Yeast strains and vectors
Yeast strains used in this study are shown in Table 1.
Selection of yeast strains carrying cab1 mutations
cab1Δ/pFL38-CAB1 wild type and mutant strains were generated using plasmid shuffling as previously described from the parent strain cab1Δ/pFL39-cab1G351S 16. Briefly, pFL38-CAB1 plasmids with wild type or mutant cab1 (cab1G351S, cab1N290I, and cab1S158A) were transformed into a cab1Δ/pFL39-cab1G351S strain. Transformants were selected on minimal medium lacking uracil and tryptophan. Plasmid loss of pFL39-cab1G351S from the strains were conducted by growing the strains in the minimal media supplemented with tryptophan and 5-fluoroanthranilic acid (5-FAA) but lacking uracil. The loss of the pFL39-cab1G351S plasmid was confirmed by growth tests on medium lacking tryptophan. Add-back strains were generated by introducing pFL39-CAB1 vector into yeast recipient strains.
Growth assays
Yeast strains (WT and cab1 mutants) were grown overnight at 30 °C in YPD medium and harvested (700 × g for 5 min at 4 °C), washed with water, and resuspended in 0.9% NaCl solution at OD600 of 0.5. Serial 10-fold dilutions were made and 5 µL of cell suspensions were spotted on YPD agar plates containing various antifungals (amphotericin B, caspofungin, fluconazole, terbinafine, hygromycin B and cycloheximide). For the respiratory growth assay, YP medium supplemented with ethanol, lactic acid or glycerol were used. Plates were incubated at 30 °C and the growth was monitored by image scan using the device ChemiDoc MP (Bio-Rad) every 24 h. For the liquid growth assay, the yeast strains were pre-grown as above and then diluted into 3 mL of yeast rich media supplemented with either 2% glucose (YPD), 2% glycerol (YPG) or 2% lactate (YPL) liquid media at the concentrations of 10 cells per μL and incubated at 30 °C by shaking at 230 rpm and the cell growth was monitored by optical density (OD600). A. fumigatus growth was examined on GMM (1% glucose, 6 g/L NaNO3, 0.52 g/L KCl, 0.52 g/L MgSO4·7H2O, 1.52 g/L KH2PO4 monobasic, 2.2 mg/L ZnSO4·7H2O, 1.1 mg/L H3BO3, 0.5 mg/L MnCl2·4H2O, 0.5 mg/L FeSO4·7H2O, 0.16 mg/L CoCl2·5H2O, 0.16 mg/L CuSO4·5H2O, 0.11 mg/L (NH4)6Mo7O24·4H2O, and 5 mg/L Na4EDTA; pH 6.5).
Electron microscopy analysis EM
Yeast strains (WT and cab1 mutants) were grown overnight at 30 °C in YPD medium, harvested, and refreshed in YP media with 2% glycerol until reached OD600 of 1. The cells were harvested, washed, and used for high pressure freezing and freeze substitution for electron microscopy analysis. Unfixed samples were high-pressure frozen using a Leica HMP100 at 2000 psi. The frozen samples were then freeze substituted using a Leica Freeze AFS unit starting at −95 °C using 0.1% uranyl acetate in acetone for 50 h to −60 °C, then rinsed in 100% acetone and infiltrated over 24 h to −45 °C with Lowicryl HM20 resin (Electron Microscopy Science). Samples were placed in gelatin capsules and UV hardened at −45 °C for 48 h. The blocks were allowed to cure for a further few days before trimmed and cut using a Leica UltraCut UC7. The 60 nm sections were collected on formvar/carbon-coated nickel grids and contrast stained using 2% uranyl acetate and lead citrate. The 60 nm sections on grids were viewed FEI Tecnai Biotwin TEM at 80 kV. Images were taken using AMT NanoSprint15 MK2 sCMOS camera.
