Loss-of-Function
            ROX1
            Mutations Suppress the Fluconazole Susceptibility of
            upc2A
            Δ Mutation in Candida glabrata, Implicating Additional Positive Regulators of Ergosterol Biosynthesis

Candida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole-class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway.

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Loss-of-function ROX1 mutations suppress the fluconazole susceptibility of upc2AΔ mutation in Candida glabrata, implicating additional positive regulators of ergosterol biosynthesis

10.1101/2021.10.07.463606 ◽

2021 ◽

Author(s):

Tomye L Ollinger ◽

Bao Vu ◽

Daniel Murante ◽

Josie Parker ◽

Lucia Simonicova ◽

...

Keyword(s):

Candida Glabrata ◽

Genetic Interaction ◽

Pathogenic Fungi ◽

Antifungal Drugs ◽

Negative Regulator ◽

Ergosterol Biosynthesis ◽

Loss Of Function ◽

Candida Spp ◽

Ergosterol Biosynthesis Inhibitors ◽

Positive Regulators

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress induced transcription factors such as Upc2 and Upc2A as well as homologs of Sterol Response Element Binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2AΔ deletion mutant. This led to the identification of loss of function mutants in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2AΔ mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels or ergosterol and decreased levels of the toxic sterol, 14α methyl-ergosta-8,24(28)-dien-3β, 6α-diol, relative to upc2AΔ. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata.

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Structural Insights into Binding of the Antifungal Drug Fluconazole to Saccharomyces cerevisiae Lanosterol 14α-Demethylase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00925-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4982-4989 ◽

Cited By ~ 69

Author(s):

Alia A. Sagatova ◽

Mikhail V. Keniya ◽

Rajni K. Wilson ◽

Brian C. Monk ◽

Joel D. A. Tyndall

Keyword(s):

Saccharomyces Cerevisiae ◽

Fungal Pathogens ◽

Structural Information ◽

Resistance Mechanisms ◽

Antifungal Drugs ◽

Ergosterol Biosynthesis ◽

Limiting Factor ◽

Content Type ◽

Structural Insights ◽

Insight Into

ABSTRACTInfections by fungal pathogens such asCandida albicansandAspergillus fumigatusand their resistance to triazole drugs are major concerns. Fungal lanosterol 14α-demethylase belongs to the CYP51 class in the cytochrome P450 superfamily of enzymes. This monospanning bitopic membrane protein is involved in ergosterol biosynthesis and is the primary target of azole antifungal drugs, including fluconazole. The lack of high-resolution structural information for this drug target from fungal pathogens has been a limiting factor for the design of modified triazole drugs that will overcome resistance. Here we report the X-ray structure of full-lengthSaccharomyces cerevisiaelanosterol 14α-demethylase in complex with fluconazole at a resolution of 2.05 Å. This structure shows the key interactions involved in fluconazole binding and provides insight into resistance mechanisms by revealing a water-mediated hydrogen bonding network between the drug and tyrosine 140, a residue frequently found mutated to histidine or phenylalanine in resistant clinical isolates.

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Caenorhabditis elegans as a Model System To Assess Candida glabrata, Candida nivariensis, and Candida bracarensis Virulence and Antifungal Efficacy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00824-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

Ainara Hernando-Ortiz ◽

Estibaliz Mateo ◽

Marcelo Ortega-Riveros ◽

Iker De-la-Pinta ◽

Guillermo Quindós ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Candida Glabrata ◽

Model System ◽

Antifungal Drugs ◽

Phenotypic Traits ◽

Content Type ◽

C Elegans ◽

Host Fungus ◽

Candida Bracarensis ◽

Candida Nivariensis

ABSTRACT Although Candida albicans remains the major etiological agent of invasive candidiasis, Candida glabrata and other emerging species of Candida are increasingly isolated. This species is the second most prevalent cause of candidiasis in many regions of the world. However, clinical isolates of Candida nivariensis and Candida bracarensis can be misidentified and are underdiagnosed due to phenotypic traits shared with C. glabrata. Little is known about the two cryptic species. Therefore, pathogenesis studies are needed to understand their virulence traits and their susceptibility to antifungal drugs. The susceptibility of Caenorhabditis elegans to different Candida species makes this nematode an excellent model for assessing host-fungus interactions. We evaluated the usefulness of C. elegans as a nonconventional host model to analyze the virulence of C. glabrata, C. nivariensis, and C. bracarensis. The three species caused candidiasis, and the highest virulence of C. glabrata was confirmed. Furthermore, we determined the efficacy of current antifungal drugs against the infection caused by these species in the C. elegans model. Amphotericin B and azoles showed the highest activity against C. glabrata and C. bracarensis infections, while echinocandins were more active for treating those caused by C. nivariensis. C. elegans proved to be a useful model system for assessing the pathogenicity of these closely related species.

