，毒力因子与结肠细胞相互作用。。我们测试STX本身是否会影响这些相互作用。最后，我们评估了STX，OMV的效果， EHEC和/或巨噬细胞共培养物，上皮连接络合物，上皮细胞凋亡和促炎分子表达。通过了解顺序促炎性，肠上皮 EHEC感染期间发生的破坏事件导致系统性吸收，我们希望确定这些事件中的哪一个可能是至关重要的，可以靶向毒品，从而防止HUS。