Copper Acts Synergistically with Fluconazole in Candida glabrata by Compromising Drug Efflux, Sterol Metabolism, and Zinc Homeostasis

The synergistic combinations of drugs are promising strategies to boost the effectiveness of current antifungals and thus prevent the emergence of resistance. In this work, we show that copper and the antifungal fluconazole act synergistically against Candida glabrata, an opportunistic pathogenic yeast intrinsically tolerant to fluconazole. Analyses of the transcriptomic profile of C. glabrata after the combination of copper and fluconazole showed that the expression of the multidrug transporter gene CDR1 was decreased, suggesting that fluconazole efflux could be affected. In agreement, we observed that copper inhibits the transactivation of Pdr1, the transcription regulator of multidrug transporters, and leads to the intracellular accumulation of fluconazole. Copper also decreases the transcriptional induction of ergosterol biosynthesis (ERG) genes by fluconazole, which culminates in the accumulation of toxic sterols. Co-treatment of cells with copper and fluconazole should affect the function of proteins located in the plasma membrane, as several ultrastructural alterations, including irregular cell wall and plasma membrane and loss of cell wall integrity, were observed. Finally, we show that the combination of copper and fluconazole downregulates the expression of the gene encoding the zinc-responsive transcription regulator Zap1, which possibly, together with the membrane transporters malfunction, generates zinc depletion. Supplementation with zinc reverts the toxic effect of combining copper with fluconazole, underscoring the importance of this metal in the observed synergistic effect. Overall, this work, while unveiling the molecular basis that supports the use of copper to enhance the effectiveness of fluconazole, paves the way for the development of new metal-based antifungal strategies.

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A novel class of Candida glabrata cell wall proteins with β-helix fold mediates adhesion in clinical isolates

PLoS Pathogens ◽

10.1371/journal.ppat.1009980 ◽

2021 ◽

Vol 17 (12) ◽

pp. e1009980

Author(s):

Viktoria Reithofer ◽

Jordan Fernández-Pereira ◽

María Alvarado ◽

Piet de Groot ◽

Lars-Oliver Essen

Keyword(s):

Cell Wall ◽

Candida Glabrata ◽

Sequence Similarity ◽

Domain Architecture ◽

Surface Characteristics ◽

Structural Similarity ◽

Cell Aggregation ◽

Phenotypic Analysis ◽

Pathogenic Yeast ◽

Opportunistic Pathogenic

Candida glabrata is an opportunistic pathogenic yeast frequently causing infections in humans. Though it lacks typical virulence factors such as hyphal development, C. glabrata contains a remarkably large and diverse set of putative wall adhesins that is crucial for its success as pathogen. Here, we present an analysis of putative adhesins from the homology clusters V and VI. First, sequence similarity network analysis revealed relationships between cluster V and VI adhesins and S. cerevisiae haze protective factors (Hpf). Crystal structures of A-domains from cluster VI adhesins Awp1 and Awp3b reveal a parallel right-handed β-helix domain that is linked to a C-terminal β-sandwich. Structure solution of the A-region of Awp3b via single wavelength anomalous diffraction phasing revealed the largest known lanthanide cluster with 21 Gd3+ ions. Awp1-A and Awp3b-A show structural similarity to pectate lyases but binding to neither carbohydrates nor Ca2+ was observed. Phenotypic analysis of awp1Δ, awp3Δ, and awp1,3Δ double mutants did also not confirm their role as adhesins. In contrast, deletion mutants of the cluster V adhesin Awp2 in the hyperadhesive clinical isolate PEU382 demonstrated its importance for adhesion to polystyrene or glass, biofilm formation, cell aggregation and other cell surface-related phenotypes. Together with cluster III and VII adhesins our study shows that C. glabrata CBS138 can rely on a set of 42 Awp1-related adhesins with β-helix/α-crystallin domain architecture for modifying the surface characteristics of its cell wall.

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CgCYN1, a Plasma Membrane Cystine-specific Transporter of Candida glabrata with Orthologues Prevalent among Pathogenic Yeast and Fungi

Journal of Biological Chemistry ◽

10.1074/jbc.m111.240648 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19714-19723 ◽

Cited By ~ 12

Author(s):

