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The Gilk Lab
Coxiella burnetii causes Q fever endocarditis, a disease that requires 18-24 months of antibiotic treatment and lacks an FDA-approved vaccine. The goal of our research it to understand how Coxiella survives in the host cell, in order to identify new drug targets. During infection, Coxiella infects macrophages and survives in a unique, lysosomal-like compartment called the parasitophorous vacuole (PV). PV formation is essential for bacterial survival, yet we know little about PV biogenesis and maintenance. We are especially interested in how cholesterol and other lipids contribute to PV formation and the unique properties of the PV membrane.
We utilize a combination of cell biology, biochemistry, and molecular biology techniques to address the role of cholesterol in Coxiella-host cell interactions. Using a novel cholesterol-free tissue culture system, we discovered Coxiella does not require cholesterol for PV formation and growth, and is in fact sensitive to high levels of cholesterol. In addition to understanding why cholesterol is toxic to Coxiella, we are determining how Coxiella removes cholesterol from the PV. Our studies suggest that cholesterol metabolism is an integral component of Coxiella pathogenesis, and may be a viable drug target.
Cholesterol and pH of the Coxiella PV
Using live cell imaging to directly measure the pH of individual PVs, we discovered the PV cholesterol levels are linked to PV pH. When cholesterol is trapped in the Coxiella PV membrane, the PV becomes more acidic and and kills the bacteria. We are currently trying to elucidate how cholesterol influences the PV microenvironment, and how Coxiella manipulates PV pH.
Mulye et al, MBio. 2017
Samanta et al, PLOS Pathogens, 2019
PV-ER membrane contact sites
One potential mechanism to lower PV cholesterol levels is bacterial-mediated transfer of cholesterol from the PV to another membrane. We found that Coxiella recruits the host cholesterol binding protein ORP1L to form membrane contact sites between the PV and host endoplasmic reticulum (ER). Ongoing projects are fully characterizing the structure and function of PV-ER membrane contact sites and their role in regulating PV cholesterol.
Justis et al, Cellular Microbiology 2017
Coxiella sterol modifying enzymes
Coxiella expresses two potential sterol-modifying enzymes, CBU1158 and CBU1206. We hypothesize that one or both of these bacterial proteins modify PV cholesterol, thus decreasing PV cholesterol levels. We are characterizing Coxiella sterol reductase mutants, as well as the in vitro activity of these novel bacterial proteins.
Gilk et al, J. Bacteriology 2010
Clemente et al, mBio, 2022
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