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，它通过一种名为Dot/ICM的特殊蛋白质转位系统将大量细菌蛋白质转移到宿主细胞中，以构建支持细菌繁殖的细胞内隔间。早期的研究表明，Dot/ICM蛋白转运体在感染过程中只需要几分钟就能建立复制空泡。然而，由于技术的限制，可以解释这一观察结果的其他可能性从未被提及。此外，最近对Dot/ICM功能的研究进展有力地表明，这种转运蛋白需要的时间比之前认为的要长得多。例如，在整个感染周期中，一些效应器被转移到受感染的细胞中，并且已经确定了在感染末端从宿主非溶血性释放细菌所需的效应器。在这项提议中，我们计划通过使用一种新的遗传系统来重新检查嗜肺性乳杆菌对Dot/ICM系统的时间要求，该系统允许我们在细菌摄取后的任何时间特异性地干扰靶基因(S)。我们已经成功地设计了一种基于Cre/loxP的体内可诱导基因缺失策略，适用于检测特定蛋白质的时间需求(S)。在细菌被宿主细胞摄取后，我们将使用IPTG来诱导对点/细胞间黏附分子转运蛋白功能至关重要的基因(S)的缺失。转运蛋白的功能将通过其将蛋白质底物转移到宿主细胞的能力来评估，细菌增殖将通过复制空泡的形成以及细菌总数来分析。这些研究应该使我们能够更彻底地分析病原体在感染过程中对Dot/ICM致病机制的时间需求。此外，所描述的遗传结构应该为研究特定的细菌蛋白质或蛋白质复合体对维持发育或感染状态是否重要提供技术支持。 与公共卫生相关：由嗜肺军团菌感染往往会导致一种致命的肺炎。此外，对这种细菌的研究可以为我们理解其他细胞内病原体提供帮助，因为嗜肺乳杆菌和这些病原体具有许多共同的致病机制。

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.