Pantothenate kinase (PanK) assay using 14C-labeled PA
Pantothenate kinase assay using labeled PA was performed as previously described13,14. Briefly, cell-free extracts from yeast expressing Cab1 variants were obtained by homogenization, followed by centrifugation at 700 × g for 5 min. The 40 μL enzyme reaction contained reaction buffer (100 mM Tris HCl, 2.5 mM MgCl2, 2.5 mM ATP, pH 7.4), D-[1-14C] pantothenate (2 nmol, 0.1 µCi), and 144 μg cell-free extracts. The lysates total protein content was determined using the Bradford assay. The reaction was done at 30 °C for 10 min following the addition of 4 µL of 10% acetic acid to stop the reaction. The reaction mixture was spotted on a DE-81 filter (0.6 mm in diameter) placed within a spin column with a 2 mL collection tube. Following 5 min incubation, the spotted filters were centrifuged for 20 s at 700 × g, washed twice with 1% acetic acid in ethanol, and collected for liquid scintillation spectrometry.
Cellular CoA determination
The determination of cellular CoA levels in yeast strains expressing different Cab1 variants was done using soluble metabolites extractions from S. cerevisiae as previously described48. Briefly, the yeast strains were inoculated in 3 mLs of YPD liquid media and grown overnight at 30 °C in a shaking incubator. Cells were harvested by centrifugation at 2000 × g for 5 min, resuspended in fresh media, and diluted to an OD of 0.2 in 10 mL YPD media. The cultures were grown until an OD of 0.8–1.0 was reached. Cells were harvested by centrifugation at 2000 × g for 5 min, washed twice with 60% methanol. Cell pellets were resuspended in 1 mL of 75% ethanol, extracted for 3 min at 85 °C with intermittent vortexing, then cooled rapidly without freezing. The ethanolic extract was separated from the cell debris by centrifugation at 4000 × g for 5 min, and the supernatants were evaporated to dryness in a vacuum centrifuge. The dried metabolites were resuspended in water (0.5 mL per 0.1 g cell weight), and insoluble particles were removed by centrifugation at 4000 × g at 4 °C for 10 min. The aqueous extract was stored at −80 °C. Metabolites extracts were then used in Coenzyme A detection kit (Sigma) to quantify cellular CoA.
Cellular cysteine determination
The determination of cellular cysteine levels in yeast strain producing different Cab1 variants was done using soluble metabolites extractions from S. cerevisiae as described above. Metabolites extracts were then used in fluorometric cysteine assay kit (Abcam).
RNA sequencing and data analysis
RNA samples from yeast strain expressing different Cab1 variants were extracted using YeaStar RNA kit (Zymo Research). The RNA samples utilized in this study comprised three biological replicates from each of seven different classes of yeast strains. These classes include: 1) cab1∆ + CAB1, 2) cab1∆ + cab1G351S, 3) cab1∆ + cab1G351S + CAB1, 4) cab1∆ + cab1S158A, 5) cab1∆ + cab1S158A + CAB1, 6) cab1∆ + cab1N290I, and 7) cab1∆ + cab1N290I + CAB1. RNA sequencing was conducted by Yale Center for Genome Analysis (YCGA). RNA quality and integrity was determined by nanodrop and by resolving an aliquot of the extracted RNA on Agilent Bioanalyzer gel, respectively. RNA integrity number (RIN) values of the analyzed samples ranged from 8.9 to 10, exceeding the minimum required value of 7 for library preparation. For cDNA library preparation, the mRNAs were isolated from approximately 200 ng of total RNA using KAPA mRNA HyperPrep Kit (Roche Molecular Systems, Inc). Following first-strand synthesis with random primers, second strand synthesis and A-tailing were performed with dUTP to generate strand-specific sequencing libraries. Finally, library amplification amplified fragments carrying the appropriate adapter sequences at both ends. Indexed libraries that met appropriate cut-offs for both were quantified by Kapa Biosystems qRT-PCR reagents and kits (Millipore Sigma). Samples were sequenced using 100 bp paired-end sequencing on an Illumina NovaSeq according to Illumina protocols. FastQC (0.11.9, llumina, Inc) was used to check the quality of the raw reads. TrimGalore (0.6.7, Babraham Bioinformatics) was used for the removal of low-quality reads (reads with Quality Phred score <20 or reads shorter than 20 bp in length) and adapter sequences. The filtered and trimmed paired-end reads were aligned to the reference genome of Saccharomyces cerevisiae (R64-1-1) using HISAT2 (2.2.1)49. The number of reads in the Bam files that overlap with gene features were counted using the featureCounts function in the Subread package (2.16.0). Differential gene expression was performed using DEseq2 in Bioconductor version: Release (3.18). Counts were converted into counts per million using the cpm function in edgeR in Bioconductor 3.18 release. The Bubble plot in Fig. 4e and the heatmap in Fig. S2 were produced using ggplot2 (3.5.0) and pheatmap (1.0.12), respectively, with Rstudio (2023.12.1) and R (4.3.3).
