PROJECT SUMMARY/ABSTRACT Coxiella burnetii is the causative agent of human Q fever, a zoonotic disease that can cause a debiliting, flu- like illness in acute cases, or a life-threatening endocarditis in chronic patients. Q fever patients present with few distinguishing clinical features, and chronic disease requires a minimum of 18 months of antibiotic treatment, highlighting the need for new therapeutics. An obligate intracellular pathogen, Coxiella survives inside a large, lysosome-like parasitophorous vacuole that is essential for bacterial replication and protects the bacteria from the host immune response. The Coxiella parasitophorous vacuole (CPV) forms through heterotypic fusion with host endosomes and autophagosomes, a process which also delivers cholesterol to the CPV membrane. We recently determined that accumulation of host cholesterol in the CPV is toxic to the bacteria, and hypothesize that Coxiella must deplete CPV cholesterol in order to survive within the host cell. The objective of this application is to test our hypothesis that Coxiella uses two distinct mechanisms to manipulate CPV cholesterol. Aim 1 will test the role of membrane contact sites in cholesterol transfer between the CPV and the host endoplasmic reticulum. Aim 2 will determine the role of putative Coxiella sterol reductases in modifying CPV cholesterol. At the conclusion of these studies, we will have elucidated how Coxiella manipulates host cholesterol to maintain the optimal microenvironment for bacterial surivival.