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

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，导致血性腹泻。EHEC产生志贺毒素(STX)，一种蛋白质 合成抑制剂，当从肠道吸收到血液中时，可能会导致毁灭性的 疾病，溶血性尿毒症综合征(HUS)。HUS以纤维蛋白-血小板血栓为特征。 肾脏等受影响器官的微血管和炎性损伤，导致肾功能衰竭。 在感染的腹泻期全身暴露于内毒素或其他PAMP可增强STX 有害的影响，被认为在发病机制中起重要作用。EHEC不是侵入性的，所以STX必须 穿过肠道上皮进入体循环。然而，免费的STX并不常见 HUS患者，我们缺乏对STX如何以及以什么形式进入血液并最终进入血液的了解 到达肾脏。一种潜在的未被探索的机制是通过摄取含有STX的外膜 膜小泡(OMV)，EHEC来源的微粒，可从肠腔吸收进入 血液中除了STX外，还含有过多的PAMP，如内毒素和鞭毛，以及 其他EHEC编码的毒素。在这个方案中，我们将评估外膜小泡(OMV)如何相互作用 在没有巨噬细胞存在的情况下，肾脏起源的细胞，炎症细胞渗透到 HUS动物模型的肾脏。因为OMV是内吞的，所以内毒素的感应发生在细胞内 而不是通过TLR4，因此我们希望观察到与发生的反应根本不同的反应 当STX和内毒素以外源方式共同传递时。从EHEC突变体中产生OMV的能力 改变的内毒素(或其他PAMP或毒素)提供了识别导致损害的关键特征的机会 细胞反应。此外，还假设HUS发病机制中的一个关键事件发生在 STX与循环血细胞接触，导致含有STX的血栓前微泡的形成 然后使远端器官中毒。在EHEC感染期间，血液成分可能会遇到STX、LPS和 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介导的微囊的产生。我们将比较产生的微泡的数量 从野生型EHEC或EHEC中分离纯化的STX或含STX的OMV对血细胞的暴露 PAMPS或致病因子。与OMV相关的货物，特别是促血栓/促凝血剂分子 将被表征，以及它们对上述肾脏细胞的影响。这些体外研究奠定了 为未来EHEC感染动物模型的体内研究奠定关键基础。一项基本原则 了解宿主如何和以何种形式遇到STX，以及细胞反应是如何 受STX给药方式的影响，可能会为预防HUS提供新的治疗方法。

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.