Respirometry analysis
The oxygen consumption rate (OCR) of yeast strains expressing different Cab1 variants was determined using Seahorse 96X and Mito Stress kit. Yeast strains (WT and cab1 mutants) were grown overnight at 30 °C in YPD medium, harvested, and refreshed in SC medium supplemented with 2% glucose until reached OD600 of 0.6. Then, cells were harvested, washed, and seeded (6 × 104 cells per well) in Seahorse XFp plates coated with poly-Lysine (50 µL of 0.1 mg/mL). A minimum of 8 technical replicates were performed for each experiment at 30 °C. The seeded plate was centrifuged at 500 rpm for 5 min to promote yeast adhesion and the plate was rested for 30 min at RT. A soaked and calibrated Seahorse XF96 Sensor Cartridge was prepared before loading into the Seahorse XF96 analyzer (Agilent) which determined the cells basal OCR and following the injection of mitochondrial uncoupling drugs; oligomycin (5 µM), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (10 µM), antimycin A (10 µM), and rotenone (5 µM). The readouts were normalized using nuclear Hoechst staining for the immobilized yeast cells.
Yeast growth in the presence of antifungal drugs, inhibitors, and potentiators
To investigate the effect of common AFDs (amphotericin B, caspofungin, fluconazole, and terbinafine) in combination with compounds and potentiators (concanamycin A, doxycycline, PZ-2891, α-PanAm, AR-12) the yeast growth was monitored using a liquid assay in a 96-well plate. Overnight yeast precultures (WT strains or acs-Tet-Off when mentioned) were prepared in YPD medium at 30 °C. Cells were washed and refreshed in YPD until reaching OD600 of 0.6. In a 96-well plate, cells (103 cells/mL, 100 µL final volume) were treated with decreasing concentrations (two-fold dilutions) of AFDs, and different dosages of compounds and potentiators. For reference, amorolfine (200 µM) and DMSO (0.6%) were used as positive and negative controls to determine 100% and 0% growth inhibition, respectively. Plates were incubated at 30 °C. Optical density measurements were taken using a BioTek SynergyMx microplate reader every 12 h. Data are shown as mean ± SD of four independent experiments. Growth curves where visualized and determined from a sigmoidal dose-response curve using GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, CA). Statistical significance was determined using t-test (p = 0.05) with GraphPad Prism.
Reactive oxygen species (ROS) content
Reactive oxygen species (ROS) in the cab1 mutants were determined by change of oxidative status of fluorescence dye caused by ROS inside of the cell. ROS oxidize dihydrorhodamine 123 (DHR123; Sigma-Aldrich®, Darmstadt, Germany), which in turn produces green, fluorescent R123. To monitor ROS, cells were pre-grown overnight at 30 °C in YPD to the OD600 of 0.5–1.0 and the cells were diluted to the OD of 0.4 and loaded with 1.25 µg/mL of DHR123 for 2 h at 30 °C. At the end of the incubation time, cells were harvested (2 min at 9000 × g) and re-suspended in water at the OD600 of 0.05, and the fluorescence was quantified by a plate reader. For each sample, 100 µL of cell suspension was added into each well and the fluorescence was measured (excitation/emission spectra of 488/530 nm). Emission values from the control cells untreated with the dye were used as background for each strain. ROS generation for each cab1 strains was measured as the percentage of fluorescence emission obtained from the cab1∆ strain harboring WT CAB1 gene.
