Investigating the cytoplasmic domain architecture of the Legionella pneumophila type IV secretion system using sub-tomogram averaging
EMSL Project ID
50910
Abstract
In fresh water environment, Legionella pneumophila relies on its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into its protozoan host. This process is essential for its survival and replication inside the host. Once inside the lung, L. pneumophila utilizes the same Dot/Icm T4SS molecular machine to infect and modify human macrophages, causing Legionnaires’ disease. The Dot/Icm T4SS is powered by three conserved ATPases (DotO, DotB and DotL). However, there is currently controversy on how the three ATPases organize under the inner membrane and what their functional oligomeric states are. The aim of our proposed project is to analyze the in situ architecture and organization of the cytoplasmic ATPases and dissect the function of each of the ATPases in substrate translocation using electron cryotomography and subtomogram averaging. Our project will carefully examine the cytoplasmic densities of 1) wild-type T4SS, 2) ATPase genomic deletion strains, and 3) ATPase genomic fusion strains. We will compare their sub-tomogram averages at nanometer resolution range. The aim is to provide a clear picture of where each of the cytoplasmic ATPases locate under the inner membrane and describe their biological functions. We expect the results from our project to clarify the existing controversy on the architecture and organization of the cytoplasmic ATPase-complex and serve as the cornerstones to understand the effector translocation mechanism. Since T4SS-defective L. pneumophila is degraded by the lysosome and cannot replicate inside macrophages, such information is valuable for developing L. pneumophila specific drugs to treat Legionnaires’ disease. Currently we plan to collect tomograms for three to four mutant strains. In order to reach resolutions in the nanometer range in our STA, which is the preferable for clear domain assignment, we aim to obtain ~150 tomograms per strain. This corresponds to three days of data collection using established SerialEM protocols. At the moment we have very limited access to the Titan Krios at our home institute due to instrument upgrade and increasing demand from other users. Access to the PNCC facility is critical for obtaining urgently needed data.
Project Details
Start Date
2019-05-15
End Date
2019-12-16
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members