Amit Kumar Yadav ◽

Anand Kumar Bachhawat

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Candida Glabrata ◽

Histoplasma Capsulatum ◽

Kinetic Studies ◽

Sulfur Source ◽

Amino Acid Permease ◽

Pathogenic Yeast ◽

Cystine Transporter ◽

Energy Dependent

We describe a novel plasma membrane cystine transporter, CgCYN1, from Candida glabrata, the first such transporter to be described from yeast and fungi. C. glabrata met15Δ strains, organic sulfur auxotrophs, were observed to utilize cystine as a sulfur source, and this phenotype was exploited in the discovery of CgCYN1. Heterologous expression of CgCYN1 in Saccharomyces cerevisiae met15Δ strains conferred the ability of S. cerevisiae strains to grow on cystine. Deletion of the CgCYN1 ORF (CAGL0M00154g) in C. glabrata met15Δ strains caused abrogation of growth on cystine with growth being restored when CgCYN1 was reintroduced. The CgCYN1 protein belongs to the amino acid permease family of transporters, with no similarity to known plasma membrane cystine transporters of bacteria and humans, or lysosomal cystine transporters of humans/yeast. Kinetic studies revealed a Km of 18 ± 5 μm for cystine. Cystine uptake was inhibited by cystine, but not by other amino acids, including cysteine. The structurally similar cystathionine, lanthionine, and selenocystine alone inhibited transport, confirming that the transporter was specific for cystine. CgCYN1 localized to the plasma membrane and transport was energy-dependent. Functional orthologues could be demonstrated from other pathogenic yeast like Candida albicans and Histoplasma capsulatum, but were absent in Schizosaccharomyces pombe and S. cerevisiae.

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Multiple mechanisms impact fluconazole resistance of mutant Erg11 proteins in Candida glabrata

10.1101/2021.06.23.449691 ◽

2021 ◽

Author(s):

Bao Vu ◽

W. Scott Moye-Rowley

Keyword(s):

Transcription Factor ◽

Candida Glabrata ◽

Double Mutant ◽

Antifungal Drugs ◽

Cross Resistance ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Pathogenic Yeast ◽

Fluconazole Resistance ◽

Growth Defects

Azoles remain the most common used antifungal drugs for invasive candidiasis worldwide. They specifically inhibit the fungal lanosterol a-14 demethylase enzyme, which is commonly referred to as Erg11 in fungi. Inhibition of Erg11 ultimately leads to a reduction in ergosterol production, an essential fungal membrane sterol. Many Candida species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased azole resistance. Candida glabrata is also a pathogenic yeast that has a low intrinsic susceptibility to azole drugs and easily develops elevated resistance. These azole resistant mutations are almost exclusively found to cause hyperactivity of the Pdr1 transcription factor and rarely lie within the ERG11 gene. Here, we generated C. glabrata ERG11 mutations that were analogous to azole resistance associated mutations in C. albicans ERG11. Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. The azole resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. These data support the view that C. glabrata does not typically acquire ERG11 mutations owing to growth defects associated with these lesions while hyperactive PDR1 alleles have no obvious growth issues. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata.

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UV009 The human pathogenic yeast Candida glabrata: characterisation of the cell wall architecture and identification of cell wall proteins

International Journal of Medical Microbiology Supplements ◽

10.1016/s1433-1128(03)80588-0 ◽

2003 ◽

Vol 293 ◽

pp. 127

Keyword(s):

Cell Wall ◽

Candida Glabrata ◽

Cell Wall Proteins ◽

Pathogenic Yeast ◽

Cell Wall Architecture

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Transcripts of a gene encoding a putative cell wall-plasma membrane linker protein are specifically cold-induced in Brassica napus

Plant Molecular Biology ◽

10.1007/bf00019465 ◽

1996 ◽

Vol 31 (4) ◽

pp. 771-781 ◽

Cited By ~ 35

Author(s):

William Goodwin ◽

Jacqueline A. Pallas ◽

Gareth I. Jenkins

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Brassica Napus ◽

Gene Encoding

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Evidence that Ergosterol Biosynthesis Modulates Activity of the Pdr1 Transcription Factor inCandida glabrata

mBio ◽

10.1128/mbio.00934-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 7

Author(s):

Bao Gia Vu ◽

Grace Heredge Thomas ◽

W. Scott Moye-Rowley

Keyword(s):

Transcription Factor ◽

Candida Glabrata ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Atp Binding ◽

Pathogenic Yeast ◽

Content Type ◽

Atp Binding Cassette Transporter ◽

Encoding Gene ◽

Atp Binding Cassette

ABSTRACTA crucial limitation in antifungal chemotherapy is the limited number of antifungal drugs currently available. Azole drugs represent the most commonly used chemotherapeutic, and loss of efficacy of these drugs is a major risk factor in successful treatment of a variety of fungal diseases.Candida glabratais a pathogenic yeast that is increasingly found associated with bloodstream infections, a finding likely contributed to by its proclivity to develop azole drug resistance.C. glabrataoften acquires azole resistance via gain-of-function (GOF) mutations in the transcription factor Pdr1. These GOF forms of Pdr1 drive elevated expression of target genes, including the ATP-binding cassette transporter-encodingCDR1locus. GOF alleles ofPDR1have been extensively studied, but little is known of how Pdr1 is normally regulated. Here we test the idea that reduction of ergosterol biosynthesis (as occurs in the presence of azole drugs) might trigger activation of Pdr1 function. Using two different means of genetically inhibiting ergosterol biosynthesis, we demonstrated that Pdr1 activity and target gene expression are elevated in the absence of azole drug. Blocks at different points in the ergosterol pathway lead to Pdr1 activation as well as to induction of other genes in this pathway. Delivery of the signal from the ergosterol pathway to Pdr1 involves the transcription factor Upc2A, anERGgene regulator. We show that Upc2A binds directly to thePDR1andCDR1promoters. Our studies argue for a physiological link between ergosterol biosynthesis and Pdr1-dependent gene regulation that is not restricted to efflux of azole drugs.IMPORTANCEA likely contributor to the increased incidence of non-albicanscandidemias involvingCandida glabratais the ease with which this yeast acquires azole resistance, in large part due to induction of the ATP-binding cassette transporter-encoding geneCDR1. Azole drugs lead to induction of Pdr1 transactivation, with a central model being that this factor binds these drugs directly. Here we provide evidence that Pdr1 is activated without azole drugs by the use of genetic means to inhibit expression of azole drug target-encoding geneERG11. These acute reductions in Erg11 levels lead to elevated Pdr1 activity even though no drug is present. A key transcriptional regulator of theERGpathway, Upc2A, is shown to directly bind to thePDR1andCDR1promoters. We interpret these data as support for the view that Pdr1 function is responsive to ergosterol biosynthesis and suggest that this connection reveals the normal physiological circuitry in which Pdr1 participates.