PanK activity of recombinant Cab1 in the presence of PZ-2891 and AcCoA
His-tagged Cab1 recombinant enzyme was produced and purified as was previously described16. A Kinase-Glo (Promega) assay kit for kinase activity was used to determine the activity of the purified PanK under different conditions16.
Vacuolar visualization and cell size determination
To determine the ratio of vacuolar area over cell area, different yeast strains were stained with CellTracker™ Blue CMAC as explained in the methods above. Images were captured using fluorescence microscope and analyzed using Image J software. The cell surface area (in square pixel) and vacuolar surface area (in square pixel) were calculated in Image J and percentage of vacuolar area/cell area was calculated. A total of 100 cells were analyzed from each yeast strain. The data was plotted and analyzed in GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, CA). Statistical significance was determined using Welch’s t-test with GraphPad Prism.
Radial growth assay and AFD sensitivity assays with A. fumigatus
The radial growth measurements of A. fumigatus were performed as previously described16,50. Briefly, 2 μL of a 2.5 × 106 mL−1 conidial suspension of wild-type CEA10 A. fumigatus was point inoculated onto the center of a solid GMM in the absence or presence of 50 µM PZ-2891, 20 µg/mL caspofungin, and their combination. Plates were incubated for 96 h at 35 °C, with colony diameters measured and photographs taken each day.
Acetyl CoA synthetase (ACS) activity assay
The ACS assay was performed by monitoring the formation of the adenyl acetate, the intermediate of the enzyme reaction, utilizing S. cerevisiae acetyl CoA synthetase (Sigma, A1765), following established protocols with some modifications26,29. In a 100 µL reaction volume, composed of 100 mM potassium phosphate at pH7.5, 5 mM MgCl2, 2 mM ATP, 50 mM potassium fluoride, 10 mM reduced glutathione, 0.35 mM CoA, 10 mM potassium acetate, 200 mM neutralized hydroxylamine adjusted to pH7.3, 0.005 units of the enzyme, and the inhibitors (in 1% DMSO), the components were combined. The mixture was then incubated for 30 min at 37 °C. Termination of the reaction was achieved by addition of 50 µL of a solution containing ferric chloride (12 M) and trichloroacetic acid (12%). The resultant product, acethydroxamic acid, was quantified using a BioTek SynergyMx microplate reader at OD540. The background correction was performed by utilizing a blank reaction comprising all the reaction components, which was subsequently terminated using acidified ferric chloride solution, without undergoing any incubation time.
Genetic interaction analysis of CAB1
The list of 292 genes displaying genetic interactions with a cab1 mutant was obtained from previous reports34,35,36. The gene IDs were converted into their corresponding Ensembl gene IDs using the conversion tool available at YeastMine (https://bluegenes.yeastgenome.org/yeastmine/upload/input). Enrichment analysis was performed using the ShinyGO tool (http://bioinformatics.sdstate.edu/go/), applying standard settings including an FDR cutoff of 0.05 and a minimum pathway size of two genes. Redundant pathways were removed to enhance clarity and relevance.
Statistics and reproducibility
The number of biological samples utilized in the experiments was 3 and the corresponding p-values were provided in each figure legend. Data are presented as means ± standard deviation (SD). Statistical analysis was conducted using GraphPad Prism 9 (Graphpad software, CA, US). For experiments involving two groups, we applied Student’s t test while ANOVA was employed for multiple comparisons. Fluorescence images were processed using the FiJi/Image J suite.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
