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Sequoia Research fluconazole
Beauvericin inhibits Hsp90 function ( a ) Beauvericin (BEA) reduces <t>fluconazole-induced</t> calcineurin activation. S. cerevisiae harboring 4xCDRE- lacZ reporter construct ± fluconazole (FLC, 64 μg/ml), ± geldanamycin (GdA, 5.6 μg/ml), FK506 (1.0 μg/ml), or beauvericin (20 μg/ml). Data are mean ± s.d. from technical triplicates and representative of biological replicates. * P
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1) Product Images from "Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling"

Article Title: Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling

Journal: Nature chemical biology

doi: 10.1038/nchembio.2165

Beauvericin inhibits Hsp90 function ( a ) Beauvericin (BEA) reduces fluconazole-induced calcineurin activation. S. cerevisiae harboring 4xCDRE- lacZ reporter construct ± fluconazole (FLC, 64 μg/ml), ± geldanamycin (GdA, 5.6 μg/ml), FK506 (1.0 μg/ml), or beauvericin (20 μg/ml). Data are mean ± s.d. from technical triplicates and representative of biological replicates. * P
Figure Legend Snippet: Beauvericin inhibits Hsp90 function ( a ) Beauvericin (BEA) reduces fluconazole-induced calcineurin activation. S. cerevisiae harboring 4xCDRE- lacZ reporter construct ± fluconazole (FLC, 64 μg/ml), ± geldanamycin (GdA, 5.6 μg/ml), FK506 (1.0 μg/ml), or beauvericin (20 μg/ml). Data are mean ± s.d. from technical triplicates and representative of biological replicates. * P

Techniques Used: Activation Assay, Construct

Beauvericin inhibits Pdr5, thereby increasing fluconazole intracellular accumulation, and can be effluxed by Pdr5 following substitutions that alter substrate-specificity ( a ) S. cerevisiae lacking Yor1 is sensitive to beauvericin, but acquires resistance via Pdr5 substitutions. 1×10 8 cells plated on YEPD containing beauvericin (100 μg/ml); resistant mutants recovered after 3–4 days at 30°C. Resistance was assessed in YEPD with two-fold serial dilutions of beauvericin, as in Figure 1 . Mutations identified by genome sequencing indicated as amino acid changes; those in Pdr5 shown in red. ( b ) Functional validation of PDR5 mutations. Amino acid 538 is altered in three of five mutants; expression of PDR5 G538R in yor1 Δ pdr5 Δ confers beauvericin resistance. Resistance was assessed in SD medium at 30°C for 72 hours. (a–b) Performed in biological triplicates with technical duplicates. ( c ) Beauvericin-resistant mutants exhibit altered substrate specificity, and beauvericin enhances azole accumulation. Deletion of PDR5 increased intracellular accumulation of radiolabelled fluconazole, as with beauvericin-resistant mutants yor1 ΔR1 and R5. Error bars represent standard deviation (s.d.), * P
Figure Legend Snippet: Beauvericin inhibits Pdr5, thereby increasing fluconazole intracellular accumulation, and can be effluxed by Pdr5 following substitutions that alter substrate-specificity ( a ) S. cerevisiae lacking Yor1 is sensitive to beauvericin, but acquires resistance via Pdr5 substitutions. 1×10 8 cells plated on YEPD containing beauvericin (100 μg/ml); resistant mutants recovered after 3–4 days at 30°C. Resistance was assessed in YEPD with two-fold serial dilutions of beauvericin, as in Figure 1 . Mutations identified by genome sequencing indicated as amino acid changes; those in Pdr5 shown in red. ( b ) Functional validation of PDR5 mutations. Amino acid 538 is altered in three of five mutants; expression of PDR5 G538R in yor1 Δ pdr5 Δ confers beauvericin resistance. Resistance was assessed in SD medium at 30°C for 72 hours. (a–b) Performed in biological triplicates with technical duplicates. ( c ) Beauvericin-resistant mutants exhibit altered substrate specificity, and beauvericin enhances azole accumulation. Deletion of PDR5 increased intracellular accumulation of radiolabelled fluconazole, as with beauvericin-resistant mutants yor1 ΔR1 and R5. Error bars represent standard deviation (s.d.), * P

Techniques Used: Sequencing, Functional Assay, Expressing, Standard Deviation

The combination of beauvericin and fluconazole provides a powerful therapeutic strategy ( a ) Beauvericin enhances fluconazole efficacy in a mouse model of C. albicans disseminated infection. Mice were infected with C. albicans (SC5314) and treated with vehicle, beauvericin, fluconazole, or the combination. Even with a high C. albicans inoculum, the combination of beauvericin and fluconazole significantly enhanced survival relative to either treatment alone. Each group consisted of 10 female BALB/c mice. Log-rank (Mantel-Cox) test: vehicle vs. beauvericin: P =0.3173, vehicle vs. fluconazole: P
Figure Legend Snippet: The combination of beauvericin and fluconazole provides a powerful therapeutic strategy ( a ) Beauvericin enhances fluconazole efficacy in a mouse model of C. albicans disseminated infection. Mice were infected with C. albicans (SC5314) and treated with vehicle, beauvericin, fluconazole, or the combination. Even with a high C. albicans inoculum, the combination of beauvericin and fluconazole significantly enhanced survival relative to either treatment alone. Each group consisted of 10 female BALB/c mice. Log-rank (Mantel-Cox) test: vehicle vs. beauvericin: P =0.3173, vehicle vs. fluconazole: P

Techniques Used: Infection, Mouse Assay

Beauvericin enhances fluconazole efficacy against diverse fungi and blocks the emergence of resistance ( a ) Beauvericin (BEA) reduces fluconazole resistance of reference strains of S. cerevisiae (BY4741), C. albicans (SN95), and C. neoformans (H99a), and A. fumigatus clinical isolate on rich medium (YEPD). Fluconazole (FLC) strips generate a gradient from 0.016 to 256 μg/ml, with the highest concentration at the top. Where indicated, plates contain 20 μg/ml of BEA. Experiment performed in biological triplicates. ( b ) BEA and FLC are synergistic and cidal against C. albicans (SN95). SN95 was subjected to two-fold serial dilutions of BEA and FLC in YEPD at 30°C for 48 hours. Optical densities were standardized to drug-free controls (see color bar) and FICI was calculated based on concentrations causing 60–70% growth inhibition. The drug combination is cidal, based on transferring cells to drug-free YEPD medium for 24 hours. ( c ) BEA reduced FLC resistance of C. albicans clinical isolates (CaCi) similar to geldanamycin (GdA) and cyclosporin A (CsA). CaCi are sequentially ordered with isolates recovered early at the top. MIC assays were performed in YEPD (−) with GdA (5 μM), CsA (20 μM), or BEA (25 μM), with a two-fold serial dilution of FLC. (b–c) Experiments performed in biological triplicates with technical duplicates. ( d ) BEA blocks the emergence of FLC resistance in C. albicans (SN95). 1×10 5 cells were plated on YEPD containing no inhibitor (−), 20 μg/ml BEA, 32 μg/ml FLC, or the combination. Plates were photographed after three days at 30°C. Experiment performed in biological triplicates.
Figure Legend Snippet: Beauvericin enhances fluconazole efficacy against diverse fungi and blocks the emergence of resistance ( a ) Beauvericin (BEA) reduces fluconazole resistance of reference strains of S. cerevisiae (BY4741), C. albicans (SN95), and C. neoformans (H99a), and A. fumigatus clinical isolate on rich medium (YEPD). Fluconazole (FLC) strips generate a gradient from 0.016 to 256 μg/ml, with the highest concentration at the top. Where indicated, plates contain 20 μg/ml of BEA. Experiment performed in biological triplicates. ( b ) BEA and FLC are synergistic and cidal against C. albicans (SN95). SN95 was subjected to two-fold serial dilutions of BEA and FLC in YEPD at 30°C for 48 hours. Optical densities were standardized to drug-free controls (see color bar) and FICI was calculated based on concentrations causing 60–70% growth inhibition. The drug combination is cidal, based on transferring cells to drug-free YEPD medium for 24 hours. ( c ) BEA reduced FLC resistance of C. albicans clinical isolates (CaCi) similar to geldanamycin (GdA) and cyclosporin A (CsA). CaCi are sequentially ordered with isolates recovered early at the top. MIC assays were performed in YEPD (−) with GdA (5 μM), CsA (20 μM), or BEA (25 μM), with a two-fold serial dilution of FLC. (b–c) Experiments performed in biological triplicates with technical duplicates. ( d ) BEA blocks the emergence of FLC resistance in C. albicans (SN95). 1×10 5 cells were plated on YEPD containing no inhibitor (−), 20 μg/ml BEA, 32 μg/ml FLC, or the combination. Plates were photographed after three days at 30°C. Experiment performed in biological triplicates.

Techniques Used: Concentration Assay, Inhibition, Transferring, Serial Dilution

2) Product Images from "Global Analysis of the Fungal Microbiome in Cystic Fibrosis Patients Reveals Loss of Function of the Transcriptional Repressor Nrg1 as a Mechanism of Pathogen Adaptation"

Article Title: Global Analysis of the Fungal Microbiome in Cystic Fibrosis Patients Reveals Loss of Function of the Transcriptional Repressor Nrg1 as a Mechanism of Pathogen Adaptation

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005308

Diversity of species and antifungal resistance profiles of 1,603 fungal isolates from cystic fibrosis patients. Each dot represents an isolate from the corresponding cystic fibrosis patient and colours represent species identity. The vertical dispersion of isolates within each phenotypic class is simply for visual clarity, as with the horizontal dispersion of isolates from an individual patient. (A) Summary of yeast isolates. The isolates are categorized as: “Fluconazole resistant” if their relative growth with fixed concentration of fluconazole at 128 μg/ml was greater than 2 times that of the relative growth of reference C . albicans strain SN95; “Fluconazole susceptible” if their relative growth was less than 2 times the relative growth of SN95; and “Variable” if their resistance profiles were variable over biological and technical duplicates. C . albicans is divided into two groups: isolates that show standard yeast morphology in rich medium at 30°C are in blue; and isolates that show filamentous growth under these conditions are in red. (B) Summary of mold isolates. The isolates are categorized as: “Itraconazole resistant” if their relative growth with fixed concentration of itraconazole at 0.5 μg/ml was greater than 2 times that of the relative growth of reference A . fumigatus strain AF293; and “Itraconazole susceptible” if their relative growth was less than 2 times the relative growth of AF293
Figure Legend Snippet: Diversity of species and antifungal resistance profiles of 1,603 fungal isolates from cystic fibrosis patients. Each dot represents an isolate from the corresponding cystic fibrosis patient and colours represent species identity. The vertical dispersion of isolates within each phenotypic class is simply for visual clarity, as with the horizontal dispersion of isolates from an individual patient. (A) Summary of yeast isolates. The isolates are categorized as: “Fluconazole resistant” if their relative growth with fixed concentration of fluconazole at 128 μg/ml was greater than 2 times that of the relative growth of reference C . albicans strain SN95; “Fluconazole susceptible” if their relative growth was less than 2 times the relative growth of SN95; and “Variable” if their resistance profiles were variable over biological and technical duplicates. C . albicans is divided into two groups: isolates that show standard yeast morphology in rich medium at 30°C are in blue; and isolates that show filamentous growth under these conditions are in red. (B) Summary of mold isolates. The isolates are categorized as: “Itraconazole resistant” if their relative growth with fixed concentration of itraconazole at 0.5 μg/ml was greater than 2 times that of the relative growth of reference A . fumigatus strain AF293; and “Itraconazole susceptible” if their relative growth was less than 2 times the relative growth of AF293

Techniques Used: Concentration Assay

3) Product Images from "Dynamic, Morphotype-Specific Candida albicans ?-Glucan Exposure during Infection and Drug Treatment"

Article Title: Dynamic, Morphotype-Specific Candida albicans ?-Glucan Exposure during Infection and Drug Treatment

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000227

Fluconazole treatment does not unmask β-glucan. Mice were pre-injected with fluconazole and infected as in Figure 4 . At 16 hr post-infection, kidneys were homogenized. (A) Homogenates were diluted and assayed for viable colony forming units or (B,D) homogenates were stained with anti-β-glucan monoclonal antibody and Cy3-labeled secondary antibody. Treatments of mice before infection were the following: (B) single dose of PBS (vehicle), (C) single dose of 2.5 mg/kg fluconazole in PBS, (D) single dose of 30 µg/kg caspofungin in PBS. Images and CFU data are representative of two independent experiments with 2 to 3 mice per group. Bars in (A) represent averages of triplicate counts and error bars represent standard deviations. Scale bar in (B) is 10 microns long and applies to all images.
Figure Legend Snippet: Fluconazole treatment does not unmask β-glucan. Mice were pre-injected with fluconazole and infected as in Figure 4 . At 16 hr post-infection, kidneys were homogenized. (A) Homogenates were diluted and assayed for viable colony forming units or (B,D) homogenates were stained with anti-β-glucan monoclonal antibody and Cy3-labeled secondary antibody. Treatments of mice before infection were the following: (B) single dose of PBS (vehicle), (C) single dose of 2.5 mg/kg fluconazole in PBS, (D) single dose of 30 µg/kg caspofungin in PBS. Images and CFU data are representative of two independent experiments with 2 to 3 mice per group. Bars in (A) represent averages of triplicate counts and error bars represent standard deviations. Scale bar in (B) is 10 microns long and applies to all images.

