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

肠出血性大肠O 157：H7型肠出血性大肠杆菌（EHEC）是一种食源性肠道疾病，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滋贺毒素（Stx），这是一种蛋白质合成抑制剂，可引起 严重的疾病，并在全身吸收，可导致毁灭性的疾病，溶血性尿毒综合征 （HUS）。由于Stx是由溶原性噬菌体编码的，因此排除了抗生素治疗，因为抗生素 可能会诱导噬菌体并增加毒素产生，加剧疾病。 肠出血性大肠杆菌介导的对肠上皮的损伤是HUS的关键，它促进了Stx从肠上皮的移动。 肠道进入血液在肠上皮定植后，EHEC穿透粘液层， 通过3型分泌系统将效应物注入宿主细胞，触发“附着和消除”的形成 (AE)以微绒毛消失和结合细菌下的肌动蛋白基座形成为特征的病变。 Stxs可诱导中毒细胞的促炎反应并导致细胞死亡。据推测 EHEC在注射3型肠毒素诱导细胞骨架重排时破坏肠屏障功能， 通过Stx介导的肠上皮细胞死亡和/或通过炎性细胞的流入，可以抑制分泌的效应物。 不幸的是，我们缺乏对上皮损伤和随后的毒素摄取如何发生的理解，部分原因是 因为我们缺乏模型来模拟体内发生的复杂的疾病促进相互作用， 肠出血性大肠杆菌、Stx和多种宿主细胞类型。最近开发的2-D结肠模型允许评估 细菌、宿主细胞和毒素本身在肠细胞摄取Stx和转移中的作用 穿过肠上皮来源于干细胞的类集落单层， 包括人结肠的四种细胞类型，因此可以区分细胞特异性毒力因子靶标， 并且它们的顶面和基底面是可接近的。与人类结肠一样， 存在。可以引入巨噬细胞等炎性细胞，增加研究EHEC的能力。 免疫细胞存在下的结肠上皮相互作用。2-D结肠模型允许评估 单个毒力因子如何协同工作以引起最大的肠道损伤。 在这个提议中，我们确定了Stx的肠上皮靶点和促进毒素结合的关键事件， 摄取。我们评估了Stx 2和含有Stx 2和其他EHEC的外膜囊泡（OMV） 在巨噬细胞和肠出血性大肠杆菌存在和不存在的情况下，毒力因子与结肠样细胞相互作用。.我们 测试Stx本身的中毒是否会影响这些相互作用。最后，我们评估了Stx，OMV， EHEC和/或巨噬细胞在顶端连接复合体上共培养，上皮细胞凋亡， 促炎分子表达。通过了解促炎性肠上皮细胞- 在EHEC感染过程中发生的导致全身吸收Stx的破坏性事件，我们希望确定 这些事件中哪些可能是关键的和药物靶向的，从而预防HUS。

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.