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Inhibiting Fungal Multidrug Resistance by Disrupting an Activator-Mediator Interaction With the Small Molecule iKIX1

ABSTRACT: Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. We previously identified an activator-targeted ~85 amino acid three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11 Mediator subunits in fungi. The Gal11 KIX domain is engaged by pleiotropic drug resistance transcription factor (Pdr1) orthologues, key regulators of the multidrug resistance (MDR) pathway in S. cerevisiae and in the clinically important human pathogen Candida glabrata. Drug-resistant clinical isolates of C. glabrata most commonly harbour point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway may represent a lynchpin in C. glabrata MDR. We have now carried out sequential biochemical and in vivo high-throughput screens to identify small molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation in both S. cerevisiae and C. glabrata and re-sensitizes drug-resistant C. glabrata to effective azole antifungal concentrations in vitro and in animal models for disseminated and urinary tract C. glabrata infection.

ORGANISM(S): Nakaseomyces glabratus Saccharomyces cerevisiae

PROVIDER: GSE74361 | GEO | 2015/12/15

SECONDARY ACCESSION(S): PRJNA299815

REPOSITORIES: GEO

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Inhibiting Fungal Multidrug Resistance by Disrupting an Activator-Mediator Interaction With the Small Molecule iKIX1

Project description:Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. We previously identified an activator-targeted ~85 amino acid three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11 Mediator subunits in fungi. The Gal11 KIX domain is engaged by pleiotropic drug resistance transcription factor (Pdr1) orthologues, key regulators of the multidrug resistance (MDR) pathway in S. cerevisiae and in the clinically important human pathogen Candida glabrata. Drug-resistant clinical isolates of C. glabrata most commonly harbour point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway may represent a lynchpin in C. glabrata MDR. We have now carried out sequential biochemical and in vivo high-throughput screens to identify small molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation in both S. cerevisiae and C. glabrata and re-sensitizes drug-resistant C. glabrata to effective azole antifungal concentrations in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Samples are generated in triplicate for four conditions (DMSO/vehicle-treated, iKIX1-treated, DMSO/vehicle and ketoconazole-treated. and iKIX1-ketoconazole treated) in both Saccharomyces cerevisiae and Candida glabrata

2015-12-15 | E-GEOD-74361 | biostudies-arrayexpress

Mechanisms of multidrug resistance caused by an Ipi1 mutation in the fungal pathogen Candida glabrata

Project description:Multidrug resistance in the pathogenic fungus Candida glabrata is a growing global threat. Here, we study mechanisms of multidrug resistance in this pathogen. Exposure of C. glabrata cells to micafungin (an echinocandin) leads to the isolation of a mutant exhibiting resistance to echinocandin and azole antifungals. The drug-resistant phenotype is due to a non-synonymous mutation (R70H) in gene IPI1, which is known to be involved in pre-rRNA processing in Saccharomyces cerevisiae. Azole resistance in the ipi1-R70H mutant depends on the Pdr1 transcription factor, which regulates the expression of multidrug transporters. We show that the C. glabrata Ipi1 protein physically interacts with the ribosome-related chaperones Ssb and Ssz1, both of which bind to Pdr1. The Ipi1-Ssb/Ssz1 complex inhibits Pdr1-mediated gene expression and multidrug resistance in C. glabrata, in contrast to S. cerevisiae where Ssz1 has been shown to act as a positive regulator of Pdr1. Furthermore, micafungin exposure reduces metabolic activity and cell proliferation in the ipi1-R70H mutant, which may contribute to micafungin tolerance.

