Outer Membrane Vesicles in Shiga Toxin-Mediated Inflammatory and Thrombotic Responses Leading to Systemic Disease

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，导致出血性腹泻。肠出血性大肠杆菌产生滋贺毒素（Stx）， 合成抑制剂从肠道吸收到血液中时，可能会导致毁灭性的后果 溶血性尿毒综合征（HUS）HUS的特征是在血管中的纤维蛋白-血小板血栓。 微血管和受影响器官如肾的炎性损伤，导致肾衰竭。 全身暴露于LPS或其他PAMP在感染的肠道愈合阶段可以加强Stx的 有害的影响，并被认为是重要的发病机制。EHEC不是侵入性的，所以Stx必须 穿过肠上皮进入体循环。然而，游离的Stx并不常见于 我们缺乏对Stx如何以及以何种形式进入血液并最终 到达肾脏。一种潜在的未被探索的机制是通过以含Stx的外膜形式摄取， 膜囊泡（OMV），EHEC衍生的微粒，其可以从肠腔吸收到肠内， 除了Stx外，还含有过多PAMP如LPS和鞭毛蛋白，以及 其他肠出血性大肠杆菌编码的毒素在这个提议中，我们将评估外膜囊泡（OMV）如何相互作用 在巨噬细胞存在或不存在的情况下，与肾源性细胞一起， HUS动物模型中的肾脏。因为OMV是内吞的，LPS传感发生在细胞内，而不是细胞内。 而不是通过TLR 4，因此我们希望观察到的反应是根本不同的， 当Stx和LPS共同外源性递送时。从EHEC突变体产生OMV的能力， 改变的LPS（或其他PAMP或毒素）提供了鉴定导致损伤的关键特征的机会， 细胞反应。此外，已经假设HUS发病机制中的一个关键事件发生在以下情况： Stx与循环血细胞结合，导致形成含Stx的促血栓形成微泡 然后毒害远端器官在肠出血性大肠杆菌感染期间，血液成分可能会遇到Stx，LPS， 在OMV的背景下，其他潜在的破坏性细菌衍生的毒力因子，但知之甚少 关于OMV介导的微囊泡产生。我们将比较微泡产生的数量， 将血细胞暴露于来自野生型EHEC或EHEC缺陷型的纯化Stx或含Stx的OMV， PAMP或毒力因子。OMV相关货物，特别是促血栓/促凝血分子 将被表征，以及它们对上述肾细胞的作用。这些体外研究奠定了 为今后在肠出血性大肠杆菌感染的动物模型中进行体内研究奠定了重要基础。一项基本 了解宿主如何以及以何种形式遇到Stx，以及细胞反应如何 受Stx递送模式的影响，可能会为预防HUS的新治疗方式提供信息。

Enterohemorrhagic E. coli (EHEC) serotype O157:H7 is a foodborne diarrheal illness typically transmitted by contaminated beef or produce, resulting in bloody diarrhea. EHEC produce Shiga toxin (Stx), a protein synthesis inhibitor that when absorbed from the intestine into the bloodstream, can lead to the devastating illness, hemolytic uremic syndrome (HUS). HUS is characterized by fibrin-platelet thrombi in the microvasculature and inflammatory damage of affected organs such as the kidney, resulting in renal failure. Systemic exposure to LPS or other PAMPs during the diarrheal phase of infection can potentiate Stx’s deleterious effects and is thought to be important in pathogenesis. EHEC are not invasive, so Stx must traverse the intestinal epithelium to enter the systemic circulation. However, free Stx is not commonly found in patients with HUS, and we lack an understanding of how and in what form Stx enters the blood and eventually reaches the kidney. One potential under-explored mechanism is via uptake in the form of Stx-containing outer membrane vesicles (OMV’s), EHEC-derived microparticles that can be absorbed from the intestinal lumen into the bloodstream and contain, in addition to Stx, a plethora of PAMPs such as LPS and flagellae, as well as other EHEC-encoded toxins. In this proposal, we will assess how outer membrane vesicles (OMV’s) interact with cells of renal origin in the presence of absence of macrophages, inflammatory cells that infiltrate the kidney in animal models of HUS. Because OMV’s are endocytosed, LPS sensing occurs intracellularly rather than via TLR4, thus we expect to observe responses that are fundamentally different from those occurring when Stx and LPS are co-delivered exogenously. The ability to generate OMV from EHEC mutants with altered LPS (or other PAMPs or toxins) provide the opportunity to identify key features leading to damaging cellular responses. In addition, it has been hypothesized that a key event in HUS pathogenesis occurs when Stx engages circulating blood cells, resulting in the formation of Stx-containing, prothrombotic microvesicles that then intoxicate distal organs. During EHEC infection, blood components may encounter Stx, LPS, and other potentially damaging bacterially derived virulence factors in the context of OMV’s, but little is known about OMV-mediated microvesicle production. We will compare the quantity of microvesicles produced upon exposure of blood cells to purified Stx or Stx-containing OMVs from wild type EHEC or EHEC deficient in PAMPs or virulence factors. OMV-associated cargoes, particularly the prothrombotic/pro-coagulant molecules will be characterized, and for their effect on the renal cells described above. These in vitro studies lay the critical groundwork for future in vivo studies in an animal model of EHEC infection. A fundamental understanding of how and in what form Stx is encountered by the host, as well as how cellular responses are influenced by the mode of Stx delivery, may inform new therapeutic modalities to prevent HUS.