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.

摘要： 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200万人死亡。靶向细菌毒力决定簇， 新的抗生素而不是必需的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，单核细胞增生李斯特菌感染的斑马鱼模型，以确定细菌 毒力因子将作为抗生素开发的新靶点。L.单核细胞增多症是人类， 李斯特菌病的病原体。也许更重要的是，它也是一个经过充分研究， 遗传上易于处理的模型病原体，几十年来一直用于剖析细菌感染的新机制， 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研究得很好的模式宿主 生物体具有强大的遗传工具，高繁殖力和半透明的幼虫，使它们成为理想的模型， 传染病和先天免疫研究。在这个建议中，我们将确定新的抗生素目标，在L。 使用高通量转座子插入测序（TIS）阴性选择方法对单核细胞增多症进行筛选。在 目标1我们将执行我们的L。在巨噬细胞培养物中进行单核细胞增多症TIS筛选， 细菌入侵、吞噬体逃逸、胞质存活和细胞内自主复制所必需的 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 in vivo.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需基因 在免疫系统中的感染。最后，为了充分利用遗传的易处理性和高 的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 吞噬细胞氧化酶复合物或炎性小体复合物的组分。在完成这些目标后， 我们将开发并执行一个全基因组筛选，以确定新的毒力因子在L。 单核细胞增多症所需的细胞内在毒力，体内毒力和毒力选择性必要的， 对抗活性氧和炎性体活化。未来的研究将扩大这些证据， 确定先天免疫其他方面所需的毒力因子的原理实验（抗菌 肽、营养免疫、特异性细胞因子功能等）以及利用我们筛选中鉴定的命中物 筛选小分子抑制剂作为新型抗生素。