Techniques Used: Mouse Assay, Injection, Infection, Staining, Labeling

4) Product Images from "Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs"

Article Title: Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1007319

Whole genome sequencing identifies aneuploidies that are associated with the restoration of azole resistance to a C . albicans clinical isolate lacking RGD1 . A) RGD1 was deleted from a clinical isolate obtained late in treatment from a patient undergoing fluconazole therapy (CaCi-17). Resistant mutants were obtained by plating 2x10 8 cells on YPD plates containing a high concentration of miconazole. Spontaneous mutants were selected after 5 days at 30°C. Azole resistance of the mutants was verified by MIC assay. Cells were grown as described in Fig 1 . Growth was measured after 48 hours. B) Spontaneous azole resistant mutants show fitness defect in the absence of the selective pressure but show enhanced growth in the presence of fluconazole. Strains were grown in the absence or presence of 128 μg/mL fluconazole. Growth was assessed by OD 600 measurements every 15 minutes for 48 hours in a Tecan plate reader. C) Copy number variation was analyzed using the Y MAP pipeline. Amplification of chromosome 7 as well as a portion of chromosome 3 occurred in all independent spontaneous resistant mutants. D) Chromosome 3 haplotype map analyzed using Y MAP . The monosomic region of Chr3L was generated from different haplotypes in the four azole-resistant isolates: haplotype A (Cyan) in isolates R1 and R2, and haplotype B (magenta) in isolates R3 and R4.
Figure Legend Snippet: Whole genome sequencing identifies aneuploidies that are associated with the restoration of azole resistance to a C . albicans clinical isolate lacking RGD1 . A) RGD1 was deleted from a clinical isolate obtained late in treatment from a patient undergoing fluconazole therapy (CaCi-17). Resistant mutants were obtained by plating 2x10 8 cells on YPD plates containing a high concentration of miconazole. Spontaneous mutants were selected after 5 days at 30°C. Azole resistance of the mutants was verified by MIC assay. Cells were grown as described in Fig 1 . Growth was measured after 48 hours. B) Spontaneous azole resistant mutants show fitness defect in the absence of the selective pressure but show enhanced growth in the presence of fluconazole. Strains were grown in the absence or presence of 128 μg/mL fluconazole. Growth was assessed by OD 600 measurements every 15 minutes for 48 hours in a Tecan plate reader. C) Copy number variation was analyzed using the Y MAP pipeline. Amplification of chromosome 7 as well as a portion of chromosome 3 occurred in all independent spontaneous resistant mutants. D) Chromosome 3 haplotype map analyzed using Y MAP . The monosomic region of Chr3L was generated from different haplotypes in the four azole-resistant isolates: haplotype A (Cyan) in isolates R1 and R2, and haplotype B (magenta) in isolates R3 and R4.

Techniques Used: Sequencing, Concentration Assay, Amplification, Generated

The ERG3 genetic interaction network in S . cerevisiae uncovers species-specific circuitry important for azole resistance. A) Genetic nodes highlighting the negative genetic interactions with an SGA score (ε) ≤ -0.25 in YPD medium. 100 genes were identified as having a genetic interaction with ScERG3 , and they are coloured based on their GO Slim Process category. B) Microbroth dilution minimum inhibitory concentration (MIC) assay for S . cerevisiae ERG3 genetic interactors for which gene deletion abrogates azole resistance of the erg3 mutant. Strains were complemented with their respective wild-type allele to verify the azole sensitivity phenotype was due to the specific gene deletion. MIC assays were conducted in YPD and growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements. Data was quantitatively displayed with colour using Treeview (see colour bar). C) MIC assay for C . albicans erg3 homozygous deletion mutants that also harboured homozygous deletions of genes originally identified in S . cerevisiae as being important for erg3- mediated resistance. MIC assay was performed as in part B. For the bottom panel, fluconazole MIC assays were conducted in YPD medium with 1 μg/mL doxycycline to achieve transcriptional repression of the target gene in the conditional expression strains.
Figure Legend Snippet: The ERG3 genetic interaction network in S . cerevisiae uncovers species-specific circuitry important for azole resistance. A) Genetic nodes highlighting the negative genetic interactions with an SGA score (ε) ≤ -0.25 in YPD medium. 100 genes were identified as having a genetic interaction with ScERG3 , and they are coloured based on their GO Slim Process category. B) Microbroth dilution minimum inhibitory concentration (MIC) assay for S . cerevisiae ERG3 genetic interactors for which gene deletion abrogates azole resistance of the erg3 mutant. Strains were complemented with their respective wild-type allele to verify the azole sensitivity phenotype was due to the specific gene deletion. MIC assays were conducted in YPD and growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements. Data was quantitatively displayed with colour using Treeview (see colour bar). C) MIC assay for C . albicans erg3 homozygous deletion mutants that also harboured homozygous deletions of genes originally identified in S . cerevisiae as being important for erg3- mediated resistance. MIC assay was performed as in part B. For the bottom panel, fluconazole MIC assays were conducted in YPD medium with 1 μg/mL doxycycline to achieve transcriptional repression of the target gene in the conditional expression strains.

Techniques Used: Concentration Assay, Mutagenesis, Expressing

Loss of PEP8 results in abnormal vacuolar morphology and overwhelms the functional capacity of calcineurin. A) Strains of C . albicans were grown to log-phase prior to staining with membrane dye FM4-64. Images were captured using differential interference contrast (DIC) microscopy and fluorescence microscopy with a TRITC/DsRED filter set on a Zeiss Axio Observer.Z1 (Carl Zeiss) using 100x magnification. Scale bar represents 10 μm. B) Deletion of PEP8 increases fluconazole-induction of two calcineurin-dependent transcripts, UTR2 and CRH11 . Transcript levels were monitored by qRT-PCR and normalized to ACT1 and GPD1 . Error bars represent standard error of the mean for triplicate samples. Treatment conditions were compared using a one-way ANOVA with Bonferroni post-test. Double asterisk indicates a significant difference in transcript level relative to both wildtype with fluconazole and erg3Δ/erg3Δ strain plus fluconazole. (** P
Figure Legend Snippet: Loss of PEP8 results in abnormal vacuolar morphology and overwhelms the functional capacity of calcineurin. A) Strains of C . albicans were grown to log-phase prior to staining with membrane dye FM4-64. Images were captured using differential interference contrast (DIC) microscopy and fluorescence microscopy with a TRITC/DsRED filter set on a Zeiss Axio Observer.Z1 (Carl Zeiss) using 100x magnification. Scale bar represents 10 μm. B) Deletion of PEP8 increases fluconazole-induction of two calcineurin-dependent transcripts, UTR2 and CRH11 . Transcript levels were monitored by qRT-PCR and normalized to ACT1 and GPD1 . Error bars represent standard error of the mean for triplicate samples. Treatment conditions were compared using a one-way ANOVA with Bonferroni post-test. Double asterisk indicates a significant difference in transcript level relative to both wildtype with fluconazole and erg3Δ/erg3Δ strain plus fluconazole. (** P

Techniques Used: Functional Assay, Staining, Microscopy, Fluorescence, Quantitative RT-PCR

C . albicans functional genomic screen identifies novel genetic circuitry important for azole tolerance and resistance. A) Schematic of functional genomic screen to identify genes important for fluconazole (FL) tolerance. Thirteen genes were identified and prioritized for follow-up analysis based on the robust hypersensitivity of the corresponding deletion mutants. B) The 13 genes that were identified as having important roles in fluconazole tolerance, coloured based on their GO Slim Process category. Note many genes belong to multiple categories. C) MIC assay for C . albicans erg3 homozygous deletion mutants that also harboured homozygous deletions of genes implicated in fluconazole tolerance in part B. Homozygous deletion of either RGD1 or PEP8 abrogates the erg3- mediated azole resistance phenotype. MIC assay was performed as described in Fig 1 . Growth was analyzed after 24 hours (see colour bar). D) Homozygous deletion of either RGD1 or PEP8 reduces azole resistance of two resistant clinical isolates collected from an HIV patient undergoing fluconazole therapy. CaCi-2 represents an isolate collected early in treatment and CaCi-17 represents an isolate collected later in treatment. MIC assay was performed as described in part C.
Figure Legend Snippet: C . albicans functional genomic screen identifies novel genetic circuitry important for azole tolerance and resistance. A) Schematic of functional genomic screen to identify genes important for fluconazole (FL) tolerance. Thirteen genes were identified and prioritized for follow-up analysis based on the robust hypersensitivity of the corresponding deletion mutants. B) The 13 genes that were identified as having important roles in fluconazole tolerance, coloured based on their GO Slim Process category. Note many genes belong to multiple categories. C) MIC assay for C . albicans erg3 homozygous deletion mutants that also harboured homozygous deletions of genes implicated in fluconazole tolerance in part B. Homozygous deletion of either RGD1 or PEP8 abrogates the erg3- mediated azole resistance phenotype. MIC assay was performed as described in Fig 1 . Growth was analyzed after 24 hours (see colour bar). D) Homozygous deletion of either RGD1 or PEP8 reduces azole resistance of two resistant clinical isolates collected from an HIV patient undergoing fluconazole therapy. CaCi-2 represents an isolate collected early in treatment and CaCi-17 represents an isolate collected later in treatment. MIC assay was performed as described in part C.

Techniques Used: Functional Assay

5) Product Images from "Inhibiting Fungal Echinocandin Resistance by Small-Molecule Disruption of Geranylgeranyltransferase Type I Activity"

Article Title: Inhibiting Fungal Echinocandin Resistance by Small-Molecule Disruption of Geranylgeranyltransferase Type I Activity

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.02046-19

The cdc43 mutant is susceptible to caspofungin and cell wall pressures. (A) Cells of wild-type (SC5314), cdc43 (YLC12), cdc43+CDC43 (YLC13), and cas5 mutant strains were serially diluted 10-fold and spotted onto YPD solid medium with or without 500 ng/ml caspofungin. Photographs were taken after 2 days of growth at 30°C. (B) Caspofungin susceptibility assays were conducted in YPD medium. Growth was measured by absorbance at 600 nm after 48 h at 30°C. Optical densities were averaged from duplicate measurements. Data are quantitatively displayed in heat map format (see color bar). (C) Cdc43 is required for cell wall stress tolerance. Wild-type (SC5314), cdr1 , and cdc43 strains were treated with cell wall stress agents, including 1 μg/ml fluconazole, 200 μg/ml Congo red, 1.5 M KCl, and 0.025% SDS. Photographs were taken after 2 days of growth at 30°C.
Figure Legend Snippet: The cdc43 mutant is susceptible to caspofungin and cell wall pressures. (A) Cells of wild-type (SC5314), cdc43 (YLC12), cdc43+CDC43 (YLC13), and cas5 mutant strains were serially diluted 10-fold and spotted onto YPD solid medium with or without 500 ng/ml caspofungin. Photographs were taken after 2 days of growth at 30°C. (B) Caspofungin susceptibility assays were conducted in YPD medium. Growth was measured by absorbance at 600 nm after 48 h at 30°C. Optical densities were averaged from duplicate measurements. Data are quantitatively displayed in heat map format (see color bar). (C) Cdc43 is required for cell wall stress tolerance. Wild-type (SC5314), cdr1 , and cdc43 strains were treated with cell wall stress agents, including 1 μg/ml fluconazole, 200 μg/ml Congo red, 1.5 M KCl, and 0.025% SDS. Photographs were taken after 2 days of growth at 30°C.

