Coxiella secreted proteins mediating inter-organelle membrane contact sites

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PROJECT SUMMARY/ABSTRACT Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Inside the host cell, the bacterium survives in a phagolysosome-like vacuole called the Coxiella Containing Vacuole (CCV). Coxiella is uniquely sensitive to host cholesterol levels, where elevated cholesterol, especially in the CCV and endolysosomal system, leads to bacterial death. While Coxiella directly regulates host cholesterol homeostasis through effector proteins secreted by the Coxiella Type 4B Secretory System (T4BSS), the T4BSS effector proteins responsible for manipulating host cell cholesterol have not yet been identified. Using a bioinformatic approach, we identified a new family of Coxiella T4BSS effector proteins which contain a eukaryotic FFAT motif. In eukaryotic cells, FFAT proteins bind to the VAP protein family on the endoplasmic reticulum (ER) and mediate membrane contact sites (MCS) between cell organelles and ER. These inter-organelle MCS play a critical role in regulating lipid homeostasis. The proposed experiments will test our hypothesis that Coxiella FFAT-containing T4BSS effector proteins mediate MCS between the ER and other host organelles in order to manipulate host cholesterol metabolism. Aim 1 will establish which members of the Coxiella FFAT protein family bind to VAP proteins, and determine where these proteins localize in the host cell. Aim 2 will test whether these proteins are essential for Coxiella infection, as well as whether they function in modulating host cholesterol. Completion of these studies will not only reveal a new strategy utilized by Coxiella to modulate host lipid metabolism, but significantly add to our knowledge on how pathogen-secreted proteins manipulate the host cell.