title: Reconstitution of high-level micafungin resistance detected in a clinical isolate of Candida glabrata identifies functional homozygosity in glucan synthase gene expression.
abstract: OBJECTIVE
  A mechanism for the acquisition of high-level echinocandin resistance in Candida glabrata was investigated. FKS mutants were constructed to: determine whether clinically significant micafungin resistance requires a hot-spot mutation in FKS1 and a premature stop codon in FKS2, as was observed in a clinical isolate; select for variants with reduced susceptibility and locate mutations in FKS genes; and assess the roles of FKS1 and FKS2.
  
  
  METHODS
  A panel of FKS mutants was constructed using micafungin-susceptible parents by site-directed mutagenesis. Drug susceptibility, gene expression and glucan synthase activities were compared between mutants. Mutations acquired by selection were identified by DNA sequence analysis of FKS genes from selected variants. Single FKS deletants were constructed and their phenotypes examined.
  
  
  RESULTS
  Introduction of the hot-spot mutation in FKS1 alone conferred an intermediate reduction in susceptibility, and the premature stop codon in FKS2 alone had no effect on susceptibility, while severely reduced susceptibility equivalent to that of the clinical isolate required both mutations. Exposure of susceptible strains to micafungin yielded variants with an intermediate reduction in susceptibility that possessed a hot-spot mutation in FKS1. Further exposure to micafungin yielded variants with severely reduced susceptibility that acquired various single mutations in FKS2. The phenotypes of Δfks1 and Δfks2 mutants indicate that the two FKS genes are functionally redundant, while deletion of both FKS1 and FKS2 conferred synthetic lethality.
  
  
  CONCLUSIONS
  In the laboratory mutants of C. glabrata, clinically significant reduced susceptibility to micafungin required single nucleotide changes in both FKS1 and FKS2, and both genes encoded β-1,3-glucan synthase catalytic subunits.
authors.affiliation: Department of Oral Sciences and Sir John Walsh Research Institute, School of Dentistry, University of Otago, Dunedin, New Zealand. kyoko.niimi@otago.ac.nz
authors.forename: Kyoko
authors.initials: K
authors.lastname: Niimi
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Woods
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Maki
authors.forename: Hironobu
authors.initials: H
authors.lastname: Nakayama
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Hatakenaka
authors.forename: Hiroji
authors.initials: H
authors.lastname: Chibana
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Ikeda
authors.forename: Keigo
authors.initials: K
authors.lastname: Ueno
authors.forename: Masakazu
authors.initials: M
authors.lastname: Niimi
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cannon
authors.forename: Brian C
authors.initials: BC
authors.lastname: Monk
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D041221:Candida glabrata / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D025141:Drug Resistance, Fungal; D054714:Echinocandins / Q000494:pharmacology*; D017353:Gene Deletion; D015870:Gene Expression; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D055666:Lipopeptides / Q000494:pharmacology*; D000077551:Micafungin; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D000935:Antifungal Agents; D004271:DNA, Fungal; D054714:Echinocandins; D055666:Lipopeptides; D005964:Glucosyltransferases; C040232:glucan synthase; D000077551:Micafungin