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Two Functionally Redundant FK506-Binding Proteins Regulate Multidrug Resistance Gene Expression and Govern Azole
Romila Moirangthem1, Kundan Kumar1,2, Rupinder Kaur3
1Laboratory of Fungal Pathogenesis, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India.
Abstract:
Increasing resistance to antifungal therapy is an impediment to the effective treatment of fungal infections. Candida glabrata is an opportunistic human fungal pathogen that is inherently less susceptible to cost-effective azole antifungals. Gain-of-function mutations in the Zn-finger pleiotropic drug resistance transcriptional activator-encoding gene CgPDR1 are the most prevalent causes of azole resistance in clinical settings. CgPDR1 is also transcriptionally activated upon azole exposure; however, factors governing CgPDR1 gene expression are not yet fully understood. Here, we have uncovered a novel role for two FK506-binding proteins, CgFpr3 and CgFpr4, in the regulation of the CgPDR1 regulon. We show that CgFpr3 and CgFpr4 possess a peptidyl-prolyl isomerase domain and act redundantly to control CgPDR1 expression, as a Cgfpr3Δ4Δ mutant displayed elevated expression of the CgPDR1 gene along with overexpression of its target genes, CgCDR1, CgCDR2, and CgSNQ2, which code for ATP-binding cassette multidrug transporters. Furthermore, CgFpr3 and CgFpr4 are required for the maintenance of histone H3 and H4 protein levels, and fluconazole exposure leads to elevated H3 and H4 protein levels. Consistent with the role of histone proteins in azole resistance, disruption of genes coding for the histone demethylase CgRph1 and the histone H3K36-specific methyltransferase CgSet2 leads to increased and decreased susceptibility to fluconazole, respectively, with the Cgrph1Δ mutant displaying significantly lower basal expression levels of the CgPDR1 and CgCDR1 genes. These data underscore a hitherto unknown role of histone methylation in modulating the most common azole antifungal resistance mechanism. Altogether, our findings establish a link between CgFpr-mediated histone homeostasis and CgPDR1 gene expression and implicate CgFpr in the virulence of C. glabrata.
Insights
Two FK506-binding proteins, CgFpr3 and CgFpr4, regulate azole antifungal resistance in Candida glabrata by controlling CgPDR1 gene expression and maintaining histone homeostasis. Their absence increases resistance, highlighting a novel role in fungal virulence.
Area of Science:
- Molecular Biology
- Mycology
- Antimicrobial Resistance
Background:
- Antifungal resistance is a major challenge in treating fungal infections, particularly with azole antifungals.
- Candida glabrata exhibits inherent resistance to azoles, often due to mutations in the CgPDR1 gene.
- Understanding the regulation of CgPDR1 gene expression is crucial for developing effective antifungal strategies.
Purpose of the Study:
- To investigate novel factors regulating CgPDR1 gene expression and azole resistance in Candida glabrata.
- To elucidate the role of FK506-binding proteins (Fpr) in modulating antifungal drug resistance mechanisms.
- To explore the connection between histone homeostasis and azole resistance pathways.
Main Methods:
- Genetic analysis of Candida glabrata mutants lacking CgFpr3 and CgFpr4 (Cgfpr3Δ4Δ).
- Quantitative analysis of CgPDR1, CgCDR1, CgCDR2, and CgSNQ2 gene expression.
- Assessment of histone H3 and H4 protein levels and susceptibility to fluconazole.
- Gene disruption of histone modifiers CgRph1 and CgSet2 to evaluate their impact on azole resistance.
Main Results:
- The Cgfpr3Δ4Δ mutant showed significantly elevated expression of CgPDR1 and its target genes (CgCDR1, CgCDR2, CgSNQ2), indicating increased azole resistance.
- CgFpr3 and CgFpr4 are essential for maintaining histone H3 and H4 protein levels; fluconazole exposure increased these levels.
- Disruption of CgRph1 (histone demethylase) increased fluconazole susceptibility and decreased CgPDR1/CgCDR1 expression, while CgSet2 (H3K36 methyltransferase) disruption decreased susceptibility.
Conclusions:
- CgFpr3 and CgFpr4 play a redundant, critical role in regulating CgPDR1 expression and maintaining histone homeostasis in Candida glabrata.
- Histone methylation is a novel regulatory mechanism influencing the prevalent azole antifungal resistance pathway in C. glabrata.
- These findings link CgFpr-mediated histone regulation to CgPDR1 expression, suggesting CgFpr proteins are potential targets for enhancing antifungal therapies and impacting C. glabrata virulence.
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