Post-translation regulation of Listeria monocytogenes virulence factors

描述（由申请人提供）：本研究的长期目标是了解环境细菌单核增生李斯特菌（Lm）如何适应哺乳动物细胞内的生活，成为人类病原体。Lm是一种兼性细胞内细菌，在土壤中作为腐生菌存活，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在严重的食源性感染中扮演着越来越重要的角色，并导致了美国历史上一些规模最大的食品安全召回事件。Lm致病能力的核心是毒力因子在细菌膜和坚硬细胞壁上的受调节运输；这一关键过程尚未在革兰氏阳性菌中得到很好的表征。这一建议侧重于PrsA2的分子和机制分析，PrsA2是一种易位后分泌的伴侣，具有肽基脯氨酸顺式/反式异构酶活性，对Lm毒力至关重要。缺乏PrsA2功能的Lm菌株在小鼠感染模型中严重减毒，并表现出至少两种关键Lm毒力基因产物的分泌和活性降低，这两种基因分别是胆固醇依赖性细胞溶素李斯特菌溶素O （LLO）和广泛特异性磷脂酶PC-PLC。这两种基因产物都有助于Lm进入宿主细胞质并将细菌传播到新的宿主细胞中。初步实验表明，PrsA2直接调控LLO的稳定性，并且可能需要PrsA2来分泌和/或活性其他在细菌毒力中起重要作用的Lm因子。本提案中概述的实验将确定PrsA2在调节用于确保细菌胞内复制生态位的Lm分泌蛋白活性中的作用。Aim 1将确定PrsA2调控LLO和PC-PLC分泌和活性的分子机制。目的2将研究一些潜在的PrsA2底物，这些底物在PrsA2直接相互作用的细菌毒力中起作用。实验将进一步确定部分补偿PrsA2功能的抑制突变的性质。目的3在构建PrsA2结构模型的基础上，对PrsA2功能域进行分子分析。具体目的的最终目标将是阐明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的细菌发病。

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.