Project 1. C. glabrata gene found essential for fidelity of chromosome transmission Candida glabrata Kre29p is a novel protein and a sequence homolog of Saccharomyces cerevisiae Kre29p, which has been identified as a subunit of the Smc5-Smc6 complex. Although the complex is known to be required for proper chromosome segregation and DNA repair, the function of ScKre29p is still unknown. In addition, the function of none of its homologs has been characterized. In contrast to ScKRE29, CgKRE29 was not an essential gene so that we could characterize various phenotypes of the Cgkre29 null mutant. The deletion of CgKRE29 resulted in reduced viability mainly due to the G2/M arrest even under normal growth conditions. CgKre29p-GFP was located in the nucleus, and the absence of CgKre29p caused chromosome missegregation and an increase of plasmid loss frequency. Moreover, the Cgkre29 deletant showed extremely increased sensitivity to high temperature as well as to DNA damaging agents including UV, gamma ray, 4-nitroquinoline-1-oxide and methyl methanesulfonate. All phenotypes observed in the Cgkre29 deletant were restored to the wild type level by the reintegration of an intact CgKRE29 into the CgKRE29 locus of the deletant. To our knowledge, this is the first report addressing a cellular protein involved in DNA repair in C. glabrata, also demonstrating that CgKre29p is required for mitotic chromosome transmission fidelity. Project 2. Gain-of-function mutations in CgPDR1 contributed to the clinical fluconazole resistance in C. glabrata. We have characterized CgPDR1, a transcriptional factor involved in pleiotropic drug resistance (PDR) of Candida glabrata. CgPDR1p regulates CgCDR1, encoding a fluconazole-effluxing ABC transporter. We have previously overexpressed CgPDR1 in C. glabrata, which resulted in increased fluconazole resistance. To investigate whether CgPDR1 contributes to fluconazole resistance developed at a clinical setting, two pairs of fluconazole sensitive and resistant clinical isolates (Cg1660 vs. Cg4672 and Cg12581 vs. Cg13928) were subjected to Northern blot and DNA sequence analyses. The Northern data showed that both CgPDR1 and CgCDR1 were overexpressed in the two resistant isolates, Cg4672 and Cg13928, compared to the paired sensitive isolates, Cg1660 and Cg12581. As overexpression of CgPDR1 is likely the cause of CgCDR1 overexpression and increased fluconazole resistance in the clinical resistant isolates, we have sequenced the CgPDR1 genes from both pairs of sensitive and resistant isolates. The resistant isolates, Cg4672 and Cg13928, were found to have a different point mutation in their CgPDR1, which resulted in a single amino acid substitution: W297S in Cg4672 and F575L in Cg13928. The amino acid 297 substitution was in the inhibitory domain of CgPDR1p. Several hyperactive mutations of PDR1 in Saccharomycese cerevisiae were also in the inhibitory domain. In contrast, the amino acid 575 substitution was in the fungal specific transcriptional factor domain, which has not been described previously. CgPDR1 from Cg1660, Cg4672, Cg12581, and Cg13928 were introduced into the Cgpdr1 mutant respectively to investigate whether the mutations contributed to the fluconazole resistance. Both strains complemented by the mutated CgPDR1 became resistant to fluconazole while the strains complemented by the native CgPDR1 of the sensitive isolates only restored the fluconazole susceptibility to the wild-type level. Northern blot analysis also revealed that CgPDR1 expression was upregulated in the Cg13928 complemented resistant strains, which suggested that CgPDR1p also regulated its own expression directly or indirectly. We conclude that gain-of-function mutations in CgPDR1 occurred in the clinical resistant isolates and led to the overexpression of CgPDR1, which contributed to the clinical fluconazole resistance of C. glabrata by upregulating the CgCDR1 expression. Project 3. Transposon approach identified genes involved with increased fluconazole resistance in C. glabrata. Two Candida glabrata fluconazole-resistance mutants, CgTn60R1 and CgTn131R1, were obtained by a transposon approach. In addition to the fluconazole resistant phenotype, Tn60R1 had a poor growth phenotype with non-fermentable carbon source but not Tn131R1. Northern blot analyses showed that expression of CgCDR1, an ABC transporter, was greatly increased in CgTn60R1 but not in CgTn131R1. This suggests that the fluconazole resistance mechanisms of two mutants are different. Poor growth with a non-fermentable carbon source is, in general, associated with mitochondrial dysfunction, which has been described to cause upregulated CgCDR1 expression and increased fluconazole resistance. Genomic DNA flanking the Tn-insertion site was rescued from CgTn60R1. DNA sequence analysis of the rescued DNA revealed that the 3.5-kb genomic DNA adjacent to the Tn insertion sites was deleted in CgTn60R1. Three genes were disrupted due to the 3.5-kb deletion in CgTn60R1. Based on the homology with S. cerevisiae, one gene is a probable membrane protein and another gene is a homolog of S. cerevisiae gene with unknown function. The third gene is a homolog of S. cerevisiae YTA12, which encodes a mitochondrial ATPase. It is likely that mitochondrial deficiency played a role in the increased resistance in CgTn60R1. However, staining of mitochondria in CgTn60R1 indicated that mitochondria were still present in CgTn60R1. In addition, disruption of CgYTA12 in C. glabrata did not resulted in increased fluconazole resistant phenotype. Therefore, mitochondrial dysfunction-induced drug resistance could not explain fully the case in CgTn60R1. The three genes were deleted in the laboratory wild-type strain to confirm that the resistance was not due to a secondary mutation in the CgTn60R1 genome. Correlation of the 3.5-kb deleted region with the increased fluconazole resistance was further demonstrated because the deletant showed the same mutant phenotypes with CgTn60R1, including increased fluconazole resistance, poor growth with non-fermentable carbon source, and increased CgCDR1 expression. Dissection of the deleted regions is needed in identifying which of the three genes is responsible for the fluconazole resistance. Therefore, each of the three genes as well as the DNA fragment containing all three genes were cloned into the C. glabrata shuttle vector, respectively. Complementation analysis is underway to define the genes contributing to the increased fluconazole resistance. The mechanism of how the mutation triggers the CgCDR1 overexpression will be explored. Project 4. Fluconazole resistance in paired C. glabrata isolates Paired susceptible and resistant C. glabrata isolates were obtained from 14 patients and compared for mechanisms of azole resistance. No difference was found in gene copy number or deduced amino acid sequence of ERG11, the gene coding for the azole target, C14 alpha sterol demethylase. Milbemycin was used to block fungal ABC drug transport. A strong correlation was found between fluconazole susceptibility and the extent to which milbemycin increased susceptibility. The major mechanism of resistance appeared to be drug efflux, not copy number or mutations in ERG11.
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