Clinically significant micafungin resistance in Candida albicans involves modification of a glucan synthase catalytic subunit GSC1 (FKS1) allele followed by loss of heterozygosity.
OBJECTIVE
To determine the mechanism of intermediate- and high-level echinocandin resistance, resulting from heterozygous and homozygous mutations in GSC1 (FKS1), in both laboratory-generated and clinical isolates of Candida albicans.


METHODS
The DNA sequences of the entire open reading frames of GSC1, GSL1 (FKS3) and RHO1, which may contribute to the beta-1,3-glucan synthase of a micafungin-susceptible strain and a resistant clinical isolate, were compared. A spontaneous heterozygous mutant isolated by selection for micafungin resistance, and a panel of laboratory-generated homozygous and heterozygous mutants that possessed combinations of the echinocandin-susceptible and -resistant alleles, or mutants with individual GSC1 alleles deleted, were used to compare levels of echinocandin resistance and inhibition of glucan synthase activity.


RESULTS
DNA sequence analysis identified a mutation, S645P, in both alleles of GSC1 from the clinical isolate. GSL1 had two homozygous amino acid changes and five non-synonymous nucleotide polymorphisms due to allelic variation. The predicted amino acid sequence of Rho1p was conserved between strains. Reconstruction of the heterozygous (S645/S645F) and homozygous (S645F/S645F) mutation showed that the homozygous mutation conferred a higher level of micafungin resistance (4 mg/L) than the heterozygous mutation (1 mg/L). Exposure of the heterozygous mutant to micafungin resulted in a loss of heterozygosity. Kinetic analysis of beta-1,3-glucan synthase activity showed that the homozygous and heterozygous mutations gave echinocandin susceptibility profiles that correlated with their MIC values.


CONCLUSIONS
A homozygous hot-spot mutation in GSC1, caused by mutation in one allele and then loss of heterozygosity, is required for high-level echinocandin resistance in C. albicans. Both alleles of GSC1 contribute equally and independently to beta-1,3-glucan synthase activity.
Niimi
K
K
Department of Oral Sciences, University of Otago, 310 Great King St, Dunedin 9054, New Zealand. kyoko.niimi@otago.ac.nz
Monk
B C
BC
Hirai
A
A
Hatakenaka
K
K
Umeyama
T
T
Lamping
E
E
Maki
K
K
Tanabe
K
K
Kamimura
T
T
Ikeda
F
F
Uehara
Y
Y
Kano
R
R
Hasegawa
A
A
Cannon
R D
RD
Niimi
M
M
D000328:Adult; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects* / Q000201:enzymology*; D020134:Catalytic Domain / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D025141:Drug Resistance, Fungal; D054714:Echinocandins / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D055666:Lipopeptides / Q000494:pharmacology*; D019656:Loss of Heterozygosity; D008297:Male; D000077551:Micafungin; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011499:Protein Processing, Post-Translational; D017422:Sequence Analysis, DNA
D000935:Antifungal Agents; D004271:DNA, Fungal; D054714:Echinocandins; D005656:Fungal Proteins; D055666:Lipopeptides; D005964:Glucosyltransferases; C040232:glucan synthase; D000077551:Micafungin