Techniques Used: Mutagenesis

6) Product Images from "The Hsp90 Co-Chaperone Sgt1 Governs Candida albicans Morphogenesis and Drug Resistance"

Article Title: The Hsp90 Co-Chaperone Sgt1 Governs Candida albicans Morphogenesis and Drug Resistance

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044734

Sgt1 enables basal tolerance and erg3 -mediated resistance to the azoles. (A) Reduced levels of Sgt1 renders cells sensitive to fluconazole in minimum inhibitory concentration (MIC) assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline (DOX), as indicated. Growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements and normalized relative to the no fluconazole control. Data was quantitatively displayed with colour using Treeview (see colour bar). (B) Reduced levels of Sgt1 renders azole-resistant erg3 mutants sensitive to fluconazole in MIC assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with a fixed concentration of 20 µg/ml doxycycline, as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in part A. (C) Cells with reduced levels of Sgt1 remain viable after exposure to fluconazole. MIC assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Assays were grown for 48 hours at 30°C, and cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.
Figure Legend Snippet: Sgt1 enables basal tolerance and erg3 -mediated resistance to the azoles. (A) Reduced levels of Sgt1 renders cells sensitive to fluconazole in minimum inhibitory concentration (MIC) assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline (DOX), as indicated. Growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements and normalized relative to the no fluconazole control. Data was quantitatively displayed with colour using Treeview (see colour bar). (B) Reduced levels of Sgt1 renders azole-resistant erg3 mutants sensitive to fluconazole in MIC assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with a fixed concentration of 20 µg/ml doxycycline, as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in part A. (C) Cells with reduced levels of Sgt1 remain viable after exposure to fluconazole. MIC assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Assays were grown for 48 hours at 30°C, and cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.

Techniques Used: Concentration Assay, Incubation

Hsp90 client protein Cna1 retains stability upon depletion of Sgt1, though Sgt1 is required for Cna1 activation. (A) Cna1 retains stability upon depletion of Sgt1. Sgt1 levels were reduced by growth overnight in 20 µg/ml doxycycline, followed by subculture in fresh medium with 20 µg/ml doxycycline and growth until mid-log phase. First panel, immune blot analysis of Sgt1 levels (50 µg protein loaded per well); and second panel, immune blot analysis of Cna1-TAP levels (50 µg protein loaded per well). Actin was used as a loading control. (B) Sgt1 is required for Cna1 activation in response to fluconazole or micafungin. Transcript levels of a Cna1-dependent gene, UTR2 , were measured for the wild-type ( SGT1/SGT1 ) or tetO-SGT1/sgt1 Δ strains by quantitative RT-PCR after growth in rich medium at 30°C with or without 20 µg/ml doxycycline (DOX), 16 µg/ml fluconazole (FL), and 30 ng/ml micafungin (MF), as indicated. UTR2 transcript levels were normalized to GPD1 . Data are means ± standard deviations for triplicate samples.
Figure Legend Snippet: Hsp90 client protein Cna1 retains stability upon depletion of Sgt1, though Sgt1 is required for Cna1 activation. (A) Cna1 retains stability upon depletion of Sgt1. Sgt1 levels were reduced by growth overnight in 20 µg/ml doxycycline, followed by subculture in fresh medium with 20 µg/ml doxycycline and growth until mid-log phase. First panel, immune blot analysis of Sgt1 levels (50 µg protein loaded per well); and second panel, immune blot analysis of Cna1-TAP levels (50 µg protein loaded per well). Actin was used as a loading control. (B) Sgt1 is required for Cna1 activation in response to fluconazole or micafungin. Transcript levels of a Cna1-dependent gene, UTR2 , were measured for the wild-type ( SGT1/SGT1 ) or tetO-SGT1/sgt1 Δ strains by quantitative RT-PCR after growth in rich medium at 30°C with or without 20 µg/ml doxycycline (DOX), 16 µg/ml fluconazole (FL), and 30 ng/ml micafungin (MF), as indicated. UTR2 transcript levels were normalized to GPD1 . Data are means ± standard deviations for triplicate samples.

Techniques Used: Activation Assay, Quantitative RT-PCR

Reduction of Sgt1 levels enhances susceptibility to echinocandins and creates a fungicidal combination. (A) Reduced levels of Sgt1 enhances susceptibility to micafungin in minimum inhibitory concentration (MIC) assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 2 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline (DOX), as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in Figure 4 . (B) Reduced levels of Sgt1 renders echinocandin-resistant FKS1 mutants susceptible to micafungin in MIC assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 8 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in Figure 4 . (C) Reduction of Sgt1 levels creates a fungicidal combination with micafungin. MIC assays were performed in YPD medium with a gradient of fluconazole from 0 to 2 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Assays were grown for 72 hours at 30°C, and cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.
Figure Legend Snippet: Reduction of Sgt1 levels enhances susceptibility to echinocandins and creates a fungicidal combination. (A) Reduced levels of Sgt1 enhances susceptibility to micafungin in minimum inhibitory concentration (MIC) assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 2 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline (DOX), as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in Figure 4 . (B) Reduced levels of Sgt1 renders echinocandin-resistant FKS1 mutants susceptible to micafungin in MIC assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 8 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in Figure 4 . (C) Reduction of Sgt1 levels creates a fungicidal combination with micafungin. MIC assays were performed in YPD medium with a gradient of fluconazole from 0 to 2 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Assays were grown for 72 hours at 30°C, and cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.

Techniques Used: Concentration Assay, Incubation

7) Product Images from "Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus"

Article Title: Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus

Journal: Nature Communications

doi: 10.1038/s41467-018-08248-w

CMLD013075 reverses antifungal resistance in co-culture. a Checkerboard analysis of antifungal activity for combination of FL with Hsp90 inhibitors. FL-resistant strain CaCi-2 was exposed to the indicated twofold serial dilutions of each compound for 48 hours. Relative growth inhibition is displayed in heat-map format. Each colored box represents the mean of duplicate determinations. Color scale for relative growth is provided to the right of panel, green: no inhibition to black: complete inhibition. The experiment was repeated as an independent biological replicate to confirm results. b Co-culture of fluconazole (FL)-resistant, GFP-expressing C. albicans strain with human 293T cells line stably expressing a luciferase reporter. The concentration-dependent inhibition of fungal (left) and human (right) cell growth and survival by Hsp90 inhibitors in the presence of FL was measured in 384-well format after 48-h treatment. Relative fungal burden was evaluated by plate reader using a fluorescence endpoint while human cell survival was evaluated by measuring luminescence. Values for each endpoint were normalized to wells receiving no Hsp90 inhibitor. Each point represents the mean of measurements from triplicate wells. Error bars, SD. The experiment was repeated once as an independent biological replicate to confirm results
Figure Legend Snippet: CMLD013075 reverses antifungal resistance in co-culture. a Checkerboard analysis of antifungal activity for combination of FL with Hsp90 inhibitors. FL-resistant strain CaCi-2 was exposed to the indicated twofold serial dilutions of each compound for 48 hours. Relative growth inhibition is displayed in heat-map format. Each colored box represents the mean of duplicate determinations. Color scale for relative growth is provided to the right of panel, green: no inhibition to black: complete inhibition. The experiment was repeated as an independent biological replicate to confirm results. b Co-culture of fluconazole (FL)-resistant, GFP-expressing C. albicans strain with human 293T cells line stably expressing a luciferase reporter. The concentration-dependent inhibition of fungal (left) and human (right) cell growth and survival by Hsp90 inhibitors in the presence of FL was measured in 384-well format after 48-h treatment. Relative fungal burden was evaluated by plate reader using a fluorescence endpoint while human cell survival was evaluated by measuring luminescence. Values for each endpoint were normalized to wells receiving no Hsp90 inhibitor. Each point represents the mean of measurements from triplicate wells. Error bars, SD. The experiment was repeated once as an independent biological replicate to confirm results

Techniques Used: Co-Culture Assay, Activity Assay, Inhibition, Expressing, Stable Transfection, Luciferase, Concentration Assay, Fluorescence

Species-selective antifungal activity of Hsp90 inhibitor CMLD013075. a Relative growth inhibition by Hsp90 inhibitors of reference C. albicans strain (SN95) and a clinical isolate (CaCi-2) with resistance to FL. The effect of 48-hour exposure to each inhibitor over a twofold dilution series of concentrations is displayed in heat-map format. Each colored box represents the mean of duplicate determinations. Color scale for relative growth is provided to far right of panel, green: no inhibition to black: complete inhibition. The experiment was repeated as an independent biological replicate to confirm results. b Concentration-dependent inhibition of mammalian cell growth and survival after 3-day culture with Hsp90 inhibitors. Data points depict the mean of triplicate determinations, error bars, SD. The experiment was repeated as an independent biological replicate to confirm results. c Monolayer cultures of human HEPG2 cells in 6-well format were infected with C. albicans and then incubated with the indicated compounds for 48 h (FL, conventional antifungal fluconazole). Cultures were then fixed and stained by Periodic Acid-Schiff (PAS) technique to visualize fungal burden (dark red/purple signal) and underlying human cells (light pink signal). Representative fields from conventional light photomicrographs are presented at the same magnification in all panels. The experiment was repeated in two independent biological replicates to confirm results. Scale bar, 100 μm
Figure Legend Snippet: Species-selective antifungal activity of Hsp90 inhibitor CMLD013075. a Relative growth inhibition by Hsp90 inhibitors of reference C. albicans strain (SN95) and a clinical isolate (CaCi-2) with resistance to FL. The effect of 48-hour exposure to each inhibitor over a twofold dilution series of concentrations is displayed in heat-map format. Each colored box represents the mean of duplicate determinations. Color scale for relative growth is provided to far right of panel, green: no inhibition to black: complete inhibition. The experiment was repeated as an independent biological replicate to confirm results. b Concentration-dependent inhibition of mammalian cell growth and survival after 3-day culture with Hsp90 inhibitors. Data points depict the mean of triplicate determinations, error bars, SD. The experiment was repeated as an independent biological replicate to confirm results. c Monolayer cultures of human HEPG2 cells in 6-well format were infected with C. albicans and then incubated with the indicated compounds for 48 h (FL, conventional antifungal fluconazole). Cultures were then fixed and stained by Periodic Acid-Schiff (PAS) technique to visualize fungal burden (dark red/purple signal) and underlying human cells (light pink signal). Representative fields from conventional light photomicrographs are presented at the same magnification in all panels. The experiment was repeated in two independent biological replicates to confirm results. Scale bar, 100 μm

Techniques Used: Activity Assay, Inhibition, Concentration Assay, Infection, Incubation, Staining

8) Product Images from "The Hsp90 Co-Chaperone Sgt1 Governs Candida albicans Morphogenesis and Drug Resistance"

Article Title: The Hsp90 Co-Chaperone Sgt1 Governs Candida albicans Morphogenesis and Drug Resistance

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044734

Sgt1 enables basal tolerance and erg3 -mediated resistance to the azoles. (A) Reduced levels of Sgt1 renders cells sensitive to fluconazole in minimum inhibitory concentration (MIC) assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline (DOX), as indicated. Growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements and normalized relative to the no fluconazole control. Data was quantitatively displayed with colour using Treeview (see colour bar). (B) Reduced levels of Sgt1 renders azole-resistant erg3 mutants sensitive to fluconazole in MIC assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with a fixed concentration of 20 µg/ml doxycycline, as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in part A. (C) Cells with reduced levels of Sgt1 remain viable after exposure to fluconazole. MIC assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Assays were grown for 48 hours at 30°C, and cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.
Figure Legend Snippet: Sgt1 enables basal tolerance and erg3 -mediated resistance to the azoles. (A) Reduced levels of Sgt1 renders cells sensitive to fluconazole in minimum inhibitory concentration (MIC) assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline (DOX), as indicated. Growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements and normalized relative to the no fluconazole control. Data was quantitatively displayed with colour using Treeview (see colour bar). (B) Reduced levels of Sgt1 renders azole-resistant erg3 mutants sensitive to fluconazole in MIC assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with a fixed concentration of 20 µg/ml doxycycline, as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in part A. (C) Cells with reduced levels of Sgt1 remain viable after exposure to fluconazole. MIC assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Assays were grown for 48 hours at 30°C, and cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.

