title: Coxiella burnetii expresses a functional Δ24 sterol reductase.
abstract: Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840, USA.
authors.forename: Stacey D
authors.initials: SD
authors.lastname: Gilk
authors.forename: Paul A
authors.initials: PA
authors.lastname: Beare
authors.forename: Robert A
authors.initials: RA
authors.lastname: Heinzen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis*; D003001:Cloning, Molecular; D016997:Coxiella burnetii / Q000201:enzymology*; D015870:Gene Expression; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009253:Naegleria / Q000201:enzymology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000172:deficiency; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013261:Sterols / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029701:Saccharomyces cerevisiae Proteins; D013261:Sterols; D010088:Oxidoreductases; C022673:delta 24-sterol reductase; C070590:ERG4 protein, S cerevisiae