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

• 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
• 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

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 是一种食源性腹泻疾病，通常通过以下途径传播： 受污染的牛肉或农产品。肠出血性大肠杆菌产生志贺毒素 (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 （胡斯）。由于 Stx 是由溶原性噬菌体编码的，因此无法使用抗生素治疗，因为抗生素 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。 肠道进入血液。肠出血性大肠杆菌定植于肠上皮后，会穿透粘液层并 通过3型分泌系统将效应器注入宿主细胞，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。 Stxs 可以诱导中毒细胞的促炎反应并导致细胞死亡。已假设 当注射 3 型肠出血性大肠杆菌 (EHEC) 诱导细胞骨架重排时，肠出血性大肠杆菌 (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可以评估 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。源自干细胞的集落单层，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， 并且它们的顶端和基底外侧表面是可接近的。就像人类结肠一样，顶端附着粘液层 存在。可以引入巨噬细胞等炎症细胞，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。二维结肠模型可用于评估 研究单个毒力因子如何协同作用以造成最大的肠道损伤。 在本提案中，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。 吸收。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) 如何 在巨噬细胞和肠出血性大肠杆菌存在或不存在的情况下，毒力因子与类结肠细胞相互作用。 。我们 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 Stx、OMV、 EHEC 和/或巨噬细胞共培养对顶端连接复合体、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细胞 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。