Techniques Used: Concentration Assay, Incubation

Hsp90 client protein Cna1 retains stability upon depletion of Sgt1, though Sgt1 is required for Cna1 activation. (A) Cna1 retains stability upon depletion of Sgt1. Sgt1 levels were reduced by growth overnight in 20 µg/ml doxycycline, followed by subculture in fresh medium with 20 µg/ml doxycycline and growth until mid-log phase. First panel, immune blot analysis of Sgt1 levels (50 µg protein loaded per well); and second panel, immune blot analysis of Cna1-TAP levels (50 µg protein loaded per well). Actin was used as a loading control. (B) Sgt1 is required for Cna1 activation in response to fluconazole or micafungin. Transcript levels of a Cna1-dependent gene, UTR2 , were measured for the wild-type ( SGT1/SGT1 ) or tetO-SGT1/sgt1 Δ strains by quantitative RT-PCR after growth in rich medium at 30°C with or without 20 µg/ml doxycycline (DOX), 16 µg/ml fluconazole (FL), and 30 ng/ml micafungin (MF), as indicated. UTR2 transcript levels were normalized to GPD1 . Data are means ± standard deviations for triplicate samples.
Figure Legend Snippet: Hsp90 client protein Cna1 retains stability upon depletion of Sgt1, though Sgt1 is required for Cna1 activation. (A) Cna1 retains stability upon depletion of Sgt1. Sgt1 levels were reduced by growth overnight in 20 µg/ml doxycycline, followed by subculture in fresh medium with 20 µg/ml doxycycline and growth until mid-log phase. First panel, immune blot analysis of Sgt1 levels (50 µg protein loaded per well); and second panel, immune blot analysis of Cna1-TAP levels (50 µg protein loaded per well). Actin was used as a loading control. (B) Sgt1 is required for Cna1 activation in response to fluconazole or micafungin. Transcript levels of a Cna1-dependent gene, UTR2 , were measured for the wild-type ( SGT1/SGT1 ) or tetO-SGT1/sgt1 Δ strains by quantitative RT-PCR after growth in rich medium at 30°C with or without 20 µg/ml doxycycline (DOX), 16 µg/ml fluconazole (FL), and 30 ng/ml micafungin (MF), as indicated. UTR2 transcript levels were normalized to GPD1 . Data are means ± standard deviations for triplicate samples.

Techniques Used: Activation Assay, Quantitative RT-PCR

Reduction of Sgt1 levels enhances susceptibility to echinocandins and creates a fungicidal combination. (A) Reduced levels of Sgt1 enhances susceptibility to micafungin in minimum inhibitory concentration (MIC) assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 2 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline (DOX), as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in Figure 4 . (B) Reduced levels of Sgt1 renders echinocandin-resistant FKS1 mutants susceptible to micafungin in MIC assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 8 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in Figure 4 . (C) Reduction of Sgt1 levels creates a fungicidal combination with micafungin. MIC assays were performed in YPD medium with a gradient of fluconazole from 0 to 2 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Assays were grown for 72 hours at 30°C, and cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.
Figure Legend Snippet: Reduction of Sgt1 levels enhances susceptibility to echinocandins and creates a fungicidal combination. (A) Reduced levels of Sgt1 enhances susceptibility to micafungin in minimum inhibitory concentration (MIC) assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 2 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline (DOX), as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in Figure 4 . (B) Reduced levels of Sgt1 renders echinocandin-resistant FKS1 mutants susceptible to micafungin in MIC assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 8 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in Figure 4 . (C) Reduction of Sgt1 levels creates a fungicidal combination with micafungin. MIC assays were performed in YPD medium with a gradient of fluconazole from 0 to 2 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Assays were grown for 72 hours at 30°C, and cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.

Techniques Used: Concentration Assay, Incubation

9) Product Images from "Inhibiting mitochondrial phosphate transport as an unexploited antifungal strategy"

Article Title: Inhibiting mitochondrial phosphate transport as an unexploited antifungal strategy

Journal: Nature chemical biology

doi: 10.1038/nchembio.2534

ML316 is active against azole-resistant C. albicans in mice ( a ) Mice were treated with corticosteroids to induce susceptibility and infected sublingually with strain CaCi-2. Three days later, they were randomly assigned to treatment as indicated (n=8/ group except fluconazole (Flu) n=7). ML316 was added to drinking water (2.85 µM ML316/ 1% w/v sucrose) while Flu was administered intraperitoneally (10 mg/kg/day). Antifungal activity was assessed by measuring residual colony forming units (CFU) persisting on the tongue after completing 2 days of therapy. Mean and s.d. are displayed. The statistical significance of differences between treatment groups was determined by Mann Whitney test (unpaired, 2 tailed, non-parametric). P values are indicated above the relevant comparisons (Flu vs ML316; not significant). ( b ) Photomicrographs of PAS-stained tissue sections to assess fungal morphology are presented. Arrow heads indicate fungal foci which appear dark pink against the lighter staining of the keratinized papillae of the host tongue.
Figure Legend Snippet: ML316 is active against azole-resistant C. albicans in mice ( a ) Mice were treated with corticosteroids to induce susceptibility and infected sublingually with strain CaCi-2. Three days later, they were randomly assigned to treatment as indicated (n=8/ group except fluconazole (Flu) n=7). ML316 was added to drinking water (2.85 µM ML316/ 1% w/v sucrose) while Flu was administered intraperitoneally (10 mg/kg/day). Antifungal activity was assessed by measuring residual colony forming units (CFU) persisting on the tongue after completing 2 days of therapy. Mean and s.d. are displayed. The statistical significance of differences between treatment groups was determined by Mann Whitney test (unpaired, 2 tailed, non-parametric). P values are indicated above the relevant comparisons (Flu vs ML316; not significant). ( b ) Photomicrographs of PAS-stained tissue sections to assess fungal morphology are presented. Arrow heads indicate fungal foci which appear dark pink against the lighter staining of the keratinized papillae of the host tongue.

Techniques Used: Mouse Assay, Infection, Activity Assay, MANN-WHITNEY, Staining

ML316 is a potent, highly selective fungicide ( a ) Structures of ML316, original screen hit and inactive analog. Minimal inhibitory concentrations (MIC) are indicated for the C. albicans strain CaCi-2. For ML316 1 µM = 0.35 µg/ml. ( b ) Glucose-grown S. cerevisiae are not sensitive to ML316. Standard antifungal susceptibility testing was performed in YNB-CSM media with either glucose (Glu, 2% w/v) or glycerol (Gly, 2% w/v) as the carbon source. Optical densities were measured after 24 h at 30 °C and standardized to drug-free controls. The mean of three independent wells is shown. Error bars: s.e.m. ( c ) ML316 reduces growth of fluconazole-resistant C. albicans (strain CaCi-2), an effect that is increased by fluconazole (Flu). Assays in the presence or absence of Flu (8 µg/ml) were performed as in (b) except growth medium was RPMI supplemented with 2% glucose. The mean of two independent wells is shown. ( d ) ML316 inhibits growth of increasingly azole-resistant C. albicans strains isolated from the same HIV-infected patient over a 2-year interval. Strain designations are indicated to the left of the heat maps. Sensitivity testing was performed as in ( c ). The mean of results from two independent experiments is depicted. ( e ) ML316 is not cytotoxic to human cell lines under respiratory conditions. Viability was assessed by standard resazurin dye-reduction assay after 72 h growth at 37 °C in DMEM supplemented with galactose (10 mM) and dialyzed fetal bovine serum (10%). Data points depict the mean of measurements from 2 independent wells for each condition tested. ( f ) ML316 is fungicidal. A standard growth assay was performed as in ( c ) at either 30 or 37 °C. After 24 h, aliquots were spotted onto YPD agar plates and cultured for an additional 2 days. Scale bar = 10 mm. Data in ( c ), ( e ), and ( f ) are derived from one representative experiment. Two independent experiments yielding similar results were performed.
Figure Legend Snippet: ML316 is a potent, highly selective fungicide ( a ) Structures of ML316, original screen hit and inactive analog. Minimal inhibitory concentrations (MIC) are indicated for the C. albicans strain CaCi-2. For ML316 1 µM = 0.35 µg/ml. ( b ) Glucose-grown S. cerevisiae are not sensitive to ML316. Standard antifungal susceptibility testing was performed in YNB-CSM media with either glucose (Glu, 2% w/v) or glycerol (Gly, 2% w/v) as the carbon source. Optical densities were measured after 24 h at 30 °C and standardized to drug-free controls. The mean of three independent wells is shown. Error bars: s.e.m. ( c ) ML316 reduces growth of fluconazole-resistant C. albicans (strain CaCi-2), an effect that is increased by fluconazole (Flu). Assays in the presence or absence of Flu (8 µg/ml) were performed as in (b) except growth medium was RPMI supplemented with 2% glucose. The mean of two independent wells is shown. ( d ) ML316 inhibits growth of increasingly azole-resistant C. albicans strains isolated from the same HIV-infected patient over a 2-year interval. Strain designations are indicated to the left of the heat maps. Sensitivity testing was performed as in ( c ). The mean of results from two independent experiments is depicted. ( e ) ML316 is not cytotoxic to human cell lines under respiratory conditions. Viability was assessed by standard resazurin dye-reduction assay after 72 h growth at 37 °C in DMEM supplemented with galactose (10 mM) and dialyzed fetal bovine serum (10%). Data points depict the mean of measurements from 2 independent wells for each condition tested. ( f ) ML316 is fungicidal. A standard growth assay was performed as in ( c ) at either 30 or 37 °C. After 24 h, aliquots were spotted onto YPD agar plates and cultured for an additional 2 days. Scale bar = 10 mm. Data in ( c ), ( e ), and ( f ) are derived from one representative experiment. Two independent experiments yielding similar results were performed.

Techniques Used: Isolation, Infection, Growth Assay, Cell Culture, Derivative Assay

ML316 renders respiration toxic and increases citrate levels ( a ) C. albicans strain CaCi2 with genetic deletion of mitochondrial COX1 are resistant to ML316 but are unable to respire or grow in the presence of fluconazole. Serial dilutions of CaCi-2 or CaCi-2 cox1 were spotted on media with carbon sources supporting fermentation (glucose) or enforcing respiration (glycerol) in presence or absence of ML316 (5 µM) or Flu (16 µM) as indicated. Plates were imaged after 2 days at 30 °C. ( b ) Citrate levels increase dramatically upon treatment with ML316 but not with other mitochondrial poisons. LC/MS was used to measure levels of citrate in C. albicans grown in synthetic defined media (2% glucose) following 30 or 60 min exposure to ML316 (5 µM), Antimycin (1 µM), or 2,4-dinitrophenol (25 µg/mL). All values were normalized to the mean for DMSO-treated samples. Values represent the mean and s.e.m. of determinations from 2 independent experiments, each consisting of 3 independent cultures. ( c ) ML316 increases citrate levels in wild-type Candida but not in ML316-resistant mutant strains expressing either mir1R or carrying a deletion of COX4 . Citrate levels were determined by LC/MS in strains growing in glucose after 60 min exposure to ML316 (5 µM). In the genotypes indicated on the x -axis, “V” denotes empty vector control. Citrate levels were normalized to the mean of wild type, DMSO-treated samples. Mean and s.e.m. from 2 independent experiments each consisting of 3 independent cultures are shown.
Figure Legend Snippet: ML316 renders respiration toxic and increases citrate levels ( a ) C. albicans strain CaCi2 with genetic deletion of mitochondrial COX1 are resistant to ML316 but are unable to respire or grow in the presence of fluconazole. Serial dilutions of CaCi-2 or CaCi-2 cox1 were spotted on media with carbon sources supporting fermentation (glucose) or enforcing respiration (glycerol) in presence or absence of ML316 (5 µM) or Flu (16 µM) as indicated. Plates were imaged after 2 days at 30 °C. ( b ) Citrate levels increase dramatically upon treatment with ML316 but not with other mitochondrial poisons. LC/MS was used to measure levels of citrate in C. albicans grown in synthetic defined media (2% glucose) following 30 or 60 min exposure to ML316 (5 µM), Antimycin (1 µM), or 2,4-dinitrophenol (25 µg/mL). All values were normalized to the mean for DMSO-treated samples. Values represent the mean and s.e.m. of determinations from 2 independent experiments, each consisting of 3 independent cultures. ( c ) ML316 increases citrate levels in wild-type Candida but not in ML316-resistant mutant strains expressing either mir1R or carrying a deletion of COX4 . Citrate levels were determined by LC/MS in strains growing in glucose after 60 min exposure to ML316 (5 µM). In the genotypes indicated on the x -axis, “V” denotes empty vector control. Citrate levels were normalized to the mean of wild type, DMSO-treated samples. Mean and s.e.m. from 2 independent experiments each consisting of 3 independent cultures are shown.

