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

描述（由申请方提供）：嗜肺军团菌（Legionella pneumophila）是军团病的病原体，通过称为Dot/Icm的专门蛋白质易位系统将大量细菌蛋白质转移到宿主细胞中，以构建支持细菌增殖的细胞内区室。早期的研究表明，Dot/Icm蛋白转运蛋白在感染过程中只需要几分钟就可以建立复制空泡。然而，由于技术限制，可以解释这种观察的其他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，这种转运蛋白需要的时间比以前认为的要长得多。例如，在整个感染周期中，一些效应物被转移到被感染的细胞中，并且已经鉴定了在感染的末端从宿主非裂解性释放细菌所需的效应物。在这个建议中，我们计划重新审查的时间要求点/ICM系统的L。通过使用一种新的遗传系统，使我们能够在细菌摄取后的任何时间特异性破坏靶基因，来研究嗜肺菌。我们已经成功地设计了一种基于Cre/loxP的可诱导的体内基因缺失策略，适用于检查特定蛋白质的时间要求。在细菌被宿主细胞摄取后，我们将使用IPTG来诱导对Dot/Icm转运蛋白的功能至关重要的基因的缺失。然后通过转运蛋白将蛋白质底物转运到宿主细胞中的能力来评估转运蛋白的功能，并通过复制空泡的形成以及细菌总数来分析细菌增殖。这些研究应使我们能够更彻底地分析感染过程中病原体对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或蛋白复合物对于维持发育或感染状态是否重要提供技术支持。 公共卫生相关性：嗜肺军团菌感染通常导致致命性肺炎的发展。此外，对该菌的研究也有助于我们对其他胞内病原菌的认识。嗜肺菌和这些感染因子共享许多致病机制。

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.