MOLECULAR-BIOLOGICAL CHARACTERIZATION OF AN AZOLE-RESISTANT CANDIDA-GLABRATA ISOLATE

Two isolates of Candida glabrata, one susceptible and one resistant to azole antifungals, were previously shown to differ in quantity and ac tivity of the cytochrome P-450 14 alpha-lanosteral demethylase which i s the target for azole antifungals. The resistant isolate also had a l ower intracellular level of fluconazole, but not of ketoconazole or it raconazole, than the susceptible isolate. In the present study a 3.7-f old increase in the copy number of the CYP51 gene, encoding the 14 alp ha-lanosterol demethylase, was found. The amount of CYP51 mRNA transcr ipt in the resistant isolate was eight times greater than it was in th e susceptible isolate. Hybridization experiments on chromosomal blots indicated that this increase in copy number was due to duplication of the entire chromosome containing the CYP51 gene. The phenotypic instab ility of the resistant isolate was demonstrated genotypically: a gradu al loss of the duplicated chromosome was seen in successive subculture s of the isolate in fluconazole-free medium and correlated with revers ion to susceptibility. The greater abundance of the amplified chromoso me induced pronounced differences in the protein patterns of the susce ptible and revertant isolates versus that of the resistant isolate, as demonstrated by two dimensional gel electrophoresis (2D-GE). Densitom etry of the 2D-GE product indicated upregulation of at least 25 protei ns and domnregulation of at least 76 proteins in the resistant isolate .