Reduced Susceptibility to Polyenes Associated with a Missense Mutation in the ERG6 Gene in a Clinical Isolate of Candida glabrata with Pseudohyphal Growth

Abstract

ABSTRACT Little information is available about the molecular mechanisms responsible for polyene resistance in pathogenic yeasts. A clinical isolate of Candida glabrata with a poor susceptibility to polyenes, as determined by disk diffusion method and confirmed by determination of MIC, was recovered from a patient treated with amphotericin B. Quantitative analysis of sterols revealed a lack of ergosterol and an accumulation of late sterol intermediates, suggesting a defect in the final steps of the ergosterol pathway. Sequencing of Cg ERG11 , Cg ERG6 , Cg ERG5 , and Cg ERG4 genes revealed exclusively a unique missense mutation in Cg ERG6 leading to the substitution of a cysteine by a phenylalanine in the corresponding protein. In addition, real-time reverse transcription-PCR demonstrated an overexpression of genes encoding enzymes involved in late steps of the ergosterol pathway. Moreover, this isolate exhibited a pseudohyphal growth whatever the culture medium used, and ultrastructural changes of the cell wall of blastoconidia were seen consisting in a thinner inner layer. Cell wall alterations were also suggested by the higher susceptibility of growing cells to Calcofluor white. Additionally, complementation of this isolate with a wild-type copy of the Cg ERG6 gene restored susceptibility to polyenes and a classical morphology. Together, these results demonstrated that mutation in the Cg ERG6 gene may lead to a reduced susceptibility to polyenes and to a pseudohyphal growth due to the subsequent changes in sterol content of the plasma membrane.