Role of ZBP1 in pathogenesis of Salmonella biofilms

项目总结 感染肠道病原体，如沙门氏菌、弯曲杆菌、志贺氏菌或耶尔森氏菌是主要原因 世界范围内的发病率和死亡率。尽管在大多数人中，感染会消失，但大约5%的人 患者随后会发展成一种称为反应性关节炎(REA)的痛苦的慢性炎症性疾病。多么 沙门氏菌感染引发REA的机制尚不清楚。使用鼠伤寒沙门氏菌(STM) 作为一种模式生物，我们发现沙门氏菌蛋白Curli是炎症的主要煽动者 在沙门氏菌感染之后。Curli是一种分泌性蛋白质，也是细菌STM生物膜的主要成分。 胃肠道。卷曲的纤维结合了生物膜内挤出的细菌DNA。就是这些卷曲：DNA复合体， 而不是单独的卷曲，这是I型干扰素、IL-17和抗双链DNA的有效触发因素 自身抗体的产生，导致ReA。然而，尚不清楚为什么卷曲：DNA复合体如此具有煽动性。 我们在这个提案中报告了一个惊人的发现，即在curli：dna复合体中存在的dna不是 纯B-DNA，经典的右撇子(沃森-克里克)双螺旋结构，但包含大量的左撇子 Z-DNA也是如此。Z-型核酸，如Z-DNA和Z-RNA，被认为在自然界中不容易出现， 直到我们去年证明了Z-RNA确实是在病毒感染过程中产生的，并且是 坏死下垂激活宿主传感器蛋白ZBP1。我们的初步结果显示，体内的Z-DNA Curli：DNA复合体激活肠上皮细胞和成纤维细胞中的ZBP1，导致RIPK3- 这些细胞的依赖性坏死性下垂。这些发现允许我们提出这样的假设，即Z-DNA在 Curli：沙门氏菌生物膜中的DNA纤维激活ZBP1引发肠道RIPK3依赖型坏死性下垂 上皮细胞(IECs)和其他细胞类型。反过来，坏死性下垂会导致细胞丢失，破坏肠道屏障的完整性， 释放炎症介质，最终导致自身免疫和REA。因为坏死性上睑下垂是一种高度 细胞死亡的炎性模式，这些发现最终为卷曲的原因提供了一个可信的机制：DNA 复合体是高度炎症的，因此，对于沙门氏菌如何触发ReA。他们还识别Z-DNA 作为一种新的PAMP，将ZBP1--目前为止被认为是一种抗病毒蛋白--作为细菌感染的传感器， 并将RIPK3抑制剂定位为治疗REA的意想不到的新疗法。在目标1中，我们将 确定Z-DNA如何在Curli：DNA复合体中形成，以及Z-DNA如何激活ZBP1。在目标2中，我们将 确定ZBP1驱动的促进凝血的免疫途径：DNA诱导的炎症和ReA，以及 确定ZBP1信号在发病机制中起重要作用的细胞类型。在目标3中，我们将评估 用RIPK3激酶抑制剂阻断坏死性下垂对REA有预防或治疗作用。一个 这些研究的成功结果将勾勒出沙门氏菌引发的一种全新的机制 发炎。他们还将为沙门氏菌诱导的REA开辟令人兴奋的新治疗途径， 对这种目前无法治愈的疾病可能产生改变游戏规则的后果。

PROJECT SUMMARY Infections with enteric pathogens such as Salmonella, Campylobacter, Shigella, or Yersinia are leading causes of morbidity and mortality worldwide. Although in most individuals the infection resolves, approximately 5% of patients subsequently develop a painful chronic inflammatory condition known as Reactive Arthritis (ReA). How Salmonella infections trigger ReA is not well understood. Using Salmonella enterica serovar Typhimurium (STm) as a model organism, we discovered that a Salmonella protein, curli, is a dominant instigator of inflammation following Salmonella infection. Curli is a secreted protein and major component of the STm biofilm in the gastrointestinal tract. Curli fibrils bind extruded bacterial DNA within the biofilm. It is these curli:DNA complexes, rather than curli alone, that are potent triggers of type I interferon, IL-17, and anti-double stranded DNA autoantibody production, leading to ReA. Unknown, however, is why curli:DNA complexes are so inflammatory. We report in this proposal the remarkable discovery that the DNA present within curli:DNA complexes is not solely B-DNA, the classic right-handed (Watson-Crick) double-helix, but includes copious amounts of left-handed Z-DNA as well. Z-form nucleic acids, such as Z-DNA and Z-RNA, were thought not to readily occur in nature, until we showed last year that Z-RNA is indeed produced during virus infections and is a ligand for the necroptosis-activating host sensor protein ZBP1. Our preliminary results now show that the Z-DNA within curli:DNA complexes activates ZBP1 in intestinal epithelial cells (IECs) and fibroblasts, resulting in RIPK3- dependent necroptosis of these cells. These findings allow us to put forward the hypothesis that Z-DNA within curli:DNA fibrils in Salmonella biofilms activates ZBP1 to instigate RIPK3-dependent necroptosis in intestinal epithelial cells (IECs) and other cell types. Necroptosis, in turn, causes cell loss and disrupts gut barrier integrity, releasing inflammatory mediators that eventually result in autoimmunity and ReA. As necroptosis is a highly inflammatory mode of cell death, these findings, at long last, supply a plausible mechanism for why curli:DNA complexes are hyperinflammatory, and, therefore, for how Salmonella triggers ReA. They also identify Z-DNA as a new PAMP, implicate ZBP1 – until now considered an antiviral protein – as a sensor of bacterial infections, and position RIPK3 inhibitors as unanticipated new therapeutics for the treatment of ReA. In aim 1, we will determine how Z-DNA forms within curli:DNA complexes, and how Z-DNA activates ZBP1. In aim 2, we will identify the ZBP1-driven immune pathways that promote curli:DNA induced inflammation and ReA, and determine the cell types in which ZBP1 signaling is important for pathogenesis. In aim 3, we will evaluate whether necroptosis blockade with RIPK3 kinase inhibitors will have preventive or therapeutic benefit in ReA. A successful outcome to these studies will outline an entirely new mechanism of Salmonella-triggered inflammation. They also stand to open up exciting new therapeutic avenues for Salmonella-induced ReA, with potentially game-changing ramifications for this currently incurable disease.