An in vivo Gene Deletion System for Analyzing Temporal Requirement of the Dot/Icm

描述（由申请人提供）：嗜肺军团菌是军团病的病原体，通过称为Dot/Icm的特殊蛋白质转运系统将大量细菌蛋白转移到宿主细胞中，以构建支持细菌增殖的细胞内隔室。早期的研究表明，在感染过程中，Dot/Icm蛋白转运体只需要几分钟就可以建立复制液泡。然而，由于技术上的限制，其他能够解释这一观察结果的可能性从未得到解决。此外，Dot/Icm功能研究的最新进展强烈表明，这种转运体所需的时间比以前认为的要长得多。例如，在整个感染周期中，一些效应器被转移到被感染的细胞中，并且在感染的末端，已经确定了从宿主非裂解性释放细菌所需的效应器。在本提案中，我们计划通过使用一种新的遗传系统来重新检查嗜肺乳杆菌对Dot/Icm系统的时间要求，该遗传系统允许我们在细菌摄取后的任何时间特异性地破坏目标基因。我们成功设计了一种基于Cre/ loxp的体内诱导基因缺失策略，适用于检测特定蛋白的时间需求。在宿主细胞被细菌摄取后，我们将使用IPTG诱导Dot/Icm转运体功能所需基因的缺失。转运体的功能将通过其将蛋白质底物转运到宿主细胞中的能力来评估，细菌增殖将通过形成复制液泡以及细菌总数来分析。这些研究将使我们能够更彻底地分析病原体在感染过程中Dot/Icm致病机制的时间要求。此外，所描述的遗传设置应该为研究特定细菌蛋白质或蛋白质复合物对维持发育或感染状态是否重要提供技术支持。

DESCRIPTION (provided by applicant): Legionella pneumophila, the causative agent of Legionnaires' disease transfers a large number of bacterial proteins into host cells via a specialized protein translocation system termed Dot/Icm to construct an intracellular compartment that supports bacterial multiplication. Earlier studies suggest that the Dot/Icm protein transporter is required only for minutes during infection for the establishment the replicative vacuole. However, due to technique limitation other possibilities that can account for this observation have never been addressed. Moreover, recent progress in studies of the function of Dot/Icm strongly suggests that this transporter is required considerably longer than previously thought. For example, some effectors are transferred into the infected cells throughout the infection cycle and effectors required for non-lytic release of the bacteria from the host at the terminal end of the infection have been identified. In this proposal, we plan to reexamine the temporal requirement of the Dot/Icm system by L. pneumophila by using a novel genetic system that allows us to specifically disrupt target gene(s) as any time after bacterial uptake. We have successfully designed a Cre/loxP-based inducible in vivo gene deletion strategy suitable for examining the temporal requirement of specific protein(s). Following bacterial uptake by host cells, we will use IPTG to induce the deletion of gene(s) essential for the function of the Dot/Icm transporter. Function of the transporter then will be assessed by its ability to translocate protein substrates into host cells and bacterial multiplication will be analyzed by the formation of replicative vacuoles as well as by total bacterial counts. These studies should allow us to more thoroughly analyze the temporal requirement of the Dot/Icm pathogenic machinery by the pathogen during infection. Furthermore, the described genetic setup should provide technique support for study whether a specific bacterial protein or protein complex is important for maintaining a development or infection status. PUBLIC HEALTH RELEVANCE: Infection by Legionella pneumophila often leads to the development of a fatal form of pneumonia. In addition, study on this bacterium could share light on our understanding of other intracellular pathogens because L. pneumophila and these infectious agents share many pathogenic mechanisms.