Project 1. Study of genotypically matched fluconazole susceptible and resistant pairs showed drug efflux to be responsible for resistance developing during fluconazole treatment. Two to 5 sequential isolates of Candida glabrata were obtained from 29 patients (104 isolates) undergoing hematopoietic stem cell transplantation and receiving fluconazole prophylaxis at the Fred Hutchinson Cancer Research Center, Seattle, Washington (FHCRC). A significant trend towards increasing fluconazole resistance appeared in the isolates from individual patients, with doubling of the MIC occurring on the average of every 31 days. We compared the most fluconazole susceptible and the most resistant isolate from 14 patients, the pairs all genotypically identical to one another by CHEF gel, choosing pairs in which the MIC differed by at least four fold. We wished to determine the factors that led to isolates becoming more resistant during fluconazole exposure. Geometric mean rhodamine accumulation and fluconazole uptake were both inversely correlated with log fluconazole MIC (Spearman rank correlation coefficient p=0.02 and p<0.002, respectively). Only two of 14 pairs of isolates did not show less rhodamine accumulation and fluconazole accumulation in the more resistant isolate. Not surprisingly, fluconazole uptake also correlated with rhodamine uptake (p<0.02). These results were consistent with the hypothesis that drug efflux was a major factor in determining the difference in fluconazole susceptibility between genotypically identical paired isolates. In order to identify mechanisms for increased drug efflux, we studied transcription of CgCDR1 and PDH1 .Both transporters are among the drug efflux pumps which remove fluconazole from the C. glabrata cell. Abundance of CgDR1 and PDH1 transcripts was compared in the 14 pairs of isolates from the same patient. In 12 of 14 pairs, the more fluconazole resistant isolate had greater transcript abundance than the more susceptible isolate, though the differences were not large. This finding confirmed that these two ABC transporters were among the genes upregulated as fluconazole resistance emerged. Project 2. Fluconazole efflux can be inhibited. Moxidectin 0.5 mcg/ml and milbemycin 5 mcg/ml both decreased the fluconazole MIC in C. glabrata. The fold decrease in fluconazole MIC in the presence of milbemycin was 8 fold in 8 isolates with an MIC of 32 mcg/ml or less and was 18 fold less in isolates with an MIC of 64 to 512 mcg/ml. The greater effect on more resistant isolates was consistent with milbemycin blocking drug efflux. The same milbemycin concentration increased rhodamine 6G accumulation 5 fold in a fluconazole resistant strain but not in a mutant in which both the ABC transporter genes, CgCDR1 and PDH1, had been interrupted. Project 3. Transposon approach identified CgPDR1, a transcriptional regulator, and CgERG1, a sterol synthesis gene, as affecting fluconazole susceptibility in Candida glabrata. A Tn-inserted CgPDR1 mutant, identified by the transposon approach, exhibited a 16-fold increase in FCZ susceptibility compared to its parental strain. The CgPDR1 mutant had increased sensitivity to fluconazole and cycloheximide. Northern analysis showed that disruption of CgPDR1 led to a major reduction in the expression of the ABC transporter gene which affects fluconazole efflux, CgCDR1, but that the expression of CgCDR1 was not completely abolished. Therefore, CgPDR1 is a major, but not the only, regulator of CgCDR1 expression. CgCDR1 expression could be induced moderately by Rhodamine 6G and was not affected much by the disruption of CgPDR1. In contrast, CgCDR1 expression was induced dramatically by cycloheximide and oligomycin. Northern hybridization analyses showed that disruption of the CgPDR1 gene abolished the induced expression of CgCDR1 by cycloheximide and oligomycin. In addition, overexpression of CgPDR1 in a C. glabrata wild-type strain increased resistance to fluconazole by 8-fold compared to the transformant containing only the empty vector. The expression of CgCDR1 was greatly increased in a transformant overexpressing CgPDR1. In contrast, overexpression of CgPDR1 did not increase resistance to FCZ in a CgCDR1 and PDH1 double deletant. Taken together, CgPDR1 contributes to, but is not the only factor involved in, the basal expression of CgCDR1 and drug induced CgCDR1 expression is signaled through CgPDR1 in C. glabata. . C. glabrata CgPDR1 in a centromere based plasmid can complement fluconazole, rhodamine 6G and cycloheximide susceptibility in a S. cerevisiae pdr1 mutant. In S. cerevisiae, ERG1 encodes a squalene epoxidase that catalyzes the biosynthesis of ergosterol, the target of flucoazole. Sequence analyses of the DNA flanking the inserted Tn revealed that the Tn went into the C-terminus of the putative CgErg1p in the CgERG1 Tn mutant. The CgERG1 mutant exhibited a pleiotropic phenotype, including increased fluconazole sensitivity. Ergosterol synthesis in the CgERG1 mutant was reduced by 50% compared to its parental strain. A significant accumulation of squalene, the substrate of squalene epoxidase, was detected in the mutant. The CgERG1 mutant had a 64-fold increase in fluconazole susceptibility as well as increased 3H-fluconazole and rhodamine 6G uptake. The increases in the drug and compound uptake were likely due to a change in the membrane composition. In addition, the CgERG1 mutant was unable to grow under hypoxic conditions. It also showed a slow growth phenotype when grown on rich media at 37 oC, which is likely due to the insufficient availability of ergosterol. In contrast to its parental strain, the CgERG1 mutant was able to take up more cholesterol. Supplementing media with cholesterol or ergosterol was sufficient to restore the growth of the CgERG1 mutant under hypoxic conditions and to reduce its fluconazole susceptibility. However, supplementing sterol was not sufficient in reducing the 3H-fluconazole uptake, which may due to incomplete restoration of membrane composition and integrity. Mutation in CgERG1 also increased C. glabrata susceptibility to terbinafine, which targets Erg1p. The CgERG1 mutation resulted in reduced susceptibility to Amphotericin B, which is a polyene drug and is proposed to bind ergosterol. It is likely that the CgERG1 mutant has fewer target sites for amphotericin B because it produces less ergosterol.
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