Post-translation regulation of Listeria monocytogenes virulence factors

描述(申请人提供)：这项研究的长期目标是了解环境细菌单核细胞增生性李斯特氏菌(Listeria monuctogen，LM)是如何适应哺乳动物细胞内的生活成为人类病原体的。LM是一种兼性胞内细菌，在土壤中以腐生植物的形式存活，但在进入哺乳动物宿主后能够转变为病原体。作为一种病原体，LM仍然是严重食源性感染的日益重要的病原体，并对美国历史上一些最大的食品安全召回事件负有责任。金黄色葡萄球菌致病能力的核心是毒力因子在细菌膜和坚硬的细胞壁上的调节运输；这一关键过程在革兰氏阳性细菌中尚未得到很好的表征。这项建议侧重于PrsA2的分子和机制分析，PrsA2是一种易位后分泌伴侣，具有肽基-脯氨基顺式/反式异构酶活性，对LM毒力至关重要。在小鼠感染模型中，缺乏PrsA2功能的LM菌株严重减毒，并表现出至少两个关键的LM毒力基因产物--胆固醇依赖细胞溶血素LLO(LLO)和广谱特异性磷脂酶PC-PLC的分泌和活性降低。这两种基因产物都有助于LM进入宿主细胞胞浆，并将细菌传播到新的宿主细胞。初步实验表明，PrsA2直接调节LLO的稳定性，可能需要PrsA2来分泌和/或活性在细菌毒力中起重要作用的其他LM因子。该提案中概述的实验将定义PrsA2在调节LM分泌蛋白活性方面的作用，该蛋白用于确保细菌的细胞内复制生态位。目的1确定PrsA2调节LLO和PC-PLC分泌和活性的分子机制。目标2将研究一些潜在的PrsA2底物，它们在细菌毒力中直接与PrsA2相互作用。实验将进一步确定部分补偿PrsA2功能的抑制子突变的性质。目的3在PrsA2结构模型的基础上，对PrsA2功能结构域进行分子分析。这些特定目标的最终目的将是阐明PrsA2如何调节毒力因子的分泌和/或活性，以促进哺乳动物宿主中的细菌致病。 与公共卫生相关：单核细胞增多性李斯特菌是一个日益严重的健康威胁，因为它与过去几年中多个州通过食物传播的疫情有关，这些疫情导致数千人患病和数百人死亡。由于单核细胞增多性李氏杆菌的污染，历史上规模最大、代价最高的食品召回事件仍在发生，这种细菌通常在北美细菌性脑膜炎的最常见原因中排名第三或第四。这项建议将阐明单核细胞增多性李斯特氏菌如何调节对细菌毒力至关重要的分泌因子的活性；这一信息可能导致识别新的革兰氏阳性细菌药物靶点，并促进基于单核细胞增多性李斯特氏菌的疫苗的开发，以运送外来抗原。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand how the environmental bacterium Listeria monocytogenes (Lm) adapts to life within mammalian cells to become a human pathogen. Lm is a facultative intracellular bacterium that survives as a saprophyte in soil but is capable of transitioning into a pathogen upon entry into a mammalian host. As a pathogen, Lm remains an increasingly important agent of serious food- borne infections and has been responsible for some of the largest food safety recalls in U.S. history. Central to Lm's ability to cause disease is the regulated transport of virulence factors across the bacterial membrane and the rigid cell wall; this critical process has not been well characterized in Gram-positive bacteria. This proposal focuses on molecular and mechanistic analyses of PrsA2, a post-translocation secretion chaperone with peptidyl-prolyl cis/trans isomerase activity that is essential for Lm virulence. Lm strains lacking PrsA2 function are severely attenuated in mouse models of infection and exhibit reduced secretion and activity of at least two key Lm virulence gene products, the cholesterol-dependent cytolysin listeriolysin O (LLO) and the broad specificity phospholipase PC-PLC. Both of these gene products contribute to Lm's ability to gain access to the host cell cytosol and to spread of the bacteria into new host cells. Preliminary experiments indicate that PrsA2 directly regulates LLO stability, and that PrsA2 may be required for the secretion and/or activity of additional Lm factors with important roles in bacterial virulence. The experiments outlined within this proposal will define the role of PrsA2 in regulating the activity of Lm secreted proteins used to secure the bacterium's intracellular replication niche. Aim 1 will determine the molecular mechanism by which PrsA2 regulates the secretion and activity of LLO and PC-PLC. Aim 2 will examine a number of potential PrsA2 substrates with roles in bacterial virulence for direct PrsA2 interactions. Experiments will further determine the nature of a suppressor mutation that partially compensates for PrsA2 function. Aim 3 experiments focus on the molecular analysis of PrsA2 functional domains based on structural modeling of PrsA2. The ultimate goal of the specific aims will be to elucidate how PrsA2 modulates the secretion and/or activity of virulence factors to promote bacterial pathogenesis in mammalian hosts. PUBLIC HEALTH RELEVANCE: Listeria monocytogenes represents an increasingly significant health threat as it has been associated with several multi-state food-borne outbreaks that have resulted in thousands of illnesses and several hundred deaths within the past few years. The largest and most expensive (> $140 million) recalls of food products in history continue to occur as a result of L. monocytogenes contamination, and the bacterium generally ranks as the third or fourth most common cause of bacterial meningitis in North America. This proposal will elucidate how L. monocytogenes regulates the activity of secreted factors critical for bacterial virulence; this Information may result in the identification of new Gram-positive bacterial drug targets as well as enhance the development of L. monocytogenes-based vaccines for delivery of foreign antigens.