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细胞增生李斯特菌（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，在土壤中以腐生物形式生存，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一个越来越重要的病原体，并导致了美国历史上一些最大的食品安全召回事件。Lm致病能力的核心是毒力因子穿过细菌膜和刚性细胞壁的调节运输;这一关键过程在革兰氏阳性菌中尚未得到很好的表征。这项建议的重点是分子和机制分析的PrsA 2，易位后分泌伴侣与肽基脯氨酰顺/反异构酶的活性，是必不可少的Lm毒力。缺乏PrsA 2功能的Lm菌株在小鼠感染模型中严重减毒，并且表现出至少两种关键Lm毒力基因产物的分泌和活性降低，所述关键Lm毒力基因产物为胆固醇依赖性溶细胞素β-溶血素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进入宿主细胞胞质溶胶并将细菌传播到新的宿主细胞中的能力。初步实验表明，PrsA 2直接调节LLO稳定性，并且PrsA 2可能是在细菌毒力中具有重要作用的额外Lm因子的分泌和/或活性所需的。该提议中概述的实验将定义PrsA 2在调节用于确保细菌的细胞内复制生态位的Lm分泌蛋白的活性中的作用。目的1探讨PrsA 2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PrsA 2底物与细菌毒力的作用，直接PrsA 2相互作用。实验将进一步确定部分补偿PrsA 2功能的抑制突变的性质。目的3：基于PrsA 2的结构模型，对PrsA 2的功能结构域进行分子分析。具体目标的最终目标是阐明PrsA 2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。

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.