title: Recapitulation in Saccharomyces cerevisiae of cytochrome b mutations conferring resistance to atovaquone in Pneumocystis jiroveci.
abstract: Pneumocystis jiroveci (human-derived P. carinii) is an opportunistic pathogenic fungus which causes pneumonia and is life-threatening in immunocompromised individuals. Spontaneously acquired resistance to atovaquone, a hydroxynaphthoquinone that is used to treat P. jiroveci infections, was linked to mutations in the mitochondrially encoded cytochrome b gene. Because P. jiroveci cannot be easily cultivated, we have developed Saccharomyces cerevisiae as an alternative system to study atovaquone resistance mutations. In this work, we introduced seven mutations linked with atovaquone resistance in P. jiroveci into the S. cerevisiae cytochrome b gene. The effects of the mutations on the respiratory function and on the sensitivity to the inhibitor were then characterized. Six of the reported mutations lowered the sensitivity of the S. cerevisiae bc(1) complex to atovaquone, while one mutation had no effect on the drug resistance. These results were confirmed by monitoring the in vivo resistance of S. cerevisiae mutants which carried both the cytochrome b mutations and a deletion of the ABC transporter genes, allowing the drug to bypass the weakened efflux pump system. S. cerevisiae thus provides an easy-to-use system to characterize in vivo and in vitro cytochrome b mutations reported in pathogens and to assess their role in drug resistance.
authors.affiliation: Wolfson Institute for Biomedical Research, University College London, London, United Kingdom.
authors.forename: Philip
authors.initials: P
authors.lastname: Hill
authors.forename: Jacques
authors.initials: J
authors.lastname: Kessl
authors.forename: Nicholas
authors.initials: N
authors.lastname: Fisher
authors.forename: Steven
authors.initials: S
authors.lastname: Meshnick
authors.forename: Bernard L
authors.initials: BL
authors.lastname: Trumpower
authors.forename: Brigitte
authors.initials: B
authors.lastname: Meunier
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D053626:Atovaquone; D003573:Cytochrome b Group / Q000235:genetics*; D025141:Drug Resistance, Fungal; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics* / Q000502:physiology; D009285:Naphthoquinones / Q000494:pharmacology*; D010101:Oxygen Consumption / Q000187:drug effects; D011010:Pneumocystis / Q000187:drug effects*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D003573:Cytochrome b Group; D009285:Naphthoquinones; D053626:Atovaquone