Azole-resistant alleles of ERG11 in Candida glabrata trigger activation of the Pdr1 and Upc2A transcription factors.

Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-14 demethylase enzyme, which is referred to as Erg11. 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 resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. In C. glabrata , these azole resistant mutations typically 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 alleles from 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. Resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. Crucially, the presence of the DM ERG11 allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in ERG11 activity can involve cellular effects beyond an alteration in this key azole target enzyme. 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 .

The Gβ-like Protein AfCpcB Affects Sexual Development, Response to Oxidative Stress and Phagocytosis by Alveolar Macrophages in Aspergillus fumigatus

G-protein signaling is important for signal transduction, allowing various stimuli that are external to a cell to affect its internal molecules. In Aspergillus fumigatus, the roles of Gβ-like protein CpcB on growth, asexual development, drug sensitivity, and virulence in a mouse model have been previously reported. To gain a deeper insight into Aspergillus fumigatus sexual development, the ΔAfcpcB strain was generated using the supermater AFB62 strain and crossed with AFIR928. This cross yields a decreased number of cleistothecia, including few ascospores. The sexual reproductive organ-specific transcriptional analysis using RNAs from the cleistothecia (sexual fruiting bodies) indicated that the CpcB is essential for the completion of sexual development by regulating the transcription of sexual genes, such as veA, steA, and vosA. The ΔAfcpcB strain revealed increased resistance to oxidative stress by regulating genes for catalase, peroxiredoxin, and ergosterol biosynthesis. The ΔAfcpcB strain showed decreased uptake by alveolar macrophages in vitro, decreased sensitivity to Congo red, decreased expression of cell wall genes, and increased expression of the hydrophobin genes. Taken together, these findings indicate that AfCpcB plays important roles in sexual development, phagocytosis by alveolar macrophages, biosynthesis of the cell wall, and oxidative stress response.

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

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.

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.

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.

Role for the phosphatidylinositol 3-phosphate 5-kinase in antifungal tolerance in Candida glabrata

Candida glabrata is an opportunistic fungal pathogen of humans, which is intrinsically less susceptible to widely used azole antifungals, that block ergosterol biosynthesis. The major azole resistance mechanisms include mitochondrial dysfunction and multidrug efflux pump overexpression. In the current study, we have uncovered an essential role for the actin cytoskeletal network reorganization in survival of the azole stress. We demonstrate for the first time that the azole antifungal fluconazole induces remodelling of the actin cytoskeleton in C. glabrata, and genetic or chemical perturbation of actin structures results in intracellular sterol accumulation and azole susceptibility. Further, we showed that the vacuolar membrane-resident phosphatidylinositol 3-phosphate 5-kinase (CgFab1) is pivotal to this process, as CgFAB1 disruption impaired vacuole homeostasis and actin organization. We also showed that the actin depolymerization factor CgCof1 binds to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), and CgCof1 distribution along with the actin filament-capping protein CgCap2 is altered upon both CgFAB1disruption and fluconazole exposure. Additionally, while the F-actin-stabilizing compound jasplakinolide rescued azole toxicity in cytoskeleton defective-mutants, the actin polymerization inhibitor latrunculin B rendered fluconazole fully and partially fungicidal in azole-susceptible and azole-resistant C. glabrata clinical isolates, respectively. These data underscore the essentiality of actin cytoskeleton reorganization for azole stress survival. Lastly, we have also shown a pivotal role of CgFab1 kinase activity regulators, CgFig4, CgVac7 and CgVac14, through genetic analysis, in azole and echinocandin antifungal tolerance. Altogether, I shall present our findings on functions and metabolism of the PI(3,5)P2 lipid in antifungal tolerance and virulence of C. glabrata.

Characterisation of a Novel Benzopyran Library Using High-Throughput Microscopy

<p>Drug discovery is a multi-disciplinary field incorporating both chemistry and biology to create novel pharmaceuticals. Nature synthesizes a diverse range of chemical entities that can demonstrate a wide range of biological interactions, though often produces these compounds in small amounts. Using natures structural diversity as a template, organic synthetic chemistry can tap into the structures of natural products and provide novel structures as well as overcome supply issues through large-scale synthetic chemical processes. A novel benzopyran library was synthesised by Sandile Simelane by reacting 3,4,6,-tri-O-acetyl-D-galactal with various phenols to create a novel focused library of bridged benzopyrans. Each molecule has unique functional groups at defined points in the structure due to varying the functional groups on the phenol, allowing for variation within the library whilst retaining the core scaffold. In this thesis, the bioactivity of this novel benzopyran library was explored using a phenotypic screen measuring growth inhibition. A compound, S13, was determined to be the most potent in the library, therefore genome-wide screening was performed using S13. High-throughput microscopy of 4,100 strains, each with a different GFP-tagged protein, was utilized to determine proteins that increased in abundance or changed localization in response to perturbation with S13. Following treatment with S13, the yeast vacuole increased in size due to an aggregation of proteins in the vacuolar lumen. The increase in vacuole size was coincident with a decrease in vacuolar acidity, potentially disrupted autophagy and the upregulation of several proteins involved in ergosterol biosynthesis. Together, these results reveal a novel bridged benzopyran that increases vacuolar size and pH through an epistatic mechanism involving ergosterol biosynthesis.</p>

