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

Research output: Contribution to journalArticle

23 Citations (Scopus)

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

Fingerprint

Lanosterol
Pneumocystis carinii
Azoles
Itraconazole
Fluconazole
Candida
Mutagenesis
Site-Directed Mutagenesis
Molecular Structure
Northern Blotting
Yeast
Molecular structure
Saccharomyces cerevisiae
Pneumonia

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Pulmonary and Respiratory Medicine

Cite this

Characterization of a lanosterol 14α-demethylase from Pneumocystis carinii. / Morales, Ian J.; Vohra, Pawan K.; Puri, Veenu; Kottom, Theodore J.; Limper, Andrew Harold; Thomas, Charles F.

In: American Journal of Respiratory Cell and Molecular Biology, Vol. 29, No. 2, 01.08.2003, p. 232-238.

Research output: Contribution to journalArticle

Morales, Ian J. ; Vohra, Pawan K. ; Puri, Veenu ; Kottom, Theodore J. ; Limper, Andrew Harold ; Thomas, Charles F. / Characterization of a lanosterol 14α-demethylase from Pneumocystis carinii. In: American Journal of Respiratory Cell and Molecular Biology. 2003 ; Vol. 29, No. 2. pp. 232-238.
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