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

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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.