Identification and Characterization of Genes Required for Listeria monocytogenes Cytosolic Survival

单核细胞增生李斯特氏菌胞浆存活所需基因的鉴定和表征

• 批准号: 9790925
• 负责人: JOHN-DEMIAN SAUER
• 金额: $ 54.41万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-24 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9790925
• 关键词: Antibiotics; Attenuated; Bacillus subtilis; Bacteria; Bacterial Genes; Bacteriolysis; Biochemical; Burkholderia; Cell Wall; Cells; Cellular Stress; Cellular biology; Collection; Complex; Cytolysis; Cytosol; Data; Defect; Defense Mechanisms; Dendritic Cells; Development; Disease; Environment; Epithelial Cells; Francisella; Francisella tularensis; Genes; Genetic; Genetic Screening; Growth; Hepatocyte; Host Defense; Immunity; Infection; Inflammasome; Innate Immune Response; Laboratories; Legionella pneumophila; Life; Listeria monocytogenes; Listeriosis; Mediating; Metabolic; Molecular; Morbidity - disease rate; Mycobacterium tuberculosis; Nutrient; Organism; Pathogenesis; Pathway interactions; Phagolysosome; Phenotype; Phosphorylation; Phosphotransferases; Property; Proteins; Regulation; Resistance; Rickettsia; Role; Salmonella enterica; Shigella flexneri; Staphylococcus aureus; Stress; Techniques; Therapeutic Intervention; Transferase; Vacuole; Virulence; antimicrobial; attenuation; base; biological adaptation to stress; cell type; design; extracellular; foodborne pathogen; in vivo; macrophage; metabolomics; mortality; mutant; new therapeutic target; novel; pathogen; small molecule; tool; trafficking; transcription factor; transcriptome sequencing

Project Summary: Intracellular pathogens are a significant cause of morbidity and mortality worldwide. Some of these pathogens, such as Listeria monocytogenes, Francisella tularensis, Burkholderia psuedomallei, Rickettsia spp. and even Mycobacterium tuberculosis, spend at least a portion of their infectious cycle in the cytosol of their host cell. For many of these organisms, access to, and survival in the cytosol, is essential for virulence. Despite the ability of these diverse pathogens to survive and thrive in this niche, it has become clear that the cytosol is an inhospitable environment for non-cytosol adapted pathogens. However, little is known about the cell autonomous defenses that bacteria encounter in the cytosol and in turn the adaptations that cytosolic pathogens have evolved to overcome these stresses. L. monocytogenes is an important facultative intracellular pathogen that causes the disease Listeriosis. L. monocytogenes is also a powerful tool to understand complex host-pathogen interactions. In this proposal we will utilize a novel genetic screen to identify L. monocytogenes genes required for survival in the host cell cytosol. Mutants identified in this screen will be used to understand bacterial strategies for evasion of host defense, to determine the importance of cytosolic survival in virulence, and to define antimicrobial defense mechanisms in the cytosol. In addition to the genetic screen, we will take a targeted approach by characterizing a subset of genes already identified as essential for cytosolic survival and virulence. Specifically we will identify the unknown function of the bacterial small molecule metabolite 1,4-Dihydroxy-2-naphthoate (DHNA) in cytosolic survival and virulence. In addition, we will determine the mechanisms by which PrkA, a highly conserved PASTA kinase, functions as a master regulator of cell wall stress responses in the host cytosol. As one example, we will characterize how PrkA regulates the newly discovered uridyl transferase activity of the highly conserved protein YvcK and whether this regulation is conserved across species. Upon completion of these aims, we will have identified a collection of genes required for L. monocytogenes cytosolic survival and will have characterized two specific pathways (DHNA and PrkA) essential for L. monocytogenes virulence. These pathways may represent novel targets for therapeutic intervention. Indeed, the PASTA kinases are already the focus of novel antibiotic development by our labs and others, and as such, understanding their role in bacterial stress response and virulence is critical.

项目总结： 细胞内病原体是全世界发病率和死亡率的重要原因。其中一些病原体， 如单核细胞增多性李斯特菌、图拉氏弗朗西斯杆菌、猪伪伯克霍尔德氏菌、立克次体等。甚至 结核分枝杆菌至少有一部分感染周期是在宿主细胞的胞浆中度过的。 对于这些生物中的许多生物来说，获得胞质并在胞质中生存对于致病力是必不可少的。尽管 这些不同的病原体在这个利基中生存和茁壮成长的能力，已经变得很明显，胞质是一种 不适宜非胞质适应的病原体的环境。然而，人们对这种细胞知之甚少 细菌在胞质中遇到的自主防御，反过来又是胞质中 病原体已经进化来克服这些压力。 单核细胞增多性乳杆菌是引起李斯特菌病的一种重要的兼性细胞内致病菌。L. 单核细胞增多症也是了解复杂的宿主-病原体相互作用的有力工具。在这份提案中，我们 将利用一种新的遗传筛选来鉴定在宿主细胞中生存所需的单核细胞增多性李斯特菌基因 胞浆。在此筛选中确定的突变体将用于了解细菌逃避宿主的策略 防御，确定胞质存活在毒力中的重要性，并定义抗菌素防御 细胞质中的机制。除了基因筛查外，我们还将采取有针对性的方法，通过 表征已经被识别为胞质生存和毒力所必需的基因的子集。特指 我们将鉴定细菌小分子代谢物1，4-二羟基-2-萘甲酸酯的未知功能 (DHNA)在胞质存活和毒力中。此外，我们将确定PrkA，a，a 高度保守的意大利面激酶，在宿主中作为细胞壁应激反应的主要调节器发挥作用 胞浆。作为一个例子，我们将描述PrkA如何调节新发现的尿酰转移酶 高度保守的蛋白YvcK的活性以及这种调节是否在物种之间保守。 在完成这些目标后，我们将确定单核细胞增多性李斯特菌所需的基因集合。 胞质存活，并将表征两个特定的途径(DHNA和PrkA)是L. 单核细胞增多性毒力。这些通路可能是治疗干预的新靶点。的确， 意大利面激酶已经是我们的实验室和其他实验室开发新型抗生素的重点，因此， 了解它们在细菌应激反应和毒力中的作用是至关重要的。