Identification of Listeria monocytogenes immune evasion mechanisms

单增李斯特菌免疫逃避机制的鉴定

• 批准号: 10566979
• 负责人: JOHN-DEMIAN SAUER
• 金额: $ 22.66万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-04 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10566979
• 关键词: Animal Model; Animals; Antibiotic Therapy; Antibiotics; Antimicrobial Resistance; Biological Models; Bone Marrow; Cell Culture Techniques; Cells; Cellular biology; Cessation of life; Combined Modality Therapy; Complex; Development; Disease; Essential Genes; Fertility; Future; Genes; Genetic; Genome; Host Defense; Human; Immune; Immune Evasion; Immune response; Immune signaling; Immune system; In Vitro; Incidence; Infection; Inflammasome; Innate Immune System; Intervention; Invaded; Larva; Libraries; Listeria monocytogenes; Listeriosis; Livestock; Macrophage; Mediating; Microbial Physiology; Modeling; Natural Immunity; Nature; Nutritional Immunity; Organism; Oxidases; Pathogenesis; Pathway interactions; Persons; Phagocytes; Phagosomes; Poverty; Production; Proteins; Public Health; Reactive Oxygen Species; Reporting; Resistance; Resistance development; Signal Pathway; Signal Transduction; Study models; Transgenic Organisms; Validation; Virulence; Virulence Factors; Work; Zebrafish; Zoonoses; antibiotic resistant infections; antimicrobial; antimicrobial peptide; combat; cytokine; emerging antibiotic resistance; experimental study; foodborne pathogen; gene function; genetic manipulation; genome-wide; global health; immune activation; in vivo; infectious disease model; lead candidate; mutant; novel; novel strategies; pathogen; pressure; screening; small molecule inhibitor; synergism; tool; transposon sequencing; virulence gene; whole genome

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.

抽象的： 抗生素耐药性的持续出现导致了全球健康危机，预计将导致 10 到 2050 年，如果不采取重大干预措施，每年将有 100 万人死亡。针对细菌毒力决定因素 新型抗生素而非必需蛋白质将限制耐药性的发展。我们将利用两个强大的 模型系统，斑马鱼模型中的单核细胞增生李斯特菌感染，以识别细菌 毒力因子将作为抗生素开发的新靶标。单核细胞增生利斯特氏菌是一种人类并且 家畜病原体，是李斯特菌病的病原体。也许更重要的是，它也是经过充分研究的， 遗传易处理的模型病原体，几十年来一直被用来剖析细菌的新机制 发病机制、先天免疫激活和细胞生物学。斑马鱼也是一种类似的经过充分研究的模型宿主 具有强大遗传工具、高繁殖力和半透明幼虫的生物体，使它们成为理想的模型 传染病和先天免疫研究。在本提案中，我们将确定乳杆菌中的新抗生素靶标。 使用高通量转座子插入测序 (TIS) 阴性选择方法检测单核细胞增生李斯特菌。在 目标 1 我们将在巨噬细胞培养物中执行单增李斯特菌 TIS 筛选，识别以下基因： 细菌入侵、吞噬体逃逸以及细胞自主生存和复制所需的 方式。在目标 2 中，我们将在完整的斑马鱼幼虫中重复 TIS 筛选，识别感染所需的基因 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显示那些必需基因 用于免疫系统背景下的感染。最后，充分利用遗传易处理性和高 斑马鱼的吞吐量性质，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，我们将在缺乏宿主免疫反应的转基因斑马鱼中进行TIS 吞噬细胞氧化酶复合物或炎性体复合物的成分。完成这些目标后 我们将开发并执行全基因组筛选，以鉴定李斯特菌中的新毒力因子。 单核细胞增多症是细胞内在毒力、体内毒力和选择性毒力所必需的 对抗活性氧和炎症小体激活。未来的研究将扩展这些证据 确定先天免疫其他方面所需的毒力因子的原理实验（抗菌药物 肽、营养免疫、特定细胞因子功能等）以及利用我们筛选中确定的命中 筛选小分子抑制剂作为新型抗生素。