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Characterization of a lanosterol 14 alpha-demethylase from Pneumocystis carinii

Ian J Morales1, Pawan K Vohra, Veenu Puri

  • 1Thoracic Diseases Research Unit, Division of Pulmonary, Critical Care and Internal Medicine, Department of Medicine, Mayo Clinic and Foundation, Rochester, MN 55905, USA.

American Journal of Respiratory Cell and Molecular Biology
|February 28, 2003
PubMed

Insights

Pneumocystis carinii (PC) exhibits inherent azole antifungal resistance due to its lanosterol 14 alpha-demethylase (Erg11) enzyme structure. Genetic modifications to PC Erg11 improved susceptibility to fluconazole and voriconazole.

Area of Science:

  • Medical Mycology
  • Antifungal Drug Resistance
  • Molecular Biology

Background:

  • Pneumocystis carinii (PC) causes severe pneumonia in immunocompromised individuals.
  • PC is intrinsically resistant to azole antifungal medications.
  • The enzyme lanosterol 14 alpha-demethylase (Erg11) is the target for azole antifungals.

Purpose of the Study:

  • To investigate the molecular basis of azole resistance in Pneumocystis carinii.
  • To compare the PC Erg11 enzyme sequence with those from azole-resistant and sensitive organisms.
  • To assess the impact of specific amino acid changes on azole susceptibility.

Main Methods:

  • Cloning and sequencing of the PCERG11 gene.
  • Chromosomal and Northern blot analysis of PCERG11.
  • Site-directed mutagenesis to alter key amino acid residues in PCERG11.
  • Susceptibility testing of engineered yeast strains expressing PCERG11 variants against fluconazole, itraconazole, and voriconazole.

Main Results:

  • PCERG11 showed inherent resistance to voriconazole (2.2-fold higher dose) and fluconazole (3.5-fold higher dose) compared to Saccharomyces cerevisiae Erg11.
  • No significant difference in itraconazole sensitivity was observed.
  • Mutated PCERG11 (PCERG11-SDM) demonstrated increased susceptibility to fluconazole and voriconazole.

Conclusions:

  • The molecular structure of Pneumocystis carinii lanosterol 14 alpha-demethylase confers inherent azole antifungal resistance.
  • Specific amino acid residues in PC Erg11 contribute significantly to this resistance.
  • Understanding these structural elements is crucial for developing effective azole therapies against PC infections.

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