A PWWP domain-containing protein Crf4-3 specifically modulates fungal azole susceptibility by regulating sterol C-14 demethylase ERG11
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ABSTRACT:
PROVIDER: PRJNA1135472 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29819210_1.fastq.gz | Fastqsanger.gz | |||
| SRR29819210_2.fastq.gz | Fastqsanger.gz | |||
| SRR29819211_1.fastq.gz | Fastqsanger.gz | |||
| SRR29819211_2.fastq.gz | Fastqsanger.gz | |||
| SRR29819212_1.fastq.gz | Fastqsanger.gz |
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Project description:The widespread use of azole antifungal drugs in agriculture and clinical settings has led to serious drug resistance issues. Under azole treatment, resistant strains can upregulate the expression of the azole drug target 14α-demethylase ERG11 through transcription factors to alleviate the stress induced by sterol synthesis inhibition, which is a common resistance mechanism. Additionally, the currently reported regulatory factors related to resistance are not sufficient to explain all resistance issues. In this study, we constructed a GFP gene reporter system based on the erg11 promoter in the model filamentous fungus, Neurospora crassa, and identified a key region of the promoter that governs the erg11 response to azole drug by stepwise deletion. Using specific probes for DNA pulldown and combined with phenotype analysis, we identified a protein, Crf4-3, containing a PWWP domain that has a positive regulatory effect. Specific deletion of Crf4-3 leads to hypersensitivity to azole drugs and loss of transcriptional response of erg11 and erg6 to ketoconazole. Furthermore, the basal expression of erg1, erg11, erg25, and erg3A is affected by the deletion of crf4-3. Crf4-3 homologs are widely present in the Pezizomycotina fungi. Deletion of the homologous protein of Crf4-3 in Aspergillus fumigatus also significantly reduced sensitivity to azole drugs like voriconazole by reducing the transcriptional response of erg11. In summary, our study revealed a new regulatory factor Crf4-3 involved in the azole stress response in filamentous fungi and its mechanism, providing new insights into understanding the mechanisms of azole drug resistance.
2024-09-15 | GSE272185 | GEO
Project description:The present study describes a novel mechanism of antifungal resistance affecting the susceptibility of both the azole and echinocandin antifungals in an azole-resistant isolate from a matched pair of C. parapsilosis isolates obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate including upregulation of ERG1, ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, ERG27, DAP1 and UPC2, of the ergosterol biosynthesis pathway. Whole genome sequencing revealed a mutation in the ERG3 gene leading to a G111R amino acid substitution in the resistant isolate. Subsequent introduction of this allele in the native ERG3 locus in the susceptible isolate resulted in a fluconazole MIC of >64 mg/ml and a caspofungin MIC of 8 mg/ml. Corresponding allelic replacement of the wildtype allele for the mutant allele in the resistant isolate resulted in a drop in MIC to 1 mg/ml for both fluconazole and caspofungin. Sterol profiles indicated a loss of sterol demethylase activity as a result of this mutation. This work demonstrate that this G111R mutation is wholly responsible for the resistant phenotype in the C. parapsilosis resistant isolate and is the first report of this multidrug resistance mechanism.
2017-05-25 | GSE98986 | GEO
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
Project description:Ergosterol, a pivotal constituent of the fungal cell membrane, plays a crucial role in diverse cellular activities. Fungi inherently regulate ergosterol homeostasis, a process traditionally associated with the control of a major transcription factor, like SREBP, activated in response to ergosterol depletion, regardless of which ergosterol biosynthesis step is affected. Contrary to the established paradigm, our investigation demonstrates that the inhibition of ergosterol biosynthesis at specific steps triggers distinct transcriptional responses in erg genes across fungi, including Neurospora crassa, Aspergillus fumigatus, and Fusarium verticillioides. In N. crassa, the targeted responses are orchestrated by several transcription factors that have undergone functional rewiring. Specifically, ERG24 inhibition by amorolfine activates transcription factors SAH-2 and AtrR, resulting in the upregulation of erg24, erg2, erg25 and erg3. Additionally, ERG11 inhibition by azoles activates transcription factor NcSR, leading to the upregulation of erg11 and erg6. Our findings unveil a novel sophisticated regulation model of sterol biosynthesis in fungi which potentially enhances fungal survival in complex environments, thus providing new insights into sterol homeostasis maintenance and a deeper understanding of drug resistance mechanisms in fungi.
2024-03-24 | GSE261900 | GEO
Project description:Purified Candida albicans sterol 14-α demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with K(s) values of 11 and 25 μM, respectively, and a K(m) value of 6 μM for lanosterol. Azole binding to CaCYP51 was "tight" with both the type II spectral intensity (ΔA(max)) and the azole concentration required to obtain a half-ΔA(max) being proportional to the CaCYP51 concentration. Tight binding of fluconazole and itraconazole was confirmed by 50% inhibitory concentration determinations from CYP51 reconstitution assays. CaCYP51 had similar affinities for clotrimazole, econazole, itraconazole, ketoconazole, miconazole, and voriconazole, with K(d) values of 10 to 26 μM under oxidative conditions, compared with 47 μM for fluconazole. The affinities of CaCYP51 for fluconazole and itraconazole appeared to be 4- and 2-fold lower based on CO displacement studies than those when using direct ligand binding under oxidative conditions. Econazole and miconazole were most readily displaced by carbon monoxide, followed by clotrimazole, ketoconazole, and fluconazole, and then voriconazole (7.8 pmol min(-1)), but itraconzole could not be displaced by carbon monoxide. This work reports in depth the characterization of the azole binding properties of wild-type C. albicans CYP51, including that of voriconazole, and will contribute to effective screening of new therapeutic azole antifungal agents. Preliminary comparative studies with the I471T CaCYP51 protein suggested that fluconazole resistance conferred by this mutation was through a combination of increased turnover, increased affinity for substrate, and a reduced affinity for fluconazole in the presence of substrate, allowing the enzyme to remain functionally active, albeit at reduced velocity, at higher fluconazole concentrations.
