title: The amino acid substitution N136Y in Candida albicans sterol 14alpha-demethylase is involved in fluconazole resistance.
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.
authors.affiliation: Département de Parasitologie et de Mycologie Médicale.
authors.forename: Nidia
authors.initials: N
authors.lastname: Alvarez-Rueda
authors.affiliation: Département de Parasitologie et de Mycologie Médicale.
authors.forename: Audrey
authors.initials: A
authors.lastname: Fleury
authors.affiliation: Laboratoire de Chimie Thérapeutique, Université de Nantes, Nantes Atlantique Universités, EA1155 - IICiMed, UFR des Sciences Pharmaceutiques et Biologiques, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Logé
authors.affiliation: Département de Parasitologie et de Mycologie Médicale.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Pagniez
authors.affiliation: Département de Parasitologie et de Mycologie Médicale.
authors.forename: Estelle
authors.initials: E
authors.lastname: Robert
authors.affiliation: Département de Parasitologie et de Mycologie Médicale.|Laboratoire de Parasitologie-Mycologie, CHU de Nantes, Nantes, France.
authors.forename: Florent
authors.initials: F
authors.lastname: Morio
authors.affiliation: Département de Parasitologie et de Mycologie Médicale.|Laboratoire de Parasitologie-Mycologie, CHU de Nantes, Nantes, France.
authors.forename: Patrice
authors.initials: P
authors.lastname: Le Pape
mesh_terms: D058888:14-alpha Demethylase Inhibitors / Q000494:pharmacology; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000494:pharmacology*; D001665:Binding Sites; D002176:Candida albicans / Q000187:drug effects* / Q000201:enzymology*; D025141:Drug Resistance, Fungal; D004875:Ergosterol / Q000096:biosynthesis; D015725:Fluconazole / Q000494:pharmacology*; D008401:Gas Chromatography-Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D058886:Sterol 14-Demethylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D058888:14-alpha Demethylase Inhibitors; D000935:Antifungal Agents; D050505:Mutant Proteins; D015725:Fluconazole; D058886:Sterol 14-Demethylase; D004875:Ergosterol
keywords: 14alpha-demethylase; 14alpha-demethylase homology model; CaCYP51; CaErg11p; Candida albicans; Fluconazole resistance