RNA-seq Analysis of Wild-Type and G898D UPC2A Transcriptomes [G898D UPC2A transcriptome]
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ABSTRACT:
ORGANISM(S): Nakaseomyces glabratus
PROVIDER: GSE182490 | GEO | 2021/09/23
REPOSITORIES: GEO
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Project description:Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress induced transcription factors such as Upc2 and Upc2A as well as homologs of Sterol Response Element Binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2A∆ deletion mutant. This led to the identification of loss of function mutants in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2A∆ mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels or ergosterol and decreased levels of the toxic sterol, 14α methyl-ergosta-8,24(28)-dien-3β, 6α-diol, relative to upc2A∆. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata.
2021-12-20 | GSE191145 | GEO
Project description:Purpose: Compare the fluconazole-induced transcriptome of isogenic wild-type and upc2A null cells. Methods: Prepare total RNA from wild-type and upc2A null cells grown under unstressed conditions or challenged with 50 microgram/ml fluconazole for 6 hours. Use standard RNA-seq to evaluate and compare the transcriptomic changes caused by fluconazole in the presence or absence of UPC2A. Results: These data established that over 100 genes were induced by fluconazole in a UPC2A-dependent manner. However, most fluconazole-induced genes (>500) did not require the presence of UPC2A. Fluconazole-induced genes included the expected genes involved in ergosterol biosynthesis but also large number of plasma membrane-localized transporters. Conclusions: These data suggest that Upc2A provided transcriptional co-regulation between membrane transporters and the lipid environment in which these proteins function. A significant portion of the fluconazole-induced transcriptome does not require the presence of Upc2A, suggesting the presence of other transcriptional circuits responsive to this pharmacologic block of ergosterol biosynthesis.
2021-09-23 | GSE182489 | GEO
Project description:To determine the genomic binding sites for the Candida glabrata transcription factor Upc2A, we utilized three different strains. One was the wild-type KKY2001 which contains a wild-type copy of UPC2A but lacks any HA tag. The other two are both derived from KKY2001 but contain a 3X HA tag immediately after the start codon of UPC2A. These strains are BVGC82 and BVGC84, respectively. Both contain a single copy of a loxP element located 252 bp downstream from the UPC2A stop codon. BVGC82 contains a wild-type copy of the UPC2A gene while BVGC84 contains a mutant form of UPC2A (G898D) that confers elevated resistance to fluconazole. ChIP was performed with anti-HA antibodies in all cases. Strains containing the wild-type UPC2A gene (no HA tag) are referred to as C1 and C2. Strains containing the 3X HA epitope tag at the N-terminus of the wild-type UPC2A gene are referred to as wt1 and wt2. These same strains challenged with fluconazole prior to ChIP are referred to as wtf1 and wtf2. The strain containing the G898D UPC2A allele are referred to as M1 and M2 (no fluconazole challenge) and MF1 and MF2 (with fluconazole challenge.
2021-09-23 | GSE182488 | GEO
Project description:Candida glabrata is a human-associated opportunistic fungal pathogen. It shares its niche with Lactobacillus spp. in the gastrointestinal and vaginal tract. In fact, Lactobacillus species are thought to competitively prevent Candida overgrowth. We investigated the molecular aspects of this antifungal effect by analyzing the interaction of C. glabrata strains with Limosilactobacillus fermentum. From a collection of clinical C. glabrata isolates, we identified strains with different sensitivities to L. fermentum in coculture. We analyzed the variation of their expression pattern to isolate the specific response to L. fermentum. C. glabrata-L. fermentum coculture induced genes associated with ergosterol biosynthesis, weak acid stress, and drug/chemical stress. L. fermentum coculture depleted C. glabrata ergosterol. The reduction of ergosterol was dependent on the Lactobacillus species, even in coculture with different Candida species. We found a similar ergosterol-depleting effect with other lactobacillus strains (Lactobacillus crispatus and Lactobacillus rhamosus) on Candida albicans, Candida tropicalis, and Candida krusei. The addition of ergosterol improved C. glabrata growth in the coculture. Blocking ergosterol synthesis with fluconazole increased the susceptibility against L. fermentum, which was again mitigated by the addition of ergosterol. In accordance, a C. glabrata Derg11 mutant, defective in ergosterol biosynthesis, was highly sensitive to L. fermentum. In conclusion, our analysis indicates an unexpected direct function of ergosterol for C. glabrata proliferation in coculture with L. fermentum.