| S-EPMC2944560 | biostudies-literature
Project description:In Candida albicans, the transcription factor Upc2 is central to the regulation of ergosterol biosynthesis. UPC2-activating mutations contribute to azole resistance, whereas disruption increases azole susceptibility. In the present study, we investigated the relationship of UPC2 to fluconazole susceptibility, particularly in azole-resistant strains. In addition to the reduced fluconazole MIC previously observed with UPC2 disruption, we observed a lower minimum fungicidal concentration (MFC) for a upc2Δ/Δ mutant than for its azole-susceptible parent, SC5314. Moreover, the upc2Δ/Δ mutant was unable to grow on a solid medium containing 10 µg/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed higher fungistatic activity against the upc2Δ/Δ mutant than against SC5314. UPC2 disruption in strains carrying specific resistance mutations also resulted in reduced MICs and MFCs. UPC2 disruption in a highly azole resistant clinical isolate containing multiple resistance mechanisms likewise resulted in a reduced MIC and MFC. This mutant was unable to grow on a solid medium containing 10 µg/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed increased fungistatic activity against the upc2Δ/Δ mutant in the resistant background. Microarray analysis showed attenuated induction by fluconazole of genes involved in sterol biosynthesis, iron transport, or iron homeostasis in the absence of UPC2. Taken together, these data demonstrate that the UPC2 transcriptional network is universally essential for azole resistance in C. albicans and represents an attractive target for enhancing azole antifungal activity.
2014-05-24 | GSE57929 | GEO
Project description:Recent studies have demonstrated that some morphologically atypical Aspergillus fumigatus strains are different species belonging to the section Fumigati. Aspergillus lentulus, one of these sibling species, is increasingly reported in patients under corticosteroid treatment. MICs of most antifungals in clinical use are elevated against A. lentulus, and it shows primary resistance to azole drugs. Two A. lentulus cytochrome P450 14-? sterol demethylases, encoded by A. lentulus cyp51A (Alcyp51A) and Alcyp51B genes, were identified. Targeted cyp51A gene knockout in A. lentulus showed that the intrinsic azole resistance of this species is cyp51A dependent. The ?cyp51A strain was morphologically indistinguishable from the A. lentulus wild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice. The heterologous expression of A. lentulus cyp51A was performed in an A. fumigatus cyp51A-deficient strain, confirming that Cyp51A is responsible for the differences in A. lentulus-azole drug interaction.
| S-EPMC3232793 | biostudies-literature
Project description:Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of L. donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme is a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.
2024-06-21 | PXD052472 | Pride
Project description:With some advances in modern medicine (such as cancer chemotherapy, broad exposure to antibiotics, and immunosuppression), the incidence of opportunistic fungal pathogens such as Candida albicans has increased. Cases of drug resistance among these pathogens have become more frequent, requiring the development of new drugs and a better understanding of the targeted enzymes. Sterol 14?-demethylase (CYP51) is a cytochrome P450 enzyme required for biosynthesis of sterols in eukaryotic cells and is the major target of clinical drugs for managing fungal pathogens, but some of the CYP51 key features important for rational drug design have remained obscure. We report the catalytic properties, ligand-binding profiles, and inhibition of enzymatic activity of C. albicans CYP51 by clinical antifungal drugs that are used systemically (fluconazole, voriconazole, ketoconazole, itraconazole, and posaconazole) and topically (miconazole and clotrimazole) and by a tetrazole-based drug candidate, VT-1161 (oteseconazole: (R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol). Among the compounds tested, the first-line drug fluconazole was the weakest inhibitor, whereas posaconazole and VT-1161 were the strongest CYP51 inhibitors. We determined the X-ray structures of C. albicans CYP51 complexes with posaconazole and VT-1161, providing a molecular mechanism for the potencies of these drugs, including the activity of VT-1161 against Candida krusei and Candida glabrata, pathogens that are intrinsically resistant to fluconazole. Our comparative structural analysis outlines phylum-specific CYP51 features that could direct future rational development of more efficient broad-spectrum antifungals.
| S-EPMC5399120 | biostudies-literature
Project description:The sterol 14-demethylase P450 (CYP51) of a fluconazole-resistant isolate of Candida albicans, DUMC136, showed reduced susceptibility to this azole but with little change in its catalytic activity. Twelve nucleotide substitutions, resulting in four amino acid changes, were identified in the DUMC136 CYP51 gene in comparison with a reported CYP51 sequence from a wild-type, fluconazole-susceptible C. albicans strain. Seven of these substitutions, including all of those causing amino acid changes, were located within a region covering one of the putative substrate recognition sites of the enzyme (SRS-1). Polymorphisms within this region were observed in several C. albicans isolates, and some were found to be CYP51 heterozygotes. Among the amino acid changes occurring in this region, only an alteration of Y132 was common among these fluconazole-resistant isolates, which suggests the importance of this residue to the fluconazole resistance of the target enzyme. DUMC136 and another fluconazole-resistant isolate were homozygotes with respect to CYP51, although the typical wild-type, fluconazole-susceptible C. albicans was a CYP51 heterozygote. These findings suggest that part of the fluconazole-resistant phenotype of C. albicans DUMC136 was acquired through a mutation-prone area of CYP51, an area which might promote the formation of fluconazole-resistant CYP51, along with a mechanism(s) which allows the formation of a homozygote of this altered CYP51 in this diploid pathogenic yeast.
| S-EPMC89127 | biostudies-literature