Candida dubliniensis is a recently identified yeast species primarily associated with oral carriage and infection in HIV-infected individuals. Previous studies have shown that stable resistance to fluconazole can be readily induced in vitro in this species. The aim of this study was to induce resistance to itraconazole in C. dubliniensis and to investigate the mechanisms involved. In order to induce resistance to itraconazole, two azole-susceptible strains, one containing a functional CDR1 gene and the other with a naturally occuring truncated version of the gene, were sequentially exposed to increasing concentrations of itraconazole on agar medium. This resulted in the recovery of derivatives exhibiting stable itraconazole resistance phenotype which was associated with cross-resistance to other azoles. In order to identify the mechanisms responsible for the resistance phenotype, the most common causes of azole resistance in Candida species were investigated. Northern hybridisation analysis indicated that there were small increases in expression of ERG11 and CDR1 in the resistant derivatives. In addition, sequence analysis of the ERG11 gene revealed that there were no mutations present. To further investigate efflux mechanisms, glucose-mediated efflux of the fluorescent compound rhodamine 6G (R6G), a substrate of efflux pumps, was measured. The results suggested that R6G efflux mediated by drug extrusion pumps was more efficient in the parental strains than in the azole-resistant derivatives. These findings suggested that the derivatives had altered membranes. Analysis of membrane sterol content by GC/MS revealed that the derivatives did not produce ergosterol but instead accumulated C5-saturated sterols, indicating that they lacked C5,6-desaturase activity. To investigate this, ERG3 alleles (encoding C5,6-desaturase enzyme) from the derivatives and their parent were sequenced and mutations affecting the amino acid sequence of the desaturase were identified. Heterologous expression of the WT and mutated alleles in •erg3 S. cerevisiae showed that the WT ERG3 alleles were able to complement the desaturase function while the mutated alleles could not. We therefore conclude that the primary mechanism of itraconazole resistance in C. dubliniensis derivatives, with or without a functional CDR1 gene, is loss of function of ERG3.
Full conference title:
The 15 th Congress of the International Society for Human and Animal Mycology
- ISHAM 15th (2003)