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Characterization of a Lanosterol 14-Demethylase from Pneumocystis carinii

Thoracic Diseases Research Unit, Division of Pulmonary, Critical Care and Internal Medicine, Department of Medicine; and the Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota

Address correspondence to: Charles F. Thomas, Jr., Thoracic Diseases Research Unit, 826 Stabile Building, Mayo Clinic and Foundation, Rochester, MN 55905. E-mail: thomas.charles{at}mayo.edu

Pneumocystis carinii (PC) causes severe pneumonia in immunocompromised patients. PC is intrinsically resistant to treatment with azole antifungal medications. The enzyme lanosterol 14-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-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.

Abbreviations: enzyme lanosterol 14-demethylase, Erg11 • Pneumocystis carinii, PC • P. carinii pneumonia, PCP • polymerase chain reaction, PCR • Saccharomyces cerevisiae, SC • substrate recognition site-1, SRS-1

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