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:The widespread use of azole antifungals in agriculture and clinical settings has led to serious drug resistance. Overexpression of the azole drug target 14α-demethylase ERG11 (CYP51) is the most common fungal resistance mechanism. However, the presence of additional regulatory proteins in the transcriptional response of erg11 is not yet fully elucidated. In this study, leveraging the identified key promoter region of erg11 that controls its response to azoles in Neurospora crassa, we pinpointed a protein, Crf4-3, which harbors a PWWP domain and exerts a positive regulatory influence on azole resistance, as determined by DNA pulldown assays. The removal of Crf4-3 results in heightened sensitivity to azoles while remaining unaffected by other stressors tested. Additionally, the deletion leads to the abolition of transcriptional responses of genes such as erg11 and erg6 to ketoconazole. Interestingly, the basal expression of erg1, erg11, erg25, and erg3A is also affected by the deletion of crf4-3, indicating its role in sterol homeostasis. Crf4-3 homologs are broadly distributed across the Pezizomycotina fungi. The gene deletion for its homologous protein in Aspergillus fumigatus also significantly improves sensitivity to azoles such as voriconazole, primarily through the attenuation of the transcriptional response of erg11. Our data, for the first time, identified Crf4-3 as a novel regulatory protein in the azole stress response of filamentous fungi, offering fresh insights into the mechanisms of azole resistance.IMPORTANCETranscriptional control of pivotal genes, such as erg11, stands as the primary driver of azole resistance. Although considerable effort has been dedicated to identifying transcription factors involved, our knowledge regarding the use of transcriptional regulation strategies to combat azole resistance is currently limited. In this study, we reveal that a PWWP domain-containing protein Crf4-3, which is conserved in Pezizomycotina fungi, modulates fungal azole sensitivity by transcriptionally regulating sterol biosynthetic genes, including erg11. These results also broaden the understanding of fungal PWWP domain-containing proteins regarding their roles in regulating resistance against azole antifungals. Considering research on small molecules targeting the PWWP domain in humans, Crf4-3 homolog emerges as a promising target for designing fungal-specific drugs to combat azole resistance.
| S-EPMC11774033 | biostudies-literature
Project description:Ergosterol, a key lipid component of fungal membranes, has its homeostasis predominantly maintained through membrane-associated mechanisms, yet the role of the many transmembrane proteins in regulating sterol homeostasis remains poorly understood. Here, we systematically screened 1,042 viable transmembrane protein mutants in Neurospora crassa, and identified 180 that exhibited altered sensitivity to one or more sterol biosynthesis inhibitors. Further analysis pinpointed three genes, cax-4, chol-6 and NCU01826, as required for the upregulation of erg11 upon azole treatment. CAX-4, a conserved dolichyl pyrophosphate phosphatase, was further characterized. Deletion of this gene leads to hypersensitivity to ketoconazole and amorolfine, and impairs the proper expression of membrane and extracellular proteins. Notably, CAX-4 was required for the transcriptional induction of core ergosterol biosynthetic genes (e.g., erg11, erg6, erg24) and sterol metabolism under azole stress. This phenotype was linked to reduced levels of NcSR, a key transcription factor in sterol biosynthesis regulation, at both the mRNA and protein level. Mechanistically, CAX-4 maintains protein homeostasis via N-glycosylation, as Tunicamycin treatment phenocopied its deletion. Above all, by characterizing key transmembrane protein in sterol biosynthesis regulation, our study unveils a CAX-4 integrated crosstalk between dolichol-mediated protein N-glycosylation and sterol biosynthesis regulation, thus offering novel insights into sterol homeostasis and pointing to potential targets for the development of antifungal strategies.
