Characterization of a lanosterol 14α-demethylase from Pneumocystis carinii

Ian J. Morales, Pawan K. Vohra, Veenu Puri, Theodore J. Kottom, Andrew Harold Limper, Charles F. Thomas

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23 Scopus citations

Abstract

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

Original languageEnglish (US)
Pages (from-to)232-238
Number of pages7
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume29
Issue number2
DOIs
StatePublished - Aug 1 2003

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ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Pulmonary and Respiratory Medicine

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