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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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Novel Antifungal Drug Discovery Based on Targeting Pathways Regulating the Fungus-Conserved Upc2 Transcription Factor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01677-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 258-266 ◽

Cited By ~ 24

Author(s):

Christina Gallo-Ebert ◽

Melissa Donigan ◽

Ilana L. Stroke ◽

Robert N. Swanson ◽

Melissa T. Manners ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Dna Binding ◽

Fungal Pathogens ◽

Direct Interaction ◽

Antifungal Drugs ◽

Sterol Biosynthesis ◽

Content Type ◽

Stressed Cells

ABSTRACTInfections byCandida albicansand related fungal pathogens pose a serious health problem for immunocompromised patients. Azole drugs, the most common agents used to combat infections, target the sterol biosynthetic pathway. Adaptation to azole therapy develops as drug-stressed cells compensate by upregulating several genes in the pathway, a process mediated in part by the Upc2 transcription factor. We have implemented a cell-based high-throughput screen to identify small-molecule inhibitors of Upc2-dependent induction of sterol gene expression in response to azole drug treatment. The assay is designed to identify not only Upc2 DNA binding inhibitors but also compounds impeding the activation of gene expression by Upc2. An AlphaScreen assay was developed to determine whether the compounds identified interact directly with Upc2 and inhibit DNA binding. Three compounds identified by the cell-based assay inhibited Upc2 protein level andUPC2-LacZgene expression in response to a block in sterol biosynthesis. The compounds were growth inhibitory and attenuated antifungal-induced sterol gene expressionin vivo. They did so by reducing the level of Upc2 protein and Upc2 DNA binding in the presence of drug. The mechanism by which the compounds restrict Upc2 DNA binding is not through a direct interaction, as demonstrated by a lack of DNA binding inhibitory activity using the AlphaScreen assay. Rather, they likely inhibit a novel pathway activating Upc2 in response to a block in sterol biosynthesis. We suggest that the compounds identified represent potential precursors for the synthesis of novel antifungal drugs.

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Amino Acid-Derived 1,2-Benzisothiazolinone Derivatives as Novel Small-Molecule Antifungal Inhibitors: Identification of Potential Genetic Targets

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00477-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4630-4639 ◽

Cited By ~ 18

Author(s):

Deepu Alex ◽

Francoise Gay-Andrieu ◽

Jared May ◽

Linta Thampi ◽

Dengfeng Dou ◽

...

Keyword(s):

Amino Acid ◽

Fungal Pathogens ◽

Structural Similarity ◽

Homozygous Deletion ◽

Antifungal Drugs ◽

Respiratory Pathway ◽

Phenotypic Data ◽

Content Type ◽

Mitochondrial Functions ◽

Phenotype Analysis

ABSTRACTWe have identified four synthetic compounds (DFD-VI-15, BD-I-186, DFD-V-49, and DFD-V-66) from an amino acid-derived 1,2-benzisothiazolinone (BZT) scaffold that have reasonable MIC50values against a panel of fungal pathogens. These compounds have no structural similarity to existing antifungal drugs. Three of the four compounds have fungicidal activity againstCandidaspp.,Cryptococcus neoformans, and several dermatophytes, while one is fungicidal toAspergillus fumigatus. The kill rates of our compounds are equal to those in clinical usage. The BZT compounds remain active against azole-, polyene-, and micafungin-resistant strains ofCandidaspp. A genetics-based approach, along with phenotype analysis, was used to begin mode of action (MOA) studies of one of these compounds, DFD-VI-15. The genetics-based screen utilized a homozygous deletion collection of approximately 4,700Saccharomyces cerevisiaemutants. We identified mutants that are both hypersensitive and resistant. Using FunSpec, the hypersensitive mutants and a resistantace2mutant clustered within a category of genes related directly or indirectly to mitochondrial functions. InCandida albicans, the functions of the Ace2p transcription factor include the regulation of glycolysis. Our model is that DFD-VI-15 targets a respiratory pathway that limits energy production. Supporting this hypothesis are phenotypic data indicating that DFD-VI-15 causes increased cell-reactive oxidants (ROS) and a decrease in mitochondrial membrane potential. Also, the same compound has activity when cells are grown in a medium containing glycerol (mitochondrial substrate) but is much less active when cells are grown anaerobically.