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Expressing, Plasmid Preparation

10) Product Images from "Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs"

Article Title: Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1007319

The ERG3 genetic interaction network in S . cerevisiae uncovers species-specific circuitry important for azole resistance. A) Genetic nodes highlighting the negative genetic interactions with an SGA score (ε) ≤ -0.25 in YPD medium. 100 genes were identified as having a genetic interaction with ScERG3 , and they are coloured based on their GO Slim Process category. B) Microbroth dilution minimum inhibitory concentration (MIC) assay for S . cerevisiae ERG3 genetic interactors for which gene deletion abrogates azole resistance of the erg3 mutant. Strains were complemented with their respective wild-type allele to verify the azole sensitivity phenotype was due to the specific gene deletion. MIC assays were conducted in YPD and growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements. Data was quantitatively displayed with colour using Treeview (see colour bar). C) MIC assay for C . albicans erg3 homozygous deletion mutants that also harboured homozygous deletions of genes originally identified in S . cerevisiae as being important for erg3- mediated resistance. MIC assay was performed as in part B. For the bottom panel, fluconazole MIC assays were conducted in YPD medium with 1 μg/mL doxycycline to achieve transcriptional repression of the target gene in the conditional expression strains.
Figure Legend Snippet: The ERG3 genetic interaction network in S . cerevisiae uncovers species-specific circuitry important for azole resistance. A) Genetic nodes highlighting the negative genetic interactions with an SGA score (ε) ≤ -0.25 in YPD medium. 100 genes were identified as having a genetic interaction with ScERG3 , and they are coloured based on their GO Slim Process category. B) Microbroth dilution minimum inhibitory concentration (MIC) assay for S . cerevisiae ERG3 genetic interactors for which gene deletion abrogates azole resistance of the erg3 mutant. Strains were complemented with their respective wild-type allele to verify the azole sensitivity phenotype was due to the specific gene deletion. MIC assays were conducted in YPD and growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements. Data was quantitatively displayed with colour using Treeview (see colour bar). C) MIC assay for C . albicans erg3 homozygous deletion mutants that also harboured homozygous deletions of genes originally identified in S . cerevisiae as being important for erg3- mediated resistance. MIC assay was performed as in part B. For the bottom panel, fluconazole MIC assays were conducted in YPD medium with 1 μg/mL doxycycline to achieve transcriptional repression of the target gene in the conditional expression strains.

Techniques Used: Concentration Assay, Mutagenesis, Expressing

11) Product Images from "Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus"

Article Title: Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus

Journal: Nature Communications

doi: 10.1038/s41467-018-08248-w

CMLD013075 reverses antifungal resistance in co-culture. a Checkerboard analysis of antifungal activity for combination of FL with Hsp90 inhibitors. FL-resistant strain CaCi-2 was exposed to the indicated twofold serial dilutions of each compound for 48 hours. Relative growth inhibition is displayed in heat-map format. Each colored box represents the mean of duplicate determinations. Color scale for relative growth is provided to the right of panel, green: no inhibition to black: complete inhibition. The experiment was repeated as an independent biological replicate to confirm results. b Co-culture of fluconazole (FL)-resistant, GFP-expressing C. albicans strain with human 293T cells line stably expressing a luciferase reporter. The concentration-dependent inhibition of fungal (left) and human (right) cell growth and survival by Hsp90 inhibitors in the presence of FL was measured in 384-well format after 48-h treatment. Relative fungal burden was evaluated by plate reader using a fluorescence endpoint while human cell survival was evaluated by measuring luminescence. Values for each endpoint were normalized to wells receiving no Hsp90 inhibitor. Each point represents the mean of measurements from triplicate wells. Error bars, SD. The experiment was repeated once as an independent biological replicate to confirm results
Figure Legend Snippet: CMLD013075 reverses antifungal resistance in co-culture. a Checkerboard analysis of antifungal activity for combination of FL with Hsp90 inhibitors. FL-resistant strain CaCi-2 was exposed to the indicated twofold serial dilutions of each compound for 48 hours. Relative growth inhibition is displayed in heat-map format. Each colored box represents the mean of duplicate determinations. Color scale for relative growth is provided to the right of panel, green: no inhibition to black: complete inhibition. The experiment was repeated as an independent biological replicate to confirm results. b Co-culture of fluconazole (FL)-resistant, GFP-expressing C. albicans strain with human 293T cells line stably expressing a luciferase reporter. The concentration-dependent inhibition of fungal (left) and human (right) cell growth and survival by Hsp90 inhibitors in the presence of FL was measured in 384-well format after 48-h treatment. Relative fungal burden was evaluated by plate reader using a fluorescence endpoint while human cell survival was evaluated by measuring luminescence. Values for each endpoint were normalized to wells receiving no Hsp90 inhibitor. Each point represents the mean of measurements from triplicate wells. Error bars, SD. The experiment was repeated once as an independent biological replicate to confirm results

Techniques Used: Co-Culture Assay, Activity Assay, Inhibition, Expressing, Stable Transfection, Luciferase, Concentration Assay, Fluorescence

Species-selective antifungal activity of Hsp90 inhibitor CMLD013075. a Relative growth inhibition by Hsp90 inhibitors of reference C. albicans strain (SN95) and a clinical isolate (CaCi-2) with resistance to FL. The effect of 48-hour exposure to each inhibitor over a twofold dilution series of concentrations is displayed in heat-map format. Each colored box represents the mean of duplicate determinations. Color scale for relative growth is provided to far right of panel, green: no inhibition to black: complete inhibition. The experiment was repeated as an independent biological replicate to confirm results. b Concentration-dependent inhibition of mammalian cell growth and survival after 3-day culture with Hsp90 inhibitors. Data points depict the mean of triplicate determinations, error bars, SD. The experiment was repeated as an independent biological replicate to confirm results. c Monolayer cultures of human HEPG2 cells in 6-well format were infected with C. albicans and then incubated with the indicated compounds for 48 h (FL, conventional antifungal fluconazole). Cultures were then fixed and stained by Periodic Acid-Schiff (PAS) technique to visualize fungal burden (dark red/purple signal) and underlying human cells (light pink signal). Representative fields from conventional light photomicrographs are presented at the same magnification in all panels. The experiment was repeated in two independent biological replicates to confirm results. Scale bar, 100 μm
Figure Legend Snippet: Species-selective antifungal activity of Hsp90 inhibitor CMLD013075. a Relative growth inhibition by Hsp90 inhibitors of reference C. albicans strain (SN95) and a clinical isolate (CaCi-2) with resistance to FL. The effect of 48-hour exposure to each inhibitor over a twofold dilution series of concentrations is displayed in heat-map format. Each colored box represents the mean of duplicate determinations. Color scale for relative growth is provided to far right of panel, green: no inhibition to black: complete inhibition. The experiment was repeated as an independent biological replicate to confirm results. b Concentration-dependent inhibition of mammalian cell growth and survival after 3-day culture with Hsp90 inhibitors. Data points depict the mean of triplicate determinations, error bars, SD. The experiment was repeated as an independent biological replicate to confirm results. c Monolayer cultures of human HEPG2 cells in 6-well format were infected with C. albicans and then incubated with the indicated compounds for 48 h (FL, conventional antifungal fluconazole). Cultures were then fixed and stained by Periodic Acid-Schiff (PAS) technique to visualize fungal burden (dark red/purple signal) and underlying human cells (light pink signal). Representative fields from conventional light photomicrographs are presented at the same magnification in all panels. The experiment was repeated in two independent biological replicates to confirm results. Scale bar, 100 μm

Techniques Used: Activity Assay, Inhibition, Concentration Assay, Infection, Incubation, Staining

12) Product Images from "A Potent Plant-Derived Antifungal Acetylenic Acid Mediates Its Activity by Interfering with Fatty Acid Homeostasis"

Article Title: A Potent Plant-Derived Antifungal Acetylenic Acid Mediates Its Activity by Interfering with Fatty Acid Homeostasis

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.05663-11

Effect of 6-NDA on fluconazole activity in a clinical isolate of C. albicans . (A) A checkerboard assay was performed to evaluate the combined effects of 6-NDA and fluconazole in a fluconazole-resistant clinical isolate (isolate 17) of C. albicans (described
Figure Legend Snippet: Effect of 6-NDA on fluconazole activity in a clinical isolate of C. albicans . (A) A checkerboard assay was performed to evaluate the combined effects of 6-NDA and fluconazole in a fluconazole-resistant clinical isolate (isolate 17) of C. albicans (described

Techniques Used: Activity Assay

13) Product Images from "PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90"

Article Title: PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001069

Pkc1 enables basal tolerance to ergosterol biosynthesis inhibitors via the MAPK cascade in Saccharomyces cerevisiae . ( A ) Drug tolerance of a wild-type (WT) strain (BY4741), a derivative (pkc1-ts) with a temperature sensitive PKC1 allele, and derivatives with deletions of BCK1 and SLT2 are compared in MIC assays. Assays were performed in synthetic defined (SD) medium at 35°C. Data was analyzed after 48 hours as in Figure 1A . The minimum drug concentration that inhibits growth by 80% relative to the drug-free growth control (MIC 80 ) is indicated for each strain. ( B ) Genetic compromise of Pkc1 creates a fungicidal combination with ergosterol biosynthesis inhibitors. MIC assays with two-fold dilutions of fluconazole (FL), fenpropimorph (FN), and terbinafine (TB) were performed in SD and incubated for 48 hours at 35°C. Cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed. ( C ) Schematic of the S. cerevisiae Pkc1 cell wall integrity pathway.
Figure Legend Snippet: Pkc1 enables basal tolerance to ergosterol biosynthesis inhibitors via the MAPK cascade in Saccharomyces cerevisiae . ( A ) Drug tolerance of a wild-type (WT) strain (BY4741), a derivative (pkc1-ts) with a temperature sensitive PKC1 allele, and derivatives with deletions of BCK1 and SLT2 are compared in MIC assays. Assays were performed in synthetic defined (SD) medium at 35°C. Data was analyzed after 48 hours as in Figure 1A . The minimum drug concentration that inhibits growth by 80% relative to the drug-free growth control (MIC 80 ) is indicated for each strain. ( B ) Genetic compromise of Pkc1 creates a fungicidal combination with ergosterol biosynthesis inhibitors. MIC assays with two-fold dilutions of fluconazole (FL), fenpropimorph (FN), and terbinafine (TB) were performed in SD and incubated for 48 hours at 35°C. Cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed. ( C ) Schematic of the S. cerevisiae Pkc1 cell wall integrity pathway.

