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.

肠出血性大肠杆菌 (EHEC) 血清型 O157:H7 是一种食源性腹泻，通常通过以下途径传播：受污染的牛肉或农产品会产生志贺毒素 (Stx)，这是一种蛋白质合成抑制剂，可导致严重的疾病，经全身吸收后，可导致毁灭性的疾病，溶血性尿毒症综合征 (HUS)，因为 Stx 是由溶原性噬菌体编码的，所以不能像抗生素一样进行抗生素治疗。可能会诱导噬菌体并增加毒素的产生，从而加剧疾病。 EHEC 介导的肠上皮损伤对于 HUS 至关重要，因为它可以促进 Stx 从肠上皮的移动。 EHEC 定植于肠上皮后进入血液。通过3型分泌系统将效应物注入宿主细胞，引发“附着和抹去”的形成 (AE) 病变特征为微绒毛消失和结合细菌下方肌动蛋白基座形成。据推测，Stxs 可以诱导中毒细胞的促炎反应并导致细胞死亡。当注射 3 型肠出血性大肠杆菌（EHEC）诱导细胞骨架重排时，EHEC 会破坏肠道屏障功能通过 Stx 介导的肠上皮细胞死亡和/或炎症细胞的流入来分泌效应物。不幸的是，我们对上皮损伤和随后的毒素吸收是如何发生的部分缺乏了解。因为我们缺乏模拟体内发生的复杂的疾病促进相互作用的模型 EHEC、Stx 和多种宿主细胞类型最近开发的二维结肠模型允许评估细菌、宿主细胞和毒素本身对 Stx 摄入肠道细胞和转移的贡献穿过肠上皮，源自干细胞的结肠样单层，极化并分化为构成人类结肠的四种细胞类型，因此可以区分细胞特异性毒力因子目标，它们的顶端和基底外侧表面是可触及的，就像人类结肠一样，顶端有粘液层。可以引入巨噬细胞等炎症细胞，从而增加研究肠出血性大肠杆菌的能力。免疫细胞存在下的结肠上皮相互作用二维结肠模型可以进行评估。研究单个毒力因子如何协同作用以造成最大的肠道损伤。在这项提案中，我们确定了 Stx 的肠上皮靶标以及促进毒素结合和的关键事件。我们评估 Stx2 和含有 Stx2 和其他肠出血性大肠杆菌的外膜囊泡 (OMV) 的吸收情况。在巨噬细胞和肠出血性大肠杆菌存在和不存在的情况下，毒力因子会与类结肠细胞相互作用。测试 Stx 本身的中毒是否会影响这些相互作用。最后，我们评估了 Stx、OMV、 EHEC 和/或巨噬细胞共培养对顶端连接复合物、上皮细胞凋亡和通过了解顺序促炎性肠上皮细胞的表达。 EHEC 感染期间发生的导致 Stx 全身吸收的破坏性事件，我们希望确定这些事件中的哪一个可能是关键的并且是药物可靶向的，从而预防 HUS。