Characterisation of a Novel Benzopyran Library Using High-Throughput Microscopy

<p>Drug discovery is a multi-disciplinary field incorporating both chemistry and biology to create novel pharmaceuticals. Nature synthesizes a diverse range of chemical entities that can demonstrate a wide range of biological interactions, though often produces these compounds in small amounts. Using natures structural diversity as a template, organic synthetic chemistry can tap into the structures of natural products and provide novel structures as well as overcome supply issues through large-scale synthetic chemical processes. A novel benzopyran library was synthesised by Sandile Simelane by reacting 3,4,6,-tri-O-acetyl-D-galactal with various phenols to create a novel focused library of bridged benzopyrans. Each molecule has unique functional groups at defined points in the structure due to varying the functional groups on the phenol, allowing for variation within the library whilst retaining the core scaffold. In this thesis, the bioactivity of this novel benzopyran library was explored using a phenotypic screen measuring growth inhibition. A compound, S13, was determined to be the most potent in the library, therefore genome-wide screening was performed using S13. High-throughput microscopy of 4,100 strains, each with a different GFP-tagged protein, was utilized to determine proteins that increased in abundance or changed localization in response to perturbation with S13. Following treatment with S13, the yeast vacuole increased in size due to an aggregation of proteins in the vacuolar lumen. The increase in vacuole size was coincident with a decrease in vacuolar acidity, potentially disrupted autophagy and the upregulation of several proteins involved in ergosterol biosynthesis. Together, these results reveal a novel bridged benzopyran that increases vacuolar size and pH through an epistatic mechanism involving ergosterol biosynthesis.</p>

Identification of Six Thiolases and their Effects on Fatty Acid and Ergosterol Biosynthesis in Aspergillus oryzae

Thiolase plays important roles in lipid metabolism. It can be divided into degradative thiolases (Thioase I) and biosynthetic thiolases (thiolases II), which are involved in fatty acid β-oxidation and acetoacetyl-CoA biosynthesis, respectively. The Saccharomyces cerevisiae (S. cerevisiae) genome harbors only one gene each for thioase I and thiolase II, namely, Pot1 and Erg10, respectively. In this study, six thiolases (named AoErg10A−AoErg10F) were identified in Aspergillus oryzae (A. oryzae) genome using bioinformatics analysis. Quantitative reverse transcription–PCR (qRT-PCR) indicated that the expression of these six thiolases varied at different growth stages and under different forms of abiotic stress. Subcellular localization analysis showed that AoErg10A was located in the cytoplasm, AoErg10B and AoErg10C in the mitochondria, and AoErg10D-AoErg10F in the peroxisome. Yeast heterologous complementation assays revealed that AoErg10A, AoErg10D, AoErg10E, AoErg10F and cytoplasmic AoErg10B (AoErg10BΔMTS) recovered the phenotypes of S. cerevisiae erg10 weak and lethal mutants, and that only AoErg10D-F recovered the phenotype of the pot1 mutant that cannot use oleic acid as the carbon source. Overexpression of AoErg10s either affected the growth speed or sporulation of the transgenic strains. In addition, the fatty acid and ergosterol content changed in all the AoErg10-overexpressing strains. This study revealed the function of six thiolases in A. oryzae and their effect on growth, and fatty acid and ergosterol biosynthesis, which may lay the foundation for genetic engineering for lipid metabolism in A. oryzae or other fungi.ImportanceThiolase including thioase I and thiolase II, plays important roles in lipid metabolism. A. oryzae, one of the most industrially important filamentous fungi, has been widely used for manufacturing oriental fermented food such as sauce, miso, and sake for a long time. Besides, A. oryzae has a high capability in production of high lipid content and has been used for lipid production. Thus, it is very important to investiagte the function of thiolases in A. oryzae. In this study, six thiolase (named AoErg10A-AoErg10F) were identified by bioinformatics analysis. Unlike other reported thiolases in fungi, three of the six thiolases showed dual function of thioase I and thiolase II in S. cerevisiae, indicating the lipid metabolism is more complex in A. oryzae. The reveal of founction of these thiolases in A. oryzae can lay the foundation for genetic engineering for lipid metabolism in A. oryzae or other fungi.