title: Elevated Cholesterol in theCoxiella burnetiiIntracellular Niche Is Bacteriolytic.
abstract: Coxiella burnetiiis an intracellular bacterial pathogen and a significant cause of culture-negative endocarditis in the United States. Upon infection, the nascentCoxiellaphagosome fuses with the host endocytic pathway to form a large lysosome-like vacuole called the parasitophorous vacuole (PV). The PV membrane is rich in sterols, and drugs perturbing host cell cholesterol homeostasis inhibit PV formation and bacterial growth. Using cholesterol supplementation of a cholesterol-free cell model system, we found smaller PVs and reducedCoxiellagrowth as cellular cholesterol concentration increased. Further, we observed in cells with cholesterol a significant number of nonfusogenic PVs that contained degraded bacteria, a phenotype not observed in cholesterol-free cells. Cholesterol had no effect on axenicCoxiellacultures, indicating that only intracellular bacteria are sensitive to cholesterol. Live-cell microscopy revealed that both plasma membrane-derived cholesterol and the exogenous cholesterol carrier protein low-density lipoprotein (LDL) traffic to the PV. To test the possibility that increasing PV cholesterol levels affects bacterial survival, infected cells were treated with U18666A, a drug that traps cholesterol in lysosomes and PVs. U18666A treatment led to PVs containing degraded bacteria and a significant loss in bacterial viability. The PV pH was significantly more acidic in cells with cholesterol or cells treated with U18666A, and the vacuolar ATPase inhibitor bafilomycin blocked cholesterol-induced PV acidification and bacterial death. Additionally, treatment of infected HeLa cells with several FDA-approved cholesterol-altering drugs led to a loss of bacterial viability, a phenotype also rescued by bafilomycin. Collectively, these data suggest that increasing PV cholesterol further acidifies the PV, leading toCoxielladeath.IMPORTANCEThe intracellular Gram-negative bacteriumCoxiella burnetiiis a significant cause of culture-negative infectious endocarditis, which can be fatal if untreated. The existing treatment strategy requires prolonged antibiotic treatment, with a 10-year mortality rate of 19% in treated patients. Therefore, new clinical therapies are needed and can be achieved by better understandingC. burnetiipathogenesis. Upon infection of host cells,C. burnetiigrows within a specialized replication niche, the parasitophorous vacuole (PV). Recent data have linked cholesterol to intracellularC. burnetiigrowth and PV formation, leading us to further decipher the role of cholesterol duringC. burnetii-host interaction. We observed that increasing PV cholesterol concentration leads to increased acidification of the PV and bacterial death. Further, treatment with FDA-approved drugs that alter host cholesterol homeostasis also killedC. burnetiithrough PV acidification. Our findings suggest that targeting host cholesterol metabolism might prove clinically efficacious in controllingC. burnetiiinfection.
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Minal
authors.initials: M
authors.lastname: Mulye
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Dhritiman
authors.initials: D
authors.lastname: Samanta
authors.affiliation: Department of Medicine, Indiana Center for Biological Microscopy, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Seth
authors.initials: S
authors.lastname: Winfree
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Heinzen
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA sgilk@iupui.edu.
authors.forename: Stacey D
authors.initials: SD
authors.lastname: Gilk
mesh_terms: D001433:Bacteriolysis / Q000187:drug effects*; D002784:Cholesterol / Q000378:metabolism*; D016997:Coxiella burnetii / Q000187:drug effects* / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D014617:Vacuoles / Q000737:chemistry* / Q000382:microbiology*
chemical_list: D002784:Cholesterol