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The Candida glabrata transcription factor Stb5p is a negative regulator of ATP-binding cassette transporters

Project description:The opportunistic yeast pathogen Candida glabrata is recognized for its ability to acquire resistance during prolonged treatment with azole antifungals. Resistance to azoles is largely mediated by the transcription factor PDR1, resulting in the upregulation of ATP-binding cassette (ABC) transporter proteins and drug efflux. Studies in the related yeast Saccharomyces cerevisiae have shown Pdr1p forms a heterodimer with another transcription factor, Stb5p. In C. glabrata the ORF designated CAGL0I02552g has 38.8% amino acid identity with STB5 (YHR178w), and shares an N-terminal Zn2Cys6 binuclear cluster domain and a fungal specific transcriptional factor domain, prompting us to test for homologous function and a possible role in azole resistance. Complementation of deltayhr178w (stb5) with CAGL0I02552g resolved the increased sensitivity to cold, hydrogen peroxide and caffeine of the mutant, for which reason we designated CAGl0I02552g as CgSTB5. Overexpression of CgSTB5 in C. glabrata repressed azole resistance, whereas deletion of CgSTB5 increased resistance, both by a mechanism independent of CgPDR1. Expression analysis found that CgSTB5 shares many of the same transcriptional targets as CgPDR1 but, unlike the later, is a negative regulator of pleiotropic drug resistance, including the ABC transporter genes CDR1,PDH1, and YOR1.

2013-03-11 | GSE37071 | GEO

The Candida glabrata transcription factor Stb5p is a negative regulator of ATP-binding cassette transporters

2013-03-11 | E-GEOD-37071 | biostudies-arrayexpress

Transcription profiling of S. cerevisiae to investigate repression of pleiotrophic drug resistance genes in using chimeric transcriptional repressors

Project description:Yeast is a powerful model system for studying the action of small molecule therapeutics. An important limitation has been low efficacy of many small molecules in yeast due to limited intracellular drug accumulation. We used the DNA binding domain of the pleiotropic drug resistance regulator Pdr1 fused in-frame to transcription repressors to repress Pdr1 regulated genes. Expression of these regulators conferred dominant enhancement of drug sensitivity and led to greatly diminished levels of Pdr1p regulated transcripts, including the yeast p-glycoprotein homologue Pdr5. Enhanced sensitivity was seen for a wide range of small molecules. Biochemical measurements demonstrated enhanced accumulation of rhodamine in yeast cells carrying the chimeras. These repressors of Pdr1p regulated transcripts can be introduced into large collections of strains such as the S. cerevisiae deletion set, and enhance the utility of yeast for studying drug action and for mechanism-based drug discovery. Experiment Overall Design: We determined the profiles of gene expression in yeast strains expressing dominant-negative Pdr1-fusion transcription factors. Yeast strains expressing different Pdr1-fusion repressors were analyzed by extracting total RNA and hybridization to Affymetrix GeneChip YG-s98 microarrays

2008-06-16 | E-GEOD-8326 | biostudies-arrayexpress

Genomic targets sites of the Candida glabrata transcription factor Pdr1

2014-09-12 | GSE59839 | GEO

Repression of pleiotrophic drug resistance genes in Saccharomyces cerevisiae using chimeric transcriptional repressors

2007-06-29 | GSE8326 | GEO

Genome-wide expression profile analysis of the Candida glabrata Pdr1 regulon

Project description:The ABC-transporters CgCdr1, CgPdh1, and CgSnq2 are known to mediate azole resistance in the pathogenic fungus Candida glabrata. Activating mutations in CgPDR1, a zinc cluster transcription factor, result in constitutive up-regulation of these ABC-transporter genes but to varying degrees. We examined the genome-wide gene expression profiles of two matched azole-susceptible and M-bM-^@M-^Sresistant C. glabrata clinical isolate pairs. Of all the genes identified in the gene expression profiles for these two matched pairs, there were 28 genes that were commonly up-regulated with CgCDR1 in both isolate sets including YOR1, LCB5, RTA1, POG1, HFD1, and several members of the FLO gene family of flocculation genes. We then sequenced CgPDR1 from each susceptible and resistant isolate and found two novel activating mutations that conferred increased resistance when expressed in a common background strain in which CgPDR1 had been disrupted. Microarray analysis comparing these re-engineered strains to their respective parent strains identified a set of commonly differentially-expressed genes, including CgCDR1, YOR1, and YIM1, as well as genes uniquely regulated by specific mutations. Our results demonstrate that while CgPdr1 activates a broad repertoire of genes, specific activating mutations result in the activation of discrete subsets of this repertoire. We examined the genome-wide gene expression profiles of two matched azole-susceptible and M-bM-^@M-^Sresistant C. glabrata clinical isolate pairs to determine the core regulon of CgPDR1 as well as how different activating alleles of PDR1 can affect the differential expression of target genes. genotype: wildtype allele: SM1, 6856, SM1Dpdr1/PDR1-SM1, SM1Dpdr1/PDR1-6856 genotype: activating allele: SM3, 6955, SM1Dpdr1/PDR1-SM3, SM1Dpdr1/PDR1-6955

