Project 1. CgPDR1 and azole resistance A. CgPDR1 contributed to clinical fluconazole resistance. We reported last year that we cloned and sequenced CgPDRI, a transcriptional factor that regulates azole resistance through activation of the ABC transporter, CgCDR1. To investigate the relevance of this transcriptional factor to clinically acquired azole resistance, we studied two pairs of karyotypically identical C. glabrata isolates obtained early and late during fluconazole treatment. The resistant isolates of both pairs had increased expression of CgCDR1, the transporter, on Northern analysis. However, the transcriptional factor, CgPDR1, was upregulated in the resistant isolate of one pair but not in the other. We postulated that in this latter pair, the resistant isolate had a mutation in CgPDR1 which increased transcriptional activity. On nucleotide sequencing, CgPDR1p did show a difference in deduced amino acid sequence between the susceptible and resistant isolates: I370T and P575L. We deleted CgPDR1 from a susceptible strain and then used fluconazole to select clones with homologous integration of the gene from the resistant strain. The integrant did show increased fluconazole resistance and increased expression of the target gene, CgCDR1. In addition, rhodamine 6G accumulation was reduced in the complemented strain, consistent with increased drug efflux. Fluconazole resistance increased during therapy by different mechanisms in these two patients. One patient?s C. glabrata mutated in a manner which increased transcription of the transcriptional factor, CgPDR1, while the other patient?s strain acquired two mutations in CgPDRI which increased transcriptional activity. B. CgPDR1 contributed to the azole resistance caused by mitochondrion deficiency. In Saccharomyces cerevisiae, mitochondrion deficient mutants (petite) have increased fluconazole resistance accompanied by overexpression of PDR5, which encodes an ABC transporter. In S. cerevisiae, PDR5 expression is regulated by two transcriptional regulators, PDR1 and PDR3. PDR3 was shown to be overexpressed in the S. cerevisiae petite mutants and was essential for upregulated expression of PDR5 in petite mutants. Similarly, Candida glabrata petite mutants also were reported to exhibit increased azole resistance. However, C. glabrata is not known to have a PDR3 homologue. We postulated that CgPDR1 might be the transcriptional activator responsible for azole resistance in mitochondrion mutants of C. glabrata. We therefore isolated petite mutants from C. glabrata and showed that they were fluconazole resistant and, on Northern analyses, had increased expression of both the transcriptional activator, CgPDR1, and its transcriptional target, the transporter CgCDR1. However, when we obtained petite mutants from a strain in which the transcriptional activator, CgPDR1, had been disrupted, neither fluconazole resistance or increased expression of the transporter gene, CgCDR1 occurred, indicating that CgPDR1 is essential for mediating fluconazole resistance in petite mutants. Of interest was that the expression of the truncated CgPDR1 in the petite mutant was increased only slightly, suggesting that CgPDRI may be self-regulated, as is PDR3 in S. cerevisiae. . C. CgPDR1-associated fluconazole resistance required CgERG1. CgERG1 encodes a squalene epoxidase that catalyzes the biosynthesis of ergosterol. As we reported last year, a Cgerg1 transposon mutant showed reduced ergosterol biosynthesis and increased susceptibility to fluconazole. This year we extended these observations and found that overexpression of CgPDR1 in a cgerg1 mutant failed to increase fluconazole resistance. Membrane sterol composition can limit fluconazole resistance mediated by membrane-bound transporters such as that encoded by CgCDR1. Project 2. Microarray Transcriptional Profiling of Candida glabrata. The pleiotropic drug resistance (PDR) network is important in the fluconazole resistance of C. glabrata. Transcriptional profiling by microarray was undertaken to investigate the PDR network in C. glabrata. A total of 5908 70-mers representing the curated ORFs in C. glabrata were obtained from Institute Pasteur in Paris and used for microarray construction. To identify the putative targets of CgPDR1, the cDNAs of wild type and strains overexpessing CgPDR1 were used as probes for microarray analyses. The results showed that 25 genes are upregulated more than 5 fold in the strains overexpressing CgPDR1. Based on the homology of the genes with their S. cerevisiae homologues, these genes encode proteins with the following functions: transport of drugs, protons, neutral amino acids, phosphate, manganese ion, or a siderochrome; mitochondrion organization, biogenesis, and processing; cell wall organization and biogenesis; cell adhesion; meiosis and flocculation. Overexpression of CgPDR1 also down regulated five genes, which are involved in zinc, biotin, and ammonium transport, amino acid catabolism, gluconeogenesis, and glycolysis. Real-time PCR is being used for validating the expression of these genes affected by overexpression of CgPDR1. Project 3. Cell cycle control in C. glabrata A cell cycle deficient mutant of C. glabrata was selected because of its susceptibility to 4-nitroquinolone (4NQO). The mutant contained an ectopically integrated CgAP1 disruption cassette that led to interruption of an open reading frame with a high degree of homology to KRE29 in S. cerevisiae. Transformation with the C. glabrata ORF homologous to KRE29 restored the phenotype to that of the wild type. The Cgkre29 mutant had viability decreased to a third that of the wild-type strain , an increased number of nonviable cells based on methylene blue staining, heterogeneity of colony size, abnormal cell morphology such as giant cells and multi-budding cells, increased population of G2/M phase cells, reduced thermotolerance and increased sensitivity to some DNA damaging agents including 4NQO, methyl methanesulfonate, UV and gamma irradiation. In S. cerevisiae KRE29 is an essential gene for viability and was identified as a K1 killer toxin resistance gene based on slightly increased resistance in a heterozygotic diploid culture. The Cgkre29 mutant was not more resistant to K1 killer toxin. Tetrad analysis of a KRE29/kre29 heterozygote with a plasmid encoding CgKRE29 (pCgKRE29/YEp352) showed that CgKRE29 did not complement the defective viability of a kre29/kre29 homozygote. Therefore, CgKRE29 does not appear to be a functional homologue of S. cerevisiae KRE29, Rather, CgKRE29 is essential for cell cycling at the G2/M phase and mutation of the gene causes both decreased viability, abnormal cell morphology and an inability to repair damaged DNA.
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