The amino acid substitution N136Y in Candida albicans sterol 14alpha-demethylase is involved in fluconazole resistance

Med Mycol. 2016 Oct 1;54(7):764-775. doi: 10.1093/mmy/myw023. Epub 2016 May 3.

Abstract

Resistance to fluconazole antifungal is an ongoing impediment to a successful treatment of Candida albicans infections. One of the most prevalent mechanisms leading to azole resistance is genetic alterations of the 14α-demethylase, the target of azole antifungals, through point mutations. Site-directed mutagenesis and molecular modeling of 14α-demethylase rationalize biological data about the role of protein substitutions in the azole treatment failure. In this work, we investigated the role of N136Y substitution by site-directed mutagenesis into Pichia pastoris guided by structural analysis. Single amino acid substitutions were created by site-directed mutagenesis into P. pastoris with C. albicans ERG11 gene as template. In vitro susceptibility of P. pastoris transformants expressing wild-type and mutants to azole compounds was determined by CLSI M27-A2 and spot agar methods. The fluconazole effect on ergosterol biosynthesis was analyzed by gas chromatography-mass spectrometry. By microdilution and spot tests, N136Y transformants showed a reduced in vitro susceptibility to fluconazole compared to wild-type controls. As expected, ergosterol/lanosterol ratios were higher in N136Y transformants compared to the wild-type controls after treatment with fluconazole. Molecular modeling suggests that residue Asn136 located within the first mutation hot spot, could play a role during heme and azole binding. These results provide new insights into the structural basis for 14α-demethylase-azole interaction and could guide the design of novel azole antifungals.

Keywords: 14alpha-demethylase; 14alpha-demethylase homology model; CaCYP51; CaErg11p; Candida albicans; Fluconazole resistance.

MeSH terms

  • 14-alpha Demethylase Inhibitors / pharmacology
  • Amino Acid Substitution*
  • Antifungal Agents / pharmacology*
  • Binding Sites
  • Candida albicans / drug effects*
  • Candida albicans / enzymology*
  • Drug Resistance, Fungal*
  • Ergosterol / biosynthesis
  • Fluconazole / pharmacology*
  • Gas Chromatography-Mass Spectrometry
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Mutation, Missense
  • Pichia / enzymology
  • Pichia / genetics
  • Protein Conformation
  • Sterol 14-Demethylase / chemistry
  • Sterol 14-Demethylase / genetics*
  • Sterol 14-Demethylase / metabolism
  • Transformation, Genetic

Substances

  • 14-alpha Demethylase Inhibitors
  • Antifungal Agents
  • Mutant Proteins
  • Fluconazole
  • Sterol 14-Demethylase
  • Ergosterol