Techniques Used: Concentration Assay, Incubation

Inhibition of PKC signaling phenocopies inhibition of Hsp90 reducing azole resistance. ( A ) Fluconazole (FL) resistance of clinical isolates is abrogated by inhibition of Hsp90 or Pkc1. MIC assays were conducted in YPD medium with no inhibitor (-), with the Hsp90 inhibitor geldanamycin (5 µM), or with the Pkc1 inhibitors cercosporamide (12.5 µg/ml) or staurosporine (0.5 µg/ml). Clinical isolates (CaCi) are ordered sequentially with those recovered early in treatment at the top and those recovered late at the bottom; the FL-sensitive strain SC5314 is included as a control. Data was analyzed after growth for 48 hours at 30°C as in Figure 1A . ( B ) Inhibition of Hsp90 or Pkc1 abrogates FL resistance of both S. cerevisiae and C. albicans erg3 mutants. Inhibition of Hsp90 or Pkc1 has no effect on the FL resistance of a S. cerevisiae strain ( PDR1 R ) that overexpresses multiple drug efflux pumps due to an activating mutation in the transcription factor Pdr1. FL MIC assays were carried out in YPD medium only (-) or in YPD with fixed concentrations of: geldanamycin ( Sc : 5 µM; Ca : 0.625 µM), cercosporamide ( Sc : 50 µg/ml; Ca : 25 µg/ml), or STS ( Sc : 0.625 µg/ml; Ca : 0.3125 µg/ml). Data was analyzed after growth for 48 hours at 30°C as in Figure 1A . The minimum drug concentration that inhibits growth by 80% relative to the no-FL growth control (MIC 80 ) is indicated for each strain.
Figure Legend Snippet: Inhibition of PKC signaling phenocopies inhibition of Hsp90 reducing azole resistance. ( A ) Fluconazole (FL) resistance of clinical isolates is abrogated by inhibition of Hsp90 or Pkc1. MIC assays were conducted in YPD medium with no inhibitor (-), with the Hsp90 inhibitor geldanamycin (5 µM), or with the Pkc1 inhibitors cercosporamide (12.5 µg/ml) or staurosporine (0.5 µg/ml). Clinical isolates (CaCi) are ordered sequentially with those recovered early in treatment at the top and those recovered late at the bottom; the FL-sensitive strain SC5314 is included as a control. Data was analyzed after growth for 48 hours at 30°C as in Figure 1A . ( B ) Inhibition of Hsp90 or Pkc1 abrogates FL resistance of both S. cerevisiae and C. albicans erg3 mutants. Inhibition of Hsp90 or Pkc1 has no effect on the FL resistance of a S. cerevisiae strain ( PDR1 R ) that overexpresses multiple drug efflux pumps due to an activating mutation in the transcription factor Pdr1. FL MIC assays were carried out in YPD medium only (-) or in YPD with fixed concentrations of: geldanamycin ( Sc : 5 µM; Ca : 0.625 µM), cercosporamide ( Sc : 50 µg/ml; Ca : 25 µg/ml), or STS ( Sc : 0.625 µg/ml; Ca : 0.3125 µg/ml). Data was analyzed after growth for 48 hours at 30°C as in Figure 1A . The minimum drug concentration that inhibits growth by 80% relative to the no-FL growth control (MIC 80 ) is indicated for each strain.

Techniques Used: Inhibition, Mutagenesis, Concentration Assay

Pkc1 enables basal tolerance to ergosterol biosynthesis inhibitors in part via the MAPK cascade in Candida albicans . ( A ) Deletion of PKC1 , BCK1 or MKC1 reduces tolerance to fluconazole (FL), fenpropimorph (FN), and terbinafine (TB) in MIC assays. Assays were performed in YPD medium at 35°C with strains derived from the WT SN95. Data was analyzed after 72 hours growth as in Figure 1A . The minimum drug concentration that inhibits growth by 80% relative to the drug-free growth control (MIC 80 ) is indicated for each strain. ( B ) Deletion of PKC1 , but not MAPK components, creates a fungicidal combination with the ergosterol biosynthesis inhibitors in C. albicans . MIC assays with four-fold dilutions of FL, FN, and TB were performed in YPD and incubated for 48 hours at 35°C. Cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.
Figure Legend Snippet: Pkc1 enables basal tolerance to ergosterol biosynthesis inhibitors in part via the MAPK cascade in Candida albicans . ( A ) Deletion of PKC1 , BCK1 or MKC1 reduces tolerance to fluconazole (FL), fenpropimorph (FN), and terbinafine (TB) in MIC assays. Assays were performed in YPD medium at 35°C with strains derived from the WT SN95. Data was analyzed after 72 hours growth as in Figure 1A . The minimum drug concentration that inhibits growth by 80% relative to the drug-free growth control (MIC 80 ) is indicated for each strain. ( B ) Deletion of PKC1 , but not MAPK components, creates a fungicidal combination with the ergosterol biosynthesis inhibitors in C. albicans . MIC assays with four-fold dilutions of FL, FN, and TB were performed in YPD and incubated for 48 hours at 35°C. Cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.

Techniques Used: Derivative Assay, Concentration Assay, Incubation

Pharmacological inhibition of PKC signaling enhances the efficacy of antifungal drugs targeting the cell membrane. ( A ) A drug screen identifies compounds that abrogate fluconazole (FL) resistance of a Candida albicans clinical isolate (CaCi-2). Seven compounds from the LOPAC 1280 Navigator library had little toxicity on their own but enhanced the efficacy of FL against CaCi-2 when tested at 12.5 µM in RPMI medium with 2% glucose in the presence or absence of 8 µg/ml FL. Growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements and normalized relative to the no compound control (-) or FL-only control. Data was quantitatively displayed with colour using Treeview (see colour bar). The target or mode of action of each compound is indicated in blue. 1 CAT = choline acetyltransferase; 2 JAK3 = Janus kinase family protein; and 3 KDO-8-P = 3-deoxy-D-manno-2-octulosonate-8-phosphate. ( B ) Pharmacological inhibition of Pkc1 with cercosporamide abrogates azole resistance and reduces echinocandin tolerance of CaCi-2 in an MIC assay. Assays were done in yeast peptone dextrose (YPD) with a fixed concentration of 2 µg/ml micafungin (MF) or 8 µg/ml FL, as indicated. Data was analyzed after 48 hours at 30°C as in part A. ( C ) Pkc1 inhibitors confer increased sensitivity to other ergosterol biosynthesis inhibitors. A fixed concentration of CaCi-2 cells was incubated in YPD with no antifungal (-), 2 µg/ml MF, 8 µg/ml FL, 2.5 µg/ml fenpropimorph (FN), or 2 µg/ml terbinafine (TB) and with the PKC inhibitors cercosporamide (100 µg/ml) or staurosporine (37.5 ng/ml), as indicated. Data was analyzed after 48 hours at 30°C as in part A.
Figure Legend Snippet: Pharmacological inhibition of PKC signaling enhances the efficacy of antifungal drugs targeting the cell membrane. ( A ) A drug screen identifies compounds that abrogate fluconazole (FL) resistance of a Candida albicans clinical isolate (CaCi-2). Seven compounds from the LOPAC 1280 Navigator library had little toxicity on their own but enhanced the efficacy of FL against CaCi-2 when tested at 12.5 µM in RPMI medium with 2% glucose in the presence or absence of 8 µg/ml FL. Growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements and normalized relative to the no compound control (-) or FL-only control. Data was quantitatively displayed with colour using Treeview (see colour bar). The target or mode of action of each compound is indicated in blue. 1 CAT = choline acetyltransferase; 2 JAK3 = Janus kinase family protein; and 3 KDO-8-P = 3-deoxy-D-manno-2-octulosonate-8-phosphate. ( B ) Pharmacological inhibition of Pkc1 with cercosporamide abrogates azole resistance and reduces echinocandin tolerance of CaCi-2 in an MIC assay. Assays were done in yeast peptone dextrose (YPD) with a fixed concentration of 2 µg/ml micafungin (MF) or 8 µg/ml FL, as indicated. Data was analyzed after 48 hours at 30°C as in part A. ( C ) Pkc1 inhibitors confer increased sensitivity to other ergosterol biosynthesis inhibitors. A fixed concentration of CaCi-2 cells was incubated in YPD with no antifungal (-), 2 µg/ml MF, 8 µg/ml FL, 2.5 µg/ml fenpropimorph (FN), or 2 µg/ml terbinafine (TB) and with the PKC inhibitors cercosporamide (100 µg/ml) or staurosporine (37.5 ng/ml), as indicated. Data was analyzed after 48 hours at 30°C as in part A.

Techniques Used: Inhibition, Concentration Assay, Incubation

PKC signaling and calcineurin independently regulate tolerance to ergosterol biosynthesis inhibitors via a common target in C. albicans . ( A ) Deletion of C. albicans PKC1 does not block EBI-induced activation of calcineurin. Transcript levels of two calcineurin-dependent genes, PLC3 and UTR2 , were measured by quantitative RT-PCR after growth in rich medium at 35°C for 6 hours without any antifungal (U) or with 16 µg/mL fluconazole (FL), as indicated. Transcripts were normalized to GPD1 . Levels are expressed relative to the untreated wild-type samples, which were set to 1. Data are means ± SD for triplicate samples and are representative of two independent experiments. ( B ) Simultaneous inhibition of calcineurin and Pkc1 signaling does not synergistically decrease FL tolerance of a WT strain (SN95). A fractional inhibitory concentration (FIC) assay was carried out in YPD medium containing a fixed concentration of 0.5 µg/mL FL and gradients of the calcineurin inhibitor cyclosporin A (CsA) and the PKC inhibitor staurosporine (STS). Data was analyzed after growth at 35°C for 48 hours as in Figure 1A . The minimum concentration of STS or CsA that inhibits growth by 80% relative to the FL-only growth control (MIC 80 ) individually or in combination is indicated along with the FIC. ( C ) FL tolerance of a mutant lacking the catalytic subunit of calcineurin is not sensitive to inhibition of PKC signaling. MIC assays were performed in YPD medium only (-) or YPD with a fixed concentration of 0.5 µg/mL FL. ( D ) Simplified schematic of how C. albicans Pkc1 regulates responses to ergosterol biosynthesis inhibitors (EBIs) important for basal tolerance and resistance.
Figure Legend Snippet: PKC signaling and calcineurin independently regulate tolerance to ergosterol biosynthesis inhibitors via a common target in C. albicans . ( A ) Deletion of C. albicans PKC1 does not block EBI-induced activation of calcineurin. Transcript levels of two calcineurin-dependent genes, PLC3 and UTR2 , were measured by quantitative RT-PCR after growth in rich medium at 35°C for 6 hours without any antifungal (U) or with 16 µg/mL fluconazole (FL), as indicated. Transcripts were normalized to GPD1 . Levels are expressed relative to the untreated wild-type samples, which were set to 1. Data are means ± SD for triplicate samples and are representative of two independent experiments. ( B ) Simultaneous inhibition of calcineurin and Pkc1 signaling does not synergistically decrease FL tolerance of a WT strain (SN95). A fractional inhibitory concentration (FIC) assay was carried out in YPD medium containing a fixed concentration of 0.5 µg/mL FL and gradients of the calcineurin inhibitor cyclosporin A (CsA) and the PKC inhibitor staurosporine (STS). Data was analyzed after growth at 35°C for 48 hours as in Figure 1A . The minimum concentration of STS or CsA that inhibits growth by 80% relative to the FL-only growth control (MIC 80 ) individually or in combination is indicated along with the FIC. ( C ) FL tolerance of a mutant lacking the catalytic subunit of calcineurin is not sensitive to inhibition of PKC signaling. MIC assays were performed in YPD medium only (-) or YPD with a fixed concentration of 0.5 µg/mL FL. ( D ) Simplified schematic of how C. albicans Pkc1 regulates responses to ergosterol biosynthesis inhibitors (EBIs) important for basal tolerance and resistance.

