Identification of Listeria monocytogenes immune evasion mechanisms

摘要： 抗生素耐药性的持续出现导致了一场全球健康危机，预计将导致10 到2050年，在没有重大干预的情况下，每年有100万人死亡。以细菌毒力决定因素为靶点 新的抗生素而不是必要的蛋白质会限制耐药性的发展。我们将利用两个强大的 模型系统，单核细胞增多性李斯特菌在斑马鱼(Danio Rerio)模型中的感染，以鉴定细菌 毒力因子将成为抗生素开发的新靶点。单核细胞增多性李斯特菌是一种人类 家畜病原体是李斯特氏病的病原体。也许更重要的是，它也是一个研究得很好的， 几十年来一直被用来剖析细菌新机制的遗传可追踪的模型病原体 发病机制、先天免疫激活和细胞生物学。斑马鱼也是同样被研究得很好的模型宿主。 具有强大的遗传工具、高繁殖力和半透明幼虫的生物体，这使它们成为 传染病和先天免疫研究。在这项提案中，我们将在L. 使用高通量转座子插入测序(TIS)阴性选择方法的单核细胞增多症。在……里面 目的1我们将在巨噬细胞培养中进行单核细胞增多性李斯特氏菌的TIS筛查，鉴定哪些基因是 细菌入侵、吞噬小体逃逸、胞质存活和在细胞内自主复制所必需的 举止。在目标2中，我们将在完整的斑马鱼幼体中重复TIS筛选，识别感染所需的基因 在活体内。将细胞培养必需基因与体内鉴定的基因进行比较，将突出那些必要的基因 在免疫系统的背景下进行感染。最后，要充分挖掘基因的可驯性和高效性 斑马鱼的吞吐特性，并确定防御所需的细菌毒力决定因素 宿主免疫反应的特定成分，我们将在转基因斑马鱼中进行TIS 吞噬细胞氧化酶复合体或炎症体复合体的成分。在完成这些目标后 我们将开发并执行一项全基因组筛选，以鉴定L. 细胞内在毒力、体内毒力所需的单核细胞增多性，以及选择性地需要的 对抗活性氧和炎症小体的激活。未来的研究将在这些证据的基础上展开 确定先天免疫其他方面所需毒力因素的原理实验(抗菌药物 多肽、营养免疫、特定细胞因子功能等)以及利用我们屏幕中识别的点击 筛选作为新型抗生素的小分子抑制剂。

Abstract: The continued emergence of antibiotic resistance has led to a global health crisis, leading to a predicted 10 million deaths per year by 2050 without significant intervention. Targeting bacterial virulence determinants with novel antibiotics rather than essential proteins would limit resistance development. We will utilize two powerful model systems, Listeria monocytogenes infection in the Danio rerio (zebrafish) model, to identify bacterial virulence factors that will serve as novel targets for antibiotic development. L. monocytogenes is a human and livestock pathogen that is the causative agent of Listeriosis. Perhaps more importantly, it is also a well-studied, genetically tractable model pathogen that has been used for decades to dissect novel mechanisms of bacterial pathogenesis, innate immune activation and cell biology. The zebrafish is a similarly well studied model host organism with robust genetic tools, high fecundity and translucent larvae that make them ideal models for infectious disease and innate immunity studies. In this proposal we will identify novel antibiotic targets in L. monocytogenes using a high-throughput transposon insertion sequencing (TIS) negative selection approach. In aim 1 we will execute our L. monocytogenes TIS screen in macrophage cell culture, identifying genes that are required for bacterial invasion, phagosomal escape, and cytosolic survival and replication in a cell autonomous manner. In aim 2 we will repeat the TIS screen in intact zebrafish larvae, identifying genes required for infection in vivo. Comparing cell culture essential genes with those identified in vivo will highlight those genes necessary for infection in the context of an immune system. Finally, to fully exploit the genetic tractability and high throughput nature of the zebrafish, and to identify bacterial virulence determinants required for defense against specific components of the host immune response, we will perform TIS in transgenic zebrafish deficient in components of the phagocyte oxidase complex or the inflammasome complex. Upon completion of these aims we will have developed and executed a whole genome screen to identify novel virulence factors in L. monocytogenes required for cell intrinsic virulence, in vivo virulence, and virulence selectively necessary to combat reactive oxygen species and inflammasome activation. Future studies will expand on these proof of principle experiments to identify virulence factors required for other aspects of innate immunity (antimicrobial peptides, nutritional immunity, specific cytokine functions, etc) as well as utilizing the hits identified in our screens to screen for small molecule inhibitors as novel antibiotics.