title: Characterization of a lanosterol 14 alpha-demethylase from Pneumocystis carinii.
abstract: Pneumocystis carinii (PC) causes severe pneumonia in immunocompromised patients. PC is intrinsically resistant to treatment with azole antifungal medications. The enzyme lanosterol 14 alpha-demethylase (Erg11) is the target for azole antifungals. We cloned PCERG11 and compared its sequence to Erg11 proteins present in azole-resistant organisms, and performed chromosomal and Northern blot analysis for PCERG11. Of 13 potential sites which could confer resistance to azoles, two were identical to azole-resistant Candida. By site-directed mutagenesis we changed these two sites in PCERG11 to those present in azole-sensitive Candida to generate PCERG11-SDM (E113D, T125K). We tested the susceptibility of ERG11 deletion strains of Saccharomyces cerevisiae (SC) expressing PCERG11, PCERG11-SDM, and wild-type SCERG11 to three azole antifungals: fluconazole, itraconazole, and voriconazole. PCERG11 required a 2.2-fold higher dose of voriconazole and 3.5-fold higher dose of fluconazole than SCERG11 for a 50% reduction in growth. No difference was observed in the sensitivity to itraconazole. PCERG11-SDM has increased sensitivity to fluconazole and voriconazole, but not itraconazole. We believe that the molecular structure of the lanosterol 14 alpha-demethylase encoded by PCERG11 confers inherent resistance to azole antifungals and plays an integral part in the overall resistance of this PC to azole therapy.
authors.affiliation: Thoracic Diseases Research Unit, Division of Pulmonary, Critical Care and Internal Medicine, Department of Medicine, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Morales
authors.forename: Pawan K
authors.initials: PK
authors.lastname: Vohra
authors.forename: Veenu
authors.initials: V
authors.lastname: Puri
authors.forename: Theodore J
authors.initials: TJ
authors.lastname: Kottom
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Limper
authors.forename: Charles F
authors.initials: CF
authors.lastname: Thomas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001483:Base Sequence; D015152:Blotting, Northern; D002175:Candida / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015725:Fluconazole / Q000494:pharmacology; D017353:Gene Deletion; D015151:Immunoblotting; D017964:Itraconazole / Q000494:pharmacology; D008168:Lung / Q000382:microbiology / Q000473:pathology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics*; D011010:Pneumocystis / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D058886:Sterol 14-Demethylase; D014230:Triazoles / Q000494:pharmacology; D065819:Voriconazole
chemical_list: D000935:Antifungal Agents; D018076:DNA, Complementary; D011743:Pyrimidines; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D011994:Recombinant Proteins; D014230:Triazoles; D017964:Itraconazole; D015725:Fluconazole; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D065819:Voriconazole