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Crystal Structures of Full-Length Lanosterol 14α-Demethylases of Prominent Fungal Pathogens Candida albicans and Candida glabrata Provide Tools for Antifungal Discovery

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01134-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 9

Author(s):

Mikhail V. Keniya ◽

Manya Sabherwal ◽

Rajni K. Wilson ◽

Matthew A. Woods ◽

Alia A. Sagatova ◽

...

Keyword(s):

Candida Albicans ◽

Crystal Structures ◽

Candida Glabrata ◽

Fungal Pathogens ◽

Catalytic Domain ◽

Fungal Infections ◽

Binding Pocket ◽

Full Length ◽

Content Type ◽

Cure Rates

ABSTRACT Targeting lanosterol 14α-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are not optimal for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed the need to improve the potency, spectrum, and specificity for azoles by expressing in Saccharomyces cerevisiae functional, recombinant, hexahistidine-tagged, full-length Candida albicans LDM (CaLDM6×His) and Candida glabrata LDM (CgLDM6×His) and determining their X-ray crystal structures. The crystal structures of CaLDM6×His, CgLDM6×His, and ScLDM6×His have the same fold and bind itraconazole in nearly identical conformations. The catalytic domains of the full-length LDMs have the same fold as the CaLDM6×His catalytic domain in complex with posaconazole, with minor structural differences within the ligand binding pocket. Our structures give insight into the LDM reaction mechanism and phenotypes of single-site CaLDM mutations. This study provides a practical basis for the structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.

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Targeting Methionine Synthase in a Fungal Pathogen Causes a Metabolic Imbalance That Impacts Cell Energetics, Growth, and Virulence

mBio ◽

10.1128/mbio.01985-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Jennifer Scott ◽

Monica Sueiro-Olivares ◽

Benjamin P. Thornton ◽

Rebecca A. Owens ◽

Howbeer Muhamadali ◽

...

Keyword(s):

Beneficial Effect ◽

Aspergillus Fumigatus ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Antifungal Drugs ◽

New Drugs ◽

Methionine Synthase ◽

Content Type ◽

Metabolic Imbalance

ABSTRACT There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose to human health. Here, we have performed a comprehensive characterization and validation of the promising target methionine synthase (MetH). We show that in Aspergillus fumigatus the absence of this enzymatic activity triggers a metabolic imbalance that causes a reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine. Interestingly, growth can be recovered in the presence of certain metabolites, which shows that metH is a conditionally essential gene and consequently should be targeted in established infections for a more comprehensive validation. Accordingly, we have validated the use of the tetOFF genetic model in fungal research and improved its performance in vivo to achieve initial validation of targets in models of established infection. We show that repression of metH in growing hyphae halts growth in vitro, which translates into a beneficial effect when targeting established infections using this model in vivo. Finally, a structure-based virtual screening of methionine synthases reveals key differences between the human and fungal structures and unravels features in the fungal enzyme that can guide the design of novel specific inhibitors. Therefore, methionine synthase is a valuable target for the development of new antifungals. IMPORTANCE Fungal pathogens are responsible for millions of life-threatening infections on an annual basis worldwide. The current repertoire of antifungal drugs is very limited and, worryingly, resistance has emerged and already become a serious threat to our capacity to treat fungal diseases. The first step to develop new drugs is often to identify molecular targets in the pathogen whose inhibition during infection can prevent its growth. However, the current models are not suitable to validate targets in established infections. Here, we have characterized the promising antifungal target methionine synthase in great detail, using the prominent fungal pathogen Aspergillus fumigatus as a model. We have uncovered the underlying reason for its essentiality and confirmed its druggability. Furthermore, we have optimized the use of a genetic system to show a beneficial effect of targeting methionine synthase in established infections. Therefore, we believe that antifungal drugs to target methionine synthase should be pursued and additionally, we provide a model that permits gaining information about the validity of antifungal targets in established infections.

