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

志贺毒素介导的导致全身性疾病的炎症和血栓反应中的外膜囊泡

基本信息

批准号：
10668016
负责人：
Sivapriya Kailasan Vanaja
金额：
$ 22.26万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-05 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10668016
关键词：
Affect Animal Experiments Animal Model Animals Antibiotics Antibody Therapy Apoptosis Bacteriophages Binding Blood Blood Cells Blood Circulation Blood Platelets Cell Communication Cell model Cells Childhood Circulation Coculture Techniques Colon Complement Cytosol Data Development Diarrhea Disease Distal Electron Microscopy Endoplasmic Reticulum Epithelium Erythrocytes Escherichia coli EHEC Escherichia coli Infections Escherichia coli O157:H7 Event Exposure to Fibrin Flagellin Flow Cytometry Future Generations Glycolipids Goals Hemolytic Anemia Hemolytic-Uremic Syndrome Hemorrhagic colitis Human Image In Vitro Infection Infiltration Inflammasome Inflammatory Ingestion Injury Intestinal Absorption Intestines Intoxication Kidney Kidney Diseases Kidney Failure Knowledge Life MAP Kinase Kinase Kinase Macrophage Mammalian Cell Measures Mediating Mediator Membrane Modality Modeling Molecular Mus Organ Oryctolagus cuniculus Pathogenesis Patients Pattern Phase Predisposition Production Protein Synthesis Inhibitors Publishing Renal Tissue Ribosomes Role Serotyping Shiga Toxin Supportive care Systemic disease TLR4 gene Thrombocytopenia Thrombus Toxin Tubular formation Uremia Vascular Endothelium Vesicle Virulence Factors Western Blotting Work beef biological adaptation to stress cell injury cell type cytokine cytotoxicity diarrheal disease druggable target efficacy testing endoplasmic reticulum stress extracellular vesicles foodborne globotriaosylceramide glomerular endothelium gut colonization gut inflammation holotoxins in vivo intestinal epithelium intraperitoneal kidney cell microvesicles monocyte mouse model mutant nanoparticle neural neutrophil novel therapeutics particle pathogen pharmacologic podocyte prevent renal damage response synergism thrombotic transmission process uptake

项目摘要

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.

Enterohehemorrhagic E. coli（EHEC）血清型O157：H7是一种通常通过受污染的牛肉或农产品，导致血腥腹泻。 EHEC产生的Shiga毒素（STX），一种蛋白质合成抑制剂，当从肠中吸收到血液中时，可能导致毁灭性疾病，溶血性尿毒症综合征（HUS）。 HUS的特征是在受影响器官（例如肾脏）的微举行和炎症损伤，导致肾衰竭。在感染的腹泻阶段，全身暴露于LPS或其他大型疾病可以潜在的STX 有害作用，被认为在发病机理中很重要。 EHEC不是侵入性的，因此STX必须穿过肠上皮进入系统循环。但是，在患有HUS的患者，我们对STX如何以及以哪种形式进入血液，最终缺乏了解到达肾脏。一种潜在的探索不足的机制是通过含有STX的外部的摄取膜蔬菜（OMV），EHEC衍生的微粒，可以从肠腔吸收到除了STX外，还包含了很多弹药，例如LPS和Flagellae，以及其他EHEC编码的毒素。在此提案中，我们将评估外膜蔬菜（OMV）的相互作用在不存在巨噬细胞的情况下，具有肾脏来源的细胞，浸润的炎性细胞肾脏中的动物模型。因为OMV是内吞的，所以LPS感应是细胞内发生的比通过TLR4，因此我们希望观察到与发生的响应根本不同当STX和LP被外源交付时。从EHEC突变体中生成OMV的能力更改的LP（或其他PAMP或毒素）提供了识别关键功能的机会细胞反应。此外，已经假设在HUS发病机理中发生关键事件 STX参与循环的血细胞，导致形成含STX的促血栓形成微囊泡然后将远端器官插入。在EHEC感染期间，血液成分可能会遇到STX，LP和在OMV的背景下关于OMV介导的微泡产生。我们将比较在从野生型EHEC或EHEC中暴露于纯化的STX或含STX的OMV或EHEC缺乏的纯化OMV 弹药或病毒因素。 OMV相关的货物，尤其是促血栓形成/促凝分子将被表征，以及它们对上述肾细胞的影响。这些体外研究是 EHEC感染动物模型中未来体内研究的关键基础。基本了解宿主遇到的如何以及以哪种形式遇到的形式以及蜂窝响应的方式受STX交付方式的影响，可以告知新的治疗方式以防止HUS。