Techniques Used: Blocking Assay, Activation Assay, Quantitative RT-PCR, Inhibition, Concentration Assay, Mutagenesis

Compromising PKC-MAPK signaling blocks calcineurin activation in response to ergosterol biosynthesis inhibitors in S. cerevisiae . ( A ) Genetically compromising PKC-MAPK signaling by deleting SLT2 blocks calcineurin activation monitored with a 4XCDRE- lacZ reporter. β-galactosidase activity was measured after incubation in SD medium for 24 hours without any antifungal (U) or in the presence of ergosterol biosynthesis inhibitors at the following concentrations: 16 µg/mL fluconazole (FL), 1 µg/mL fenpropimorph (FN), or 25 µg/mL terbinafine (TB). While the WT strain exhibited increased β-galactosidase activity in response to ergosterol biosynthesis inhibitors, deletion of SLT2 or CNB1 (which encodes the regulatory subunit of calcineurin) blocked calcineurin activation. Data are means ± SD for triplicate samples and are representative of two independent experiments. ( B ) Pharmacological inhibition of PKC signaling with staurosporine (STS) blocks calcineurin activation monitored with a 4XCDRE- lacZ reporter. β-galactosidase activity was measured after incubation in SD medium (-) or in SD with 2.5 µg/mL STS. Cells were then treated with 32 µg/mL FL or were left untreated (U). Data are means ± SD for triplicate samples and are representative of two independent experiments. ( C ) Simplified schematic of how S. cerevisiae Pkc1 regulates responses to ergosterol biosynthesis inhibitors (EBIs) important for basal tolerance and resistance.
Figure Legend Snippet: Compromising PKC-MAPK signaling blocks calcineurin activation in response to ergosterol biosynthesis inhibitors in S. cerevisiae . ( A ) Genetically compromising PKC-MAPK signaling by deleting SLT2 blocks calcineurin activation monitored with a 4XCDRE- lacZ reporter. β-galactosidase activity was measured after incubation in SD medium for 24 hours without any antifungal (U) or in the presence of ergosterol biosynthesis inhibitors at the following concentrations: 16 µg/mL fluconazole (FL), 1 µg/mL fenpropimorph (FN), or 25 µg/mL terbinafine (TB). While the WT strain exhibited increased β-galactosidase activity in response to ergosterol biosynthesis inhibitors, deletion of SLT2 or CNB1 (which encodes the regulatory subunit of calcineurin) blocked calcineurin activation. Data are means ± SD for triplicate samples and are representative of two independent experiments. ( B ) Pharmacological inhibition of PKC signaling with staurosporine (STS) blocks calcineurin activation monitored with a 4XCDRE- lacZ reporter. β-galactosidase activity was measured after incubation in SD medium (-) or in SD with 2.5 µg/mL STS. Cells were then treated with 32 µg/mL FL or were left untreated (U). Data are means ± SD for triplicate samples and are representative of two independent experiments. ( C ) Simplified schematic of how S. cerevisiae Pkc1 regulates responses to ergosterol biosynthesis inhibitors (EBIs) important for basal tolerance and resistance.

Techniques Used: Activation Assay, Activity Assay, Incubation, Inhibition

14) Product Images from "Hsp90 Governs Dispersion and Drug Resistance of Fungal Biofilms"

Article Title: Hsp90 Governs Dispersion and Drug Resistance of Fungal Biofilms

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002257

Inhibition of Hsp90 function dramatically enhances the efficacy of fluconazole against C. albicans biofilms in vitro. ( A ) Strains of C. albicans were grown in 96-well microtiter plates in RPMI at 37°C. After 24 hours cells were washed with PBS to remove non-adherent cells and fresh medium was added with varying concentrations of the azole fluconazole (FL) and either the calcineurin inhibitor FK506 or the Hsp90 inhibitor geldanamycin (GdA) in a checkerboard format. Metabolic activity was measured as in Figure 1A . The FIC index was calculated as indicated in Table 1 . Bright green represents growth above the MIC 50 , dull green represents growth at the MIC 50 , and black represents growth below the MIC 50 . Data was quantitatively displayed with colour using the program Java TreeView 1.1.3 ( http://jtreeview.sourceforge.net ). Inhibiting calcineurin or Hsp90 function has synergistic activity with fluconazole. ( B ) Strains of C. albicans were grown in 96-well microtiter plates in RPMI at 37°C. When indicated, 20 µg/mL doxycycline (DOX) was added to the medium. After 24 hours cells were washed with PBS to remove non-adherent cells and fresh medium was added with varying concentrations of fluconazole. Metabolic activity was measured as in Figure 1A . Genetic depletion of Hsp90 reduces the MIC 50 of fluconazole to a greater extent than deletion of its client proteins calcineurin or Mkc1.
Figure Legend Snippet: Inhibition of Hsp90 function dramatically enhances the efficacy of fluconazole against C. albicans biofilms in vitro. ( A ) Strains of C. albicans were grown in 96-well microtiter plates in RPMI at 37°C. After 24 hours cells were washed with PBS to remove non-adherent cells and fresh medium was added with varying concentrations of the azole fluconazole (FL) and either the calcineurin inhibitor FK506 or the Hsp90 inhibitor geldanamycin (GdA) in a checkerboard format. Metabolic activity was measured as in Figure 1A . The FIC index was calculated as indicated in Table 1 . Bright green represents growth above the MIC 50 , dull green represents growth at the MIC 50 , and black represents growth below the MIC 50 . Data was quantitatively displayed with colour using the program Java TreeView 1.1.3 ( http://jtreeview.sourceforge.net ). Inhibiting calcineurin or Hsp90 function has synergistic activity with fluconazole. ( B ) Strains of C. albicans were grown in 96-well microtiter plates in RPMI at 37°C. When indicated, 20 µg/mL doxycycline (DOX) was added to the medium. After 24 hours cells were washed with PBS to remove non-adherent cells and fresh medium was added with varying concentrations of fluconazole. Metabolic activity was measured as in Figure 1A . Genetic depletion of Hsp90 reduces the MIC 50 of fluconazole to a greater extent than deletion of its client proteins calcineurin or Mkc1.

Techniques Used: Inhibition, In Vitro, Activity Assay

Compromise of Hsp90 function genetically or pharmacologically enhances the efficacy of fluconazole in vivo. ( A ) The tetO - HSP90/hsp90 Δ strain was inoculated in rat venous catheters for 24 hours with or without 20 µg/mL doxycycline (DOX) followed by intraluminal azole treatment for an additional 24 hours. Following drug exposure, catheters were removed for visualization by scanning electron microscopy. The first column represents treatment with 250 µg/mL fluconazole (FL), followed by treatment with both 20 µg/mL DOX and 250 µg/mL FL. The top row represents 50 X magnification and the bottom row represents 1,000 X magnification. The combination of FL and DOX abrogates biofilms. ( B ) Biofilms were cultured as in A with 250 µg/mL FL, 100 µg/mL 17-AAG, or the combination of drugs. Serial dilutions of the catheter fluid were plated for viable fungal colony counts. Results are expressed as the mean colony forming unit (CFU) per catheter. The combination of FL and 17-AAG reduces fungal burden in the catheter compared to individual drug treatments (Asterisk indicates P
Figure Legend Snippet: Compromise of Hsp90 function genetically or pharmacologically enhances the efficacy of fluconazole in vivo. ( A ) The tetO - HSP90/hsp90 Δ strain was inoculated in rat venous catheters for 24 hours with or without 20 µg/mL doxycycline (DOX) followed by intraluminal azole treatment for an additional 24 hours. Following drug exposure, catheters were removed for visualization by scanning electron microscopy. The first column represents treatment with 250 µg/mL fluconazole (FL), followed by treatment with both 20 µg/mL DOX and 250 µg/mL FL. The top row represents 50 X magnification and the bottom row represents 1,000 X magnification. The combination of FL and DOX abrogates biofilms. ( B ) Biofilms were cultured as in A with 250 µg/mL FL, 100 µg/mL 17-AAG, or the combination of drugs. Serial dilutions of the catheter fluid were plated for viable fungal colony counts. Results are expressed as the mean colony forming unit (CFU) per catheter. The combination of FL and 17-AAG reduces fungal burden in the catheter compared to individual drug treatments (Asterisk indicates P

Techniques Used: In Vivo, Electron Microscopy, Cell Culture

15) Product Images from "Beauvericin Potentiates Azole Activity via Inhibition of Multidrug Efflux, Blocks Candida albicans Morphogenesis, and Is Effluxed via Yor1 and Circuitry Controlled by Zcf29"

Article Title: Beauvericin Potentiates Azole Activity via Inhibition of Multidrug Efflux, Blocks Candida albicans Morphogenesis, and Is Effluxed via Yor1 and Circuitry Controlled by Zcf29

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01959-16

Beauvericin potentiates azole activity via inhibition of Cdr1. Left, synergy between beauvericin and fluconazole is abrogated in strains lacking CDR1 but not CDR2 . Checkerboard assays were performed as described in the legend to . Right, beauvericin
Figure Legend Snippet: Beauvericin potentiates azole activity via inhibition of Cdr1. Left, synergy between beauvericin and fluconazole is abrogated in strains lacking CDR1 but not CDR2 . Checkerboard assays were performed as described in the legend to . Right, beauvericin

Techniques Used: Activity Assay, Inhibition

Beauvericin enhances azole efficacy against azole-resistant Candida isolates. (a) Beauvericin abrogates fluconazole resistance of a reference strain of C. glabrata (BG2) and two azole-resistant C. albicans clinical isolates, CaCi-2 and CaCi-17, on rich
Figure Legend Snippet: Beauvericin enhances azole efficacy against azole-resistant Candida isolates. (a) Beauvericin abrogates fluconazole resistance of a reference strain of C. glabrata (BG2) and two azole-resistant C. albicans clinical isolates, CaCi-2 and CaCi-17, on rich

Techniques Used:

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    Sequoia Research fluconazole
    Beauvericin inhibits Hsp90 function ( a ) Beauvericin (BEA) reduces <t>fluconazole-induced</t> calcineurin activation. S. cerevisiae harboring 4xCDRE- lacZ reporter construct ± fluconazole (FLC, 64 μg/ml), ± geldanamycin (GdA, 5.6 μg/ml), FK506 (1.0 μg/ml), or beauvericin (20 μg/ml). Data are mean ± s.d. from technical triplicates and representative of biological replicates. * P
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    Beauvericin inhibits Hsp90 function ( a ) Beauvericin (BEA) reduces fluconazole-induced calcineurin activation. S. cerevisiae harboring 4xCDRE- lacZ reporter construct ± fluconazole (FLC, 64 μg/ml), ± geldanamycin (GdA, 5.6 μg/ml), FK506 (1.0 μg/ml), or beauvericin (20 μg/ml). Data are mean ± s.d. from technical triplicates and representative of biological replicates. * P

    Journal: Nature chemical biology

    Article Title: Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling

    doi: 10.1038/nchembio.2165

    Figure Lengend Snippet: Beauvericin inhibits Hsp90 function ( a ) Beauvericin (BEA) reduces fluconazole-induced calcineurin activation. S. cerevisiae harboring 4xCDRE- lacZ reporter construct ± fluconazole (FLC, 64 μg/ml), ± geldanamycin (GdA, 5.6 μg/ml), FK506 (1.0 μg/ml), or beauvericin (20 μg/ml). Data are mean ± s.d. from technical triplicates and representative of biological replicates. * P

    Article Snippet: Fluconazole (Sequoia Research Products) was dissolved in sterile ddH2 O, beauvericin in 100% methanol, cycloheximide (AG Scientific Inc.) in sterile ddH2 O, rhodamine-6G (Sigma-Aldrich) in sterile ddH2 O, geldanamycin (Invitrogen) in DMSO, and cyclosporin A (CalBiochem) in DMSO.

