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

肠出血性大肠杆菌(EHEC)O157：H7型是一种食源性腹泻疾病，通常由 受污染的牛肉或农产品。EHEC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，一旦全身吸收，可能会导致毁灭性的疾病，溶血性尿毒症综合征。 (HUS)。因为STX是由溶源噬菌体编码的，所以抗生素治疗是不可能的。 可能会诱导噬菌体并增加毒素的产生，从而加重疾病。 EHEC介导的肠上皮损伤是HUS的关键，它通过促进STX从 肠道进入血液。在肠道上皮细胞定植后，EHEC穿透粘液层并 通过3型分泌系统将效应物注射到宿主细胞中，触发“附着和消除”的形成 (AE)病变的特征是微绒毛消失和结合细菌下的肌动蛋白底座形成。 STXs可在中毒细胞中诱导促炎反应，导致细胞死亡。这是一种假设 当注射3型-EHEC诱导细胞骨架重排时，EHEC破坏肠道屏障功能 通过STX介导的肠道细胞死亡和/或炎症细胞的涌入而分泌效应物。 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缺乏了解，部分是 因为我们缺乏模型来模拟体内发生的复杂的致病相互作用 EHEC、STX和多种宿主细胞类型。最近开发的2-D结肠型模型允许评估 细菌、宿主细胞和毒素本身在肠道细胞摄取和转移STX中的作用 穿过肠道上皮。来源于干细胞的集落样单分子层极化并分化为 组成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(S)， 并且它们的顶面和基底侧面是可接近的。就像人类的结肠一样，顶端的粘液层 是存在的。可以引入巨噬细胞等炎性细胞，增加研究EHEC- 在免疫细胞存在的情况下，结肠上皮的相互作用。2-D结肠型模型允许评估 单个毒力因素如何协同作用，导致最大的肠道损伤。 在这项建议中，我们确定了STX的肠道上皮靶点和促进毒素结合和 领悟。我们评估了含有Stx2和其他EHEC的Stx2和外膜囊泡(OMV)是如何 在巨噬细胞和EHEC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中毒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/或巨噬细胞在顶端连接复合体上共培养，上皮细胞凋亡，以及 促炎分子的表达。通过了解顺序的促炎、肠上皮- 在EHEC感染期间发生的导致全身感染STX的破坏性事件，我们希望确定 这些事件中的哪些可能是关键的和有药物靶向的，从而防止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.