2011-02-21 | E-GEOD-24168 | biostudies-arrayexpress

Set1-mediated histone H3K4 methylation is conserved and required for azole induction of ergosterol biosynthesis genes and antifungal drug resistance in Candida glabrata

Project description:Fungal infections are a major health concern because of limited antifungal drugs and development of drug resistance. Candida can develop azole drug resistance by overexpression of drug efflux pumps or mutating ERG11, the target of azoles. However, the role of epigenetic histone modifications in azole-induced gene expression and drug resistance is poorly understood in Candida glabrata. In this study, we show that Set1 mediates histone H3K4 methylation in C. glabrata. In addition, loss of SET1 and histone H3K4 methylation increases azole susceptibility in both C. glabrata and S. cerevisiae. This increase in azole susceptibility in S. cerevisiae and C. glabrata strains lacking SET1 is due to distinct mechanisms. For S. cerevisiae, loss of SET1 decreased the expression and function of the efflux pump Pdr5, but not ERG11 expression under azole treatment. In contrast, loss of SET1 in C. glabrata does not alter expression or function of efflux pumps. However, RNA sequencing revealed that C. glabrata Set1 is necessary for azole-induced expression of all 12 genes in the late ergosterol biosynthesis pathway, including ERG11 and ERG3. Furthermore, chromatin immunoprecipitation analysis shows histone H3K4 trimethylation increases upon azole-induced ERG gene expression. In addition, high performance liquid chromatography analysis indicated Set1 is necessary for maintaining proper ergosterol levels under azole treatment. Clinical isolates lacking SET1 were also hypersusceptible to azoles which is attributed to reduced ERG11 expression but not defects in drug efflux. Overall, Set1 contributes to azole susceptibility in a species-specific manner by altering the expression and consequently disrupting pathways known for mediating drug resistance.

2022-07-28 | GSE209637 | GEO

Genomic targets sites of the Candida glabrata transcription factor Pdr1

Project description:Pdr1 is the major regulator of azole resistance in the fungal pathogen Candida glabrata. Earlier experiments demonstrated that expression of Pdr1 itself is increased when cells lose their mitochondrial genome (rho0). Here we use chromatin immunoprecipitation coupled with highthroughput sequencing (ChIP-seq) to map the genomic binding sites for Pdr1 in both normal and rho0 cells. These data provide the first look at genes that are likely to represent the direct targets of Pdr1 in this important pathogen. Examination of Pdr1 binding sites using two different antibodies in five different strains. Strains labeled TAP carry the TAP-tagged allele and capture was done with anti-TAP. The control was performed by carrying out a ChIP reaction on rho0 TAP-PDR1 cells but with no primary anti-TAP antibody. Since Pdr1 expression is the highest in this background, this was selected as the best control for antibody specificity. We also performed ChIP reactions on rho+, rho0 and cells lacking the PDR1 gene (pdr1D) cells using the anti-Pdr1 antibody. The control for these reactions was a ChIP experiment using anti-Pdr1 antibody on cells lacking the endogenous Pdr1 protein.

2014-09-12 | E-GEOD-59839 | biostudies-arrayexpress

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