    Techniques: Activation Assay, Construct

    Beauvericin inhibits Pdr5, thereby increasing fluconazole intracellular accumulation, and can be effluxed by Pdr5 following substitutions that alter substrate-specificity ( a ) S. cerevisiae lacking Yor1 is sensitive to beauvericin, but acquires resistance via Pdr5 substitutions. 1×10 8 cells plated on YEPD containing beauvericin (100 μg/ml); resistant mutants recovered after 3–4 days at 30°C. Resistance was assessed in YEPD with two-fold serial dilutions of beauvericin, as in Figure 1 . Mutations identified by genome sequencing indicated as amino acid changes; those in Pdr5 shown in red. ( b ) Functional validation of PDR5 mutations. Amino acid 538 is altered in three of five mutants; expression of PDR5 G538R in yor1 Δ pdr5 Δ confers beauvericin resistance. Resistance was assessed in SD medium at 30°C for 72 hours. (a–b) Performed in biological triplicates with technical duplicates. ( c ) Beauvericin-resistant mutants exhibit altered substrate specificity, and beauvericin enhances azole accumulation. Deletion of PDR5 increased intracellular accumulation of radiolabelled fluconazole, as with beauvericin-resistant mutants yor1 ΔR1 and R5. Error bars represent standard deviation (s.d.), * P

    Journal: Nature chemical biology

    Article Title: Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling

    doi: 10.1038/nchembio.2165

    Figure Lengend Snippet: Beauvericin inhibits Pdr5, thereby increasing fluconazole intracellular accumulation, and can be effluxed by Pdr5 following substitutions that alter substrate-specificity ( a ) S. cerevisiae lacking Yor1 is sensitive to beauvericin, but acquires resistance via Pdr5 substitutions. 1×10 8 cells plated on YEPD containing beauvericin (100 μg/ml); resistant mutants recovered after 3–4 days at 30°C. Resistance was assessed in YEPD with two-fold serial dilutions of beauvericin, as in Figure 1 . Mutations identified by genome sequencing indicated as amino acid changes; those in Pdr5 shown in red. ( b ) Functional validation of PDR5 mutations. Amino acid 538 is altered in three of five mutants; expression of PDR5 G538R in yor1 Δ pdr5 Δ confers beauvericin resistance. Resistance was assessed in SD medium at 30°C for 72 hours. (a–b) Performed in biological triplicates with technical duplicates. ( c ) Beauvericin-resistant mutants exhibit altered substrate specificity, and beauvericin enhances azole accumulation. Deletion of PDR5 increased intracellular accumulation of radiolabelled fluconazole, as with beauvericin-resistant mutants yor1 ΔR1 and R5. Error bars represent standard deviation (s.d.), * P

    Article Snippet: Fluconazole (Sequoia Research Products) was dissolved in sterile ddH2 O, beauvericin in 100% methanol, cycloheximide (AG Scientific Inc.) in sterile ddH2 O, rhodamine-6G (Sigma-Aldrich) in sterile ddH2 O, geldanamycin (Invitrogen) in DMSO, and cyclosporin A (CalBiochem) in DMSO.

    Techniques: Sequencing, Functional Assay, Expressing, Standard Deviation

    The combination of beauvericin and fluconazole provides a powerful therapeutic strategy ( a ) Beauvericin enhances fluconazole efficacy in a mouse model of C. albicans disseminated infection. Mice were infected with C. albicans (SC5314) and treated with vehicle, beauvericin, fluconazole, or the combination. Even with a high C. albicans inoculum, the combination of beauvericin and fluconazole significantly enhanced survival relative to either treatment alone. Each group consisted of 10 female BALB/c mice. Log-rank (Mantel-Cox) test: vehicle vs. beauvericin: P =0.3173, vehicle vs. fluconazole: P

    Journal: Nature chemical biology

    Article Title: Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling

    doi: 10.1038/nchembio.2165

    Figure Lengend Snippet: The combination of beauvericin and fluconazole provides a powerful therapeutic strategy ( a ) Beauvericin enhances fluconazole efficacy in a mouse model of C. albicans disseminated infection. Mice were infected with C. albicans (SC5314) and treated with vehicle, beauvericin, fluconazole, or the combination. Even with a high C. albicans inoculum, the combination of beauvericin and fluconazole significantly enhanced survival relative to either treatment alone. Each group consisted of 10 female BALB/c mice. Log-rank (Mantel-Cox) test: vehicle vs. beauvericin: P =0.3173, vehicle vs. fluconazole: P

    Article Snippet: Fluconazole (Sequoia Research Products) was dissolved in sterile ddH2 O, beauvericin in 100% methanol, cycloheximide (AG Scientific Inc.) in sterile ddH2 O, rhodamine-6G (Sigma-Aldrich) in sterile ddH2 O, geldanamycin (Invitrogen) in DMSO, and cyclosporin A (CalBiochem) in DMSO.

    Techniques: Infection, Mouse Assay

    Beauvericin enhances fluconazole efficacy against diverse fungi and blocks the emergence of resistance ( a ) Beauvericin (BEA) reduces fluconazole resistance of reference strains of S. cerevisiae (BY4741), C. albicans (SN95), and C. neoformans (H99a), and A. fumigatus clinical isolate on rich medium (YEPD). Fluconazole (FLC) strips generate a gradient from 0.016 to 256 μg/ml, with the highest concentration at the top. Where indicated, plates contain 20 μg/ml of BEA. Experiment performed in biological triplicates. ( b ) BEA and FLC are synergistic and cidal against C. albicans (SN95). SN95 was subjected to two-fold serial dilutions of BEA and FLC in YEPD at 30°C for 48 hours. Optical densities were standardized to drug-free controls (see color bar) and FICI was calculated based on concentrations causing 60–70% growth inhibition. The drug combination is cidal, based on transferring cells to drug-free YEPD medium for 24 hours. ( c ) BEA reduced FLC resistance of C. albicans clinical isolates (CaCi) similar to geldanamycin (GdA) and cyclosporin A (CsA). CaCi are sequentially ordered with isolates recovered early at the top. MIC assays were performed in YEPD (−) with GdA (5 μM), CsA (20 μM), or BEA (25 μM), with a two-fold serial dilution of FLC. (b–c) Experiments performed in biological triplicates with technical duplicates. ( d ) BEA blocks the emergence of FLC resistance in C. albicans (SN95). 1×10 5 cells were plated on YEPD containing no inhibitor (−), 20 μg/ml BEA, 32 μg/ml FLC, or the combination. Plates were photographed after three days at 30°C. Experiment performed in biological triplicates.

    Journal: Nature chemical biology

    Article Title: Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling

    doi: 10.1038/nchembio.2165

    Figure Lengend Snippet: Beauvericin enhances fluconazole efficacy against diverse fungi and blocks the emergence of resistance ( a ) Beauvericin (BEA) reduces fluconazole resistance of reference strains of S. cerevisiae (BY4741), C. albicans (SN95), and C. neoformans (H99a), and A. fumigatus clinical isolate on rich medium (YEPD). Fluconazole (FLC) strips generate a gradient from 0.016 to 256 μg/ml, with the highest concentration at the top. Where indicated, plates contain 20 μg/ml of BEA. Experiment performed in biological triplicates. ( b ) BEA and FLC are synergistic and cidal against C. albicans (SN95). SN95 was subjected to two-fold serial dilutions of BEA and FLC in YEPD at 30°C for 48 hours. Optical densities were standardized to drug-free controls (see color bar) and FICI was calculated based on concentrations causing 60–70% growth inhibition. The drug combination is cidal, based on transferring cells to drug-free YEPD medium for 24 hours. ( c ) BEA reduced FLC resistance of C. albicans clinical isolates (CaCi) similar to geldanamycin (GdA) and cyclosporin A (CsA). CaCi are sequentially ordered with isolates recovered early at the top. MIC assays were performed in YEPD (−) with GdA (5 μM), CsA (20 μM), or BEA (25 μM), with a two-fold serial dilution of FLC. (b–c) Experiments performed in biological triplicates with technical duplicates. ( d ) BEA blocks the emergence of FLC resistance in C. albicans (SN95). 1×10 5 cells were plated on YEPD containing no inhibitor (−), 20 μg/ml BEA, 32 μg/ml FLC, or the combination. Plates were photographed after three days at 30°C. Experiment performed in biological triplicates.

    Article Snippet: Fluconazole (Sequoia Research Products) was dissolved in sterile ddH2 O, beauvericin in 100% methanol, cycloheximide (AG Scientific Inc.) in sterile ddH2 O, rhodamine-6G (Sigma-Aldrich) in sterile ddH2 O, geldanamycin (Invitrogen) in DMSO, and cyclosporin A (CalBiochem) in DMSO.

    Techniques: Concentration Assay, Inhibition, Transferring, Serial Dilution

    Diversity of species and antifungal resistance profiles of 1,603 fungal isolates from cystic fibrosis patients. Each dot represents an isolate from the corresponding cystic fibrosis patient and colours represent species identity. The vertical dispersion of isolates within each phenotypic class is simply for visual clarity, as with the horizontal dispersion of isolates from an individual patient. (A) Summary of yeast isolates. The isolates are categorized as: “Fluconazole resistant” if their relative growth with fixed concentration of fluconazole at 128 μg/ml was greater than 2 times that of the relative growth of reference C . albicans strain SN95; “Fluconazole susceptible” if their relative growth was less than 2 times the relative growth of SN95; and “Variable” if their resistance profiles were variable over biological and technical duplicates. C . albicans is divided into two groups: isolates that show standard yeast morphology in rich medium at 30°C are in blue; and isolates that show filamentous growth under these conditions are in red. (B) Summary of mold isolates. The isolates are categorized as: “Itraconazole resistant” if their relative growth with fixed concentration of itraconazole at 0.5 μg/ml was greater than 2 times that of the relative growth of reference A . fumigatus strain AF293; and “Itraconazole susceptible” if their relative growth was less than 2 times the relative growth of AF293

    Journal: PLoS Pathogens

    Article Title: Global Analysis of the Fungal Microbiome in Cystic Fibrosis Patients Reveals Loss of Function of the Transcriptional Repressor Nrg1 as a Mechanism of Pathogen Adaptation

    doi: 10.1371/journal.ppat.1005308

    Figure Lengend Snippet: Diversity of species and antifungal resistance profiles of 1,603 fungal isolates from cystic fibrosis patients. Each dot represents an isolate from the corresponding cystic fibrosis patient and colours represent species identity. The vertical dispersion of isolates within each phenotypic class is simply for visual clarity, as with the horizontal dispersion of isolates from an individual patient. (A) Summary of yeast isolates. The isolates are categorized as: “Fluconazole resistant” if their relative growth with fixed concentration of fluconazole at 128 μg/ml was greater than 2 times that of the relative growth of reference C . albicans strain SN95; “Fluconazole susceptible” if their relative growth was less than 2 times the relative growth of SN95; and “Variable” if their resistance profiles were variable over biological and technical duplicates. C . albicans is divided into two groups: isolates that show standard yeast morphology in rich medium at 30°C are in blue; and isolates that show filamentous growth under these conditions are in red. (B) Summary of mold isolates. The isolates are categorized as: “Itraconazole resistant” if their relative growth with fixed concentration of itraconazole at 0.5 μg/ml was greater than 2 times that of the relative growth of reference A . fumigatus strain AF293; and “Itraconazole susceptible” if their relative growth was less than 2 times the relative growth of AF293

    Article Snippet: The same overnight cultures were diluted 20,000 fold into 200 μl of YPD and 200 μl YPD containing 128 μg/ml fluconazole (Sequoia Research Products) in clear 96-well plates.

    Techniques: Concentration Assay

    Fluconazole treatment does not unmask β-glucan. Mice were pre-injected with fluconazole and infected as in Figure 4 . At 16 hr post-infection, kidneys were homogenized. (A) Homogenates were diluted and assayed for viable colony forming units or (B,D) homogenates were stained with anti-β-glucan monoclonal antibody and Cy3-labeled secondary antibody. Treatments of mice before infection were the following: (B) single dose of PBS (vehicle), (C) single dose of 2.5 mg/kg fluconazole in PBS, (D) single dose of 30 µg/kg caspofungin in PBS. Images and CFU data are representative of two independent experiments with 2 to 3 mice per group. Bars in (A) represent averages of triplicate counts and error bars represent standard deviations. Scale bar in (B) is 10 microns long and applies to all images.

    Journal: PLoS Pathogens

    Article Title: Dynamic, Morphotype-Specific Candida albicans ?-Glucan Exposure during Infection and Drug Treatment

    doi: 10.1371/journal.ppat.1000227

    Figure Lengend Snippet: Fluconazole treatment does not unmask β-glucan. Mice were pre-injected with fluconazole and infected as in Figure 4 . At 16 hr post-infection, kidneys were homogenized. (A) Homogenates were diluted and assayed for viable colony forming units or (B,D) homogenates were stained with anti-β-glucan monoclonal antibody and Cy3-labeled secondary antibody. Treatments of mice before infection were the following: (B) single dose of PBS (vehicle), (C) single dose of 2.5 mg/kg fluconazole in PBS, (D) single dose of 30 µg/kg caspofungin in PBS. Images and CFU data are representative of two independent experiments with 2 to 3 mice per group. Bars in