Browsing by Author "Heinzen, Robert A."

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    Coxiella burnetii Sterol-Modifying Protein Stmp1 Regulates Cholesterol in the Intracellular Niche
    (American Society for Microbiology, 2022) Clemente, Tatiana M.; Ratnayake, Rochelle; Samanta, Dhritiman; Augusto, Leonardo; Beare, Paul A.; Heinzen, Robert A.; Gilk, Stacey D.; Microbiology and Immunology, School of Medicine
    Coxiella burnetii replicates in a phagolysosome-like vacuole called the Coxiella-containing vacuole (CCV). While host cholesterol readily traffics to the CCV, cholesterol accumulation leads to CCV acidification and bacterial death. Thus, bacterial regulation of CCV cholesterol content is essential for Coxiella pathogenesis. Coxiella expresses a sterol-modifying protein, Stmp1, that may function to lower CCV cholesterol through enzymatic modification. Using an Stmp1 knockout (Δstmp1), we determined that Stmp1 is not essential for axenic growth. Inside host cells, however, Δstmp1 mutant bacteria form smaller CCVs which accumulate cholesterol, preferentially fuse with lysosomes, and become more acidic, correlating with a significant growth defect. However, in cholesterol-free cells, Δstmp1 mutant bacteria grow similarly to wild-type bacteria but are hypersensitive to cholesterol supplementation. To better understand the underlying mechanism behind the Δstmp1 mutant phenotype, we performed sterol profiling. Surprisingly, we found that Δstmp1 mutant-infected macrophages accumulated the potent cholesterol homeostasis regulator 25-hydroxycholesterol (25-HC). We next determined whether dysregulated 25-HC alters Coxiella infection by treating wild-type Coxiella-infected cells with 25-HC. Similar to the Δstmp1 mutant phenotype, 25-HC increased CCV proteolytic activity and inhibited bacterial growth. Collectively, these data indicate that Stmp1 alters host cholesterol metabolism and is essential to establish a mature CCV which supports Coxiella growth.
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    Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic
    (American Society for Microbiology, 2017-02-28) Mulye, Minal; Samanta, Dhritiman; Winfree, Seth; Heinzen, Robert A.; Gilk, Stacey D.; Department of Microbiology & Immunology, IU School of Medicine
    Coxiella burnetii is an intracellular bacterial pathogen and a significant cause of culture-negative endocarditis in the United States. Upon infection, the nascent Coxiella phagosome 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 reduced Coxiella growth 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 axenic Coxiella cultures, 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 to Coxiella death.IMPORTANCE The intracellular Gram-negative bacterium Coxiella burnetii is 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 understanding C. burnetii pathogenesis. Upon infection of host cells, C. burnetii grows within a specialized replication niche, the parasitophorous vacuole (PV). Recent data have linked cholesterol to intracellular C. burnetii growth and PV formation, leading us to further decipher the role of cholesterol during C. 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 killed C. burnetii through PV acidification. Our findings suggest that targeting host cholesterol metabolism might prove clinically efficacious in controlling C. burnetii infection.
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    Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L
    (Wiley, 2017-01) Justis, Anna V.; Hansen, Bryan; Beare, Paul A.; King, Kourtney B.; Heinzen, Robert A.; Gilk, Stacey D.; Microbiology and Immunology, School of Medicine
    Coxiella burnetii is a gram-negative intracellular bacterium that forms a large, lysosome-like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol-binding protein-related protein 1 long (ORP1L) is a mammalian lipid-binding protein that plays a dual role in cholesterol-dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N-terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co-localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L-depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.