2023-02-08 | GSE202656 | GEO
Project description:In this study we used a transcriptomic approach to study the molecular determinants of the synergy between copper and fluconazole, in Candida glabrata, the second most frequent cause of invasive candidiasis. We demonstrate that copper acts together with fluconazole by downregulating three distinct pathways: azole efflux, ergosterol biosynthesis and zinc homeostasis. Coincidently, all these pathways are important for C. glabrata to cope with fluconazole stress. Therefore, when copper and fluconazole are combined cells fail to detoxify this drug and accumulate toxic methylated intermediates of sterol metabolism. This accumulation, possibly together with copper-induced lipid damages, should affect the activity of plasma membrane transporters, which impedes zinc uptake, leading to zinc depletion
2022-06-01 | GSE162741 | GEO
Project description:In Candida albicans, Upc2 is a zinc-cluster transcription factor that targets genes including those of the ergosterol biosynthesis pathway. To date there have been three documented UPC2 gain-of-function (GOF) mutations recovered from fluconazole-resistant clinical isolates that contribute to an increase in ERG11 expression and decreased fluconazole susceptibility. In a group of 62 fluconazole-resistant isolates, we found that 47 of these overexpressed ERG11 by at least two-fold over that of an average expression of 3 unrelated fluconazole susceptible strains. Of those 47 isolates, 29 contained a mutation in UPC2, whereas the remaining 18 isolates did not. Of the isolates containing mutations in UPC2, we recovered eight distinct mutations resulting in single putative amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V and W478C. Seven of these resulted in increased ERG11 expression, increased cellular ergosterol, and decreased susceptibility to fluconazole as compared to the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S and Y642F). Genes commonly upregulated in all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are more prevalent than previously thought among clinical isolates, make a significant contribution to azole antifungal resistance, but do not account for ERG11 overexpression in all such isolates of C. albicans. We examined the expression of genes in response to the presence of 4 gain-of-function alleles of the zinc-cluster transcription factor Upc2. The global gene expression of each mutant Upc2 strain was compared to that of the wildtype strain SC5314.
2013-01-31 | E-GEOD-39201 | biostudies-arrayexpress
Project description:In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Over-expression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Constitutive up-regulation of ERG11 is a major cause of resistance to fluconazole in clinical isolates of C. albicans, yet the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately up-regulated with ERG11 in a fluconazole resistant clinical isolate as compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug susceptible strain resulted in constitutive up-regulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans. Keywords: genome-wide expression profiling Clinical isolates S1 (susceptible) and S2 (resistant), genome strain SC5314, UPC2 disruption strains (UPC2M4A and UPC2M4B), UPC2 re-integrant strains of non-mutated UPC2 allele (UPC2M2K21A and UPC2M2K21B), and UPC2 re-integrant strains of mutated UPC2 allele (UPC2M2K31A and UPC2M2K31B) were grown for 3 hours until log phase. RNA was extracted for each isolate/strain. Experiments were performed in duplicate.
2008-11-04 | E-GEOD-11320 | biostudies-arrayexpress
Project description:In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Over-expression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Constitutive up-regulation of ERG11 is a major cause of resistance to fluconazole in clinical isolates of C. albicans, yet the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately up-regulated with ERG11 in a fluconazole resistant clinical isolate as compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug susceptible strain resulted in constitutive up-regulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans. Keywords: genome-wide expression profiling
2008-11-05 | GSE11320 | GEO
Project description:In Candida albicans, Upc2 is a zinc-cluster transcription factor that targets genes including those of the ergosterol biosynthesis pathway. To date there have been three documented UPC2 gain-of-function (GOF) mutations recovered from fluconazole-resistant clinical isolates that contribute to an increase in ERG11 expression and decreased fluconazole susceptibility. In a group of 62 fluconazole-resistant isolates, we found that 47 of these overexpressed ERG11 by at least two-fold over that of an average expression of 3 unrelated fluconazole susceptible strains. Of those 47 isolates, 29 contained a mutation in UPC2, whereas the remaining 18 isolates did not. Of the isolates containing mutations in UPC2, we recovered eight distinct mutations resulting in single putative amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V and W478C. Seven of these resulted in increased ERG11 expression, increased cellular ergosterol, and decreased susceptibility to fluconazole as compared to the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S and Y642F). Genes commonly upregulated in all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are more prevalent than previously thought among clinical isolates, make a significant contribution to azole antifungal resistance, but do not account for ERG11 overexpression in all such isolates of C. albicans.
2013-01-31 | GSE39201 | GEO
Project description:Fungal infections are an emerging health risk, especially those involving yeast that are resistant to antifungal agents. To understand the range of mechanisms by which yeasts can respond to anti-fungals, we compared gene expression patterns across three evolutionarily distant species - Saccharomyces cerevisiae, Candida glabrata and Kluyveromyces lactis - over time following fluconazole exposure. Conserved and diverged expression patterns were identified using a novel soft clustering algorithm that concurrently clusters data from all species while incorporating sequence orthology. The analysis suggests complementary strategies for coping with ergosterol depletion by azoles - Saccharomyces imports exogenous ergosterol, Candida exports fluconazole, while Kluyveromyces does neither leading to extreme sensitivity. In support of this hypothesis we find that only Saccharomyces becomes more azole resistant in ergosterol-supplemented media; that this depends on sterol importers Aus1 and Pdr11; and that transgenic expression of sterol importers in Kluyveromyces alleviates its drug sensitivity. We have compared the dynamic transcriptional responses of three diverse yeast species to fluconazole treatment using a novel clustering algorithm. This approach revealed significant divergence among regulatory programs associated with fluconazole sensitivity. In future, such approaches might be used to survey a wider range of species, drug concentrations and stimuli to reveal conserved and divergent molecular response pathways.
2010-07-08 | GSE15710 | GEO