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: 10461587
• 负责人: JOHN-DEMIAN SAUER
• 金额: $ 5.91万
• 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/10461587
• 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 typhimurium; 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.

项目概要： 细胞内病原体是全世界发病和死亡的重要原因。其中一些病原体， 例如单增李斯特氏菌、土拉弗朗西斯氏菌、假鼻伯克霍尔德氏菌、立克次体属。甚至 结核分枝杆菌的感染周期的至少一部分是在宿主细胞的细胞质中度过的。 对于许多此类生物体来说，进入细胞质并在其中生存对于毒力至关重要。尽管 由于这些不同的病原体在这个生态位中生存和繁衍的能力，很明显，细胞质是一种 对于不适应细胞质的病原体来说，环境不适宜。然而，人们对这种细胞知之甚少 细菌在细胞质中遇到的自主防御，以及细胞质的适应性 病原体已经进化来克服这些压力。 单核细胞增生李斯特氏菌是一种重要的兼性细胞内病原体，可引起李斯特菌病。 L。 单核细胞增多症也是了解复杂的宿主-病原体相互作用的强大工具。在这个提案中我们 将利用新型遗传筛选来鉴定单核细胞增生李斯特氏菌在宿主细胞中生存所需的基因 细胞质。本次筛选中鉴定出的突变体将用于了解细菌逃避宿主的策略 防御，确定胞质存活对毒力的重要性，并定义抗菌防御 细胞质中的机制。除了基因筛查之外，我们还将采取有针对性的方法 描述已被确定为细胞质存活和毒力所必需的基因子集的特征。具体来说 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) 影响细胞质存活和毒力。此外，我们将确定 PrkA（一种 高度保守的 PASTA 激酶，作为宿主细胞壁应激反应的主要调节因子 细胞质。作为一个例子，我们将描述 PrkA 如何调节新发现的尿苷基转移酶 高度保守的蛋白质 YvcK 的活性以及这种调节是否在物种间保守。 完成这些目标后，我们将鉴定出单增李斯特菌所需的一系列基因 细胞质存活，并具有 L.L. 必需的两个特定途径（DHNA 和 PrkA）的特征。 单核细胞增多性毒力。这些途径可能代表治疗干预的新靶点。的确， PASTA 激酶已经成为我们实验室和其他实验室开发新型抗生素的焦点，因此， 了解它们在细菌应激反应和毒力中的作用至关重要。