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Erg4A and Erg4B Are Required for Conidiation and Azole Resistance via Regulation of Ergosterol Biosynthesis in Aspergillus fumigatus

Applied and Environmental Microbiology ◽

10.1128/aem.02924-16 ◽

2016 ◽

Vol 83 (4) ◽

Cited By ~ 11

Author(s):

Nanbiao Long ◽

Xiaoling Xu ◽

Qiuqiong Zeng ◽

Hong Sang ◽

Ling Lu

Keyword(s):

Endoplasmic Reticulum ◽

Aspergillus Fumigatus ◽

Plasma Membranes ◽

Current Knowledge ◽

Hyphal Growth ◽

Antifungal Drugs ◽

Ergosterol Biosynthesis ◽

Wild Type ◽

Content Type ◽

Ergosterol Synthesis

ABSTRACT Ergosterol, a fungus-specific sterol enriched in cell plasma membranes, is an effective antifungal drug target. However, current knowledge of the ergosterol biosynthesis process in the saprophytic human fungal pathogen Aspergillus fumigatus remains limited. In this study, we found that two endoplasmic reticulum-localized sterol C-24 reductases encoded by both erg4A and erg4B homologs are required to catalyze the reaction during the final step of ergosterol biosynthesis. Loss of one homolog of Erg4 induces the overexpression of the other one, accompanied by almost normal ergosterol synthesis and wild-type colony growth. However, double deletions of erg4A and erg4B completely block the last step of ergosterol synthesis, resulting in the accumulation of ergosta-5,7,22,24(28)-tetraenol, a precursor compound of ergosterol. Further studies indicate that erg4A and erg4B are required for conidiation but not for hyphal growth. Importantly, the Δerg4A Δerg4B mutant still demonstrates wild-type virulence in a compromised mouse model but displays remarkable increased susceptibility to antifungal azoles. Our data suggest that inhibitors of Erg4A and Erg4B may serve as effective candidates for adjunct antifungal agents with azoles. IMPORTANCE Knowledge of the ergosterol biosynthesis pathway in the human opportunistic pathogen A. fumigatus is useful for designing and finding new antifungal drugs. In this study, we demonstrated that the endoplasmic reticulum-localized sterol C-24 reductases Erg4A and Erg4B are required for conidiation via regulation of ergosterol biosynthesis. Moreover, inactivation of both Erg4A and Erg4B results in hypersensitivity to the clinical guideline-recommended antifungal drugs itraconazole and voriconazole. Therefore, our finding indicates that inhibition of Erg4A and Erg4B might be an effective approach for alleviating A. fumigatus infection.

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A Computational Approach to Predict the Molecular Drug Targets Against Candida glabrata

Bioscience Biotechnology Research Communications ◽

10.21786/bbrc/14.4.84 ◽

2021 ◽

Vol 14 (4) ◽

pp. 1946-1955

Author(s):

Priyanka Sirari

Keyword(s):

Green Tea ◽

In Silico ◽

Candida Glabrata ◽

Drug Targets ◽

Antifungal Drugs ◽

High Rate ◽

Ergosterol Biosynthesis ◽

Drug Candidates ◽

Beneficial Effects ◽

Potent Drug

Non- albicans Candida (NAC) species are responsible for 35-65% of all candidaemias in the general population and are associated with a high rate of morbidity and mortality (about 15% to 35%). The availability of few commercially used antifungal drugs against candidiasis and rapid emergences of antibiotic resistance among NAC species has significantly contributed to their increased global outbreak. Green tea is known for its multi-beneficial effects including antimicrobial potential against Candida. The present study investigated the molecular drug targets of green tea phytocompounds against inhibition of ergosterol biosynthesis in Candida glabrata using in silico tools.The molecular interaction was studied between ligands and essential proteins participating in ergosterol biosynthesis in C. glabrata using autodockvina software. The protein validation and homology modeling estimation were determined by the SWISS MODEL workspace. The Drug likeness study of all the test ligands was performed using SwissADME, while the toxicity of test compounds was analyzed using the admetSAR 2.0 version.The in silico analyses identified Rutin, Chlorogenic acid, Coumaroylquinic acid, Quercetin, Epigallocatechingallate as the potent phytocompounds with significant molecular binding with Erg 6, Erg 27, Erg 8, Erg 7, Erg 24 respectively. The ADMET data suggested an absence of the CYP2 inhibitors indicating the metabolism of all the tested drug candidates in the intestine and liver.The present study highlighted the possible drug targets of green tea phytocompounds against ergosterol biosynthesis protein in C. glabrata. It is pertinent that the current study has provided preliminary breakthroughs which could lead to exploring their avenues in potent drug development against NAC species.

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