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Candida glabrata Transcription Factor Rpn4 Mediates Fluconazole Resistance through Regulation of Ergosterol Biosynthesis and Plasma Membrane Permeability

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00554-20 ◽

2020 ◽

Vol 64 (9) ◽

Cited By ~ 2

Author(s):

Pedro Pais ◽

Raquel Califórnia ◽

Mónica Galocha ◽

Romeu Viana ◽

Mihaela Ola ◽

...

Keyword(s):

Transcription Factor ◽

Plasma Membrane ◽

Candida Glabrata ◽

Cell Permeability ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Binding Motif ◽

Plasma Membrane Permeability ◽

Content Type ◽

Content Understanding

ABSTRACT The ability to acquire azole resistance is an emblematic trait of the fungal pathogen Candida glabrata. Understanding the molecular basis of azole resistance in this pathogen is crucial for designing more suitable therapeutic strategies. This study shows that the C. glabrata transcription factor (TF) CgRpn4 is a determinant of azole drug resistance. RNA sequencing during fluconazole exposure revealed that CgRpn4 regulates the expression of 212 genes, activating 80 genes and repressing, likely in an indirect fashion, 132 genes. Targets comprise several proteasome and ergosterol biosynthesis genes, including ERG1, ERG2, ERG3, and ERG11. The localization of CgRpn4 to the nucleus increases upon fluconazole stress. Consistent with a role in ergosterol and plasma membrane homeostasis, CgRpn4 is required for the maintenance of ergosterol levels upon fluconazole stress, which is associated with a role in the upkeep of cell permeability and decreased intracellular fluconazole accumulation. We provide evidence that CgRpn4 directly regulates ERG11 expression through the TTGCAAA binding motif, reinforcing the relevance of this regulatory network in azole resistance. In summary, CgRpn4 is a new regulator of the ergosterol biosynthesis pathway in C. glabrata, contributing to plasma membrane homeostasis and, thus, decreasing azole drug accumulation.

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Essential Role for the Phosphatidylinositol 3,5-Bisphosphate Synthesis Complex in Caspofungin Tolerance and Virulence in Candida glabrata

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00886-19 ◽

2019 ◽

Vol 63 (8) ◽

Cited By ~ 1

Author(s):

Deepak Kumar Choudhary ◽

Priyanka Bhakt ◽

Rupinder Kaur

Keyword(s):

Cell Wall ◽

Candida Glabrata ◽

Scaffolding Protein ◽

Pathogenic Yeast ◽

Lipid Kinase ◽

Lipid Molecule ◽

Content Type ◽

Phosphoinositide Signaling ◽

Promising Target ◽

Opportunistic Fungal Pathogen

ABSTRACT Increasing resistance of the human opportunistic fungal pathogen Candida glabrata toward the echinocandin antifungals, which target the cell wall, is a matter of grave clinical concern. Echinocandin resistance in C. glabrata has primarily been associated with mutations in the β-glucan synthase-encoding genes C. glabrata FKS1 (CgFKS1) and CgFKS2. This notwithstanding, the role of the phosphoinositide signaling in antifungal resistance is just beginning to be deciphered. The phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance lipid molecule that is pivotal to the intracellular membrane traffic. Here, we demonstrate for the first time that the PI(3,5)P2 kinase CgFab1, along with its activity regulator CgVac7 and the scaffolding protein CgVac14, is required for maintenance of the cell wall chitin content, survival of the cell wall, and caspofungin stress. Further, deletion analyses implicated the PI(3,5)P2 phosphatase CgFig4 in the regulation of PI(3,5)P2 levels and azole and echinocandin tolerance through CgVac14. We also show the localization of the CgFab1 lipid kinase to the vacuole to be independent of the CgVac7, CgVac14, and CgFig4 proteins. Lastly, our data demonstrate an essential requirement for PI(3,5)P2 signaling components, CgFab1, CgVac7, and CgVac14, in the intracellular survival and virulence in C. glabrata. Altogether, our data have yielded key insights into the functions and metabolism of PI(3,5)P2 lipid in the pathogenic yeast C. glabrata. In addition, our data highlight that CgVac7, whose homologs are absent in higher eukaryotes, may represent a promising target for antifungal therapy.

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