RRC ID 46887
Author Niimi K, Woods MA, Maki K, Nakayama H, Hatakenaka K, Chibana H, Ikeda F, Ueno K, Niimi M, Cannon RD, Monk BC.
Title Reconstitution of high-level micafungin resistance detected in a clinical isolate of Candida glabrata identifies functional homozygosity in glucan synthase gene expression.
Journal J Antimicrob Chemother
Abstract OBJECTIVES: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.
Volume 67(7)
Pages 1666-76
Published 2012-7-1
DOI 10.1093/jac/dks112
PII dks112
PMID 22514266
MeSH Antifungal Agents / pharmacology* Candida glabrata / drug effects* Candida glabrata / enzymology* Candida glabrata / genetics DNA Mutational Analysis DNA, Fungal / chemistry DNA, Fungal / genetics Drug Resistance, Fungal* Echinocandins / pharmacology* Gene Deletion Gene Expression* Glucosyltransferases / biosynthesis* Glucosyltransferases / genetics Lipopeptides / pharmacology* Micafungin Molecular Sequence Data Mutagenesis, Site-Directed Mutation Selection, Genetic Sequence Analysis, DNA
IF 5.439
Times Cited 13
Pathogenic microorganisms