2025-12-02 | GSE311794 | 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:As a major fungal pathogen, Aspergillus fumigatus can induce chronic, allergic or severe invasive infections, meanwhile its escalating resistance to azole antifungals has emerged as a global public health menace. Squalene, an essential molecule in the sterol biosynthesis pathway, can be cyclized by squalene hopene cyclase (SHC) to produce hopanoids and affect probably sterol biosynthesis modifying azole resistance; however, the physiological function of SHC in A. fumigatus pathogenesis is poorly understood.
2026-02-03 | GSE317610 | GEO
Project description:Candida tropicalis is a leading cause of invasive candidiasis in the Asia-Pacific region, with mortality rates exceeding 50%. The rising prevalence of azole resistant clinical strains, particularly in this region, presents a significant clinical challenge. In this study, we analyzed 1,032 C. tropicalis isolates, including 1,016 clinical isolates collected over nine years from 27 hospitals across India, as well as 16 environmental isolates. Fluconazole resistance was detected in 5.1% of clinical isolates, with more than half also exhibiting cross-resistance to voriconazole and itraconazole. Multilocus sequence typing (MLST) and phylogenomic analysis of 1,571 global isolates, confirmed the clonal emergence and persistence of azole- resistant MLST clade 4 strains in Indian hospitals. Genomic analysis revealed that Indian isolates cluster closely with azole resistant strains from China, Singapore, and Taiwan. Azole resistance was found to be multifactorial, involving well-characterized hotspot mutations in the ERG11 gene (Y132F, S154F), ERG11 gene amplification (2–7.5 copies), and significant overexpression of ERG11. Transcriptomic profiling showed significant up regulation of virulence-associated genes in the fluconazole-resistant isolate. Notably, ALS7, a member of the agglutinin-like sequence (ALS) family involved in cell adhesion along with Secreted Aspartyl Proteinases (SAP) genes SAP7 and SAP9, were significantly upregulated in fluconazole-resistant isolates. Additionally, fluconazole-resistant isolates exhibited significantly enhanced biofilm formation compared to fluconazole-susceptible strains, indicating a higher virulence potential. Furthermore, fluconazole-resistant isolates exhibited reduced β-glucan exposure, a trait linked to immune evasion, and showed greater survival in both neutrophil and macrophage killing assays. Together, our findings provide comprehensive genomic and phenotypic evidence supporting the emergence, persistence, and increased pathogenic potential of azole-resistant C. tropicalis clade 4 in Indian hospitals. A deeper understanding of the epidemiological trends and molecular mechanisms driving drug resistance in C. tropicalis is essential for improving diagnostic accuracy, optimising antifungal susceptibility testing, and informing effective clinical management strategies.
2026-02-23 | GSE309448 | 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:Azole-resistant Aspergillus infections are a source of serious concern, as only three classes of drugs are available for treatment. However, as most infections are still caused by azole-sensitive AF strains, there is a need to better understand fungal response to azole drugs. Here, we have identified a long non-coding RNA, afu-182, as a major regulator of erg11-independent fungal azole recalcitrant colony growth (ARC), without an apparent change in azole’s minimum inhibitory concentration. As strains are currently classified in a binary system of resistant or susceptible to drugs, we show that afu-182 is a negative regulator of azole drug response, leading to azole recalcitrant biofilms and poor disease outcomes in an animal model upon azole treatment. Furthermore, overexpression of afu-182 significantly reduced fungal burden in animals treated with azole drugs, showing a genetic basis of action. Whole transcriptome analysis revealed that azole drugs lead to upregulation of 7-transmembrane domain proteins of the RTA1 family, and these proteins are negatively regulated by afu-182. Here, we characterized two RTA1 family genes downstream of afu-182 and showed their individual and additive effects in regulating fungal ARC. Taken together, our data show a trans-acting role of long non-coding RNA afu-182 in regulating fungal response to azole drugs both in vitro and in vivo. This provides a promising novel approach to determine the treatment outcomes, given that MIC alone is a poor predictor for treatment outcomes in infections caused by ASAF strains.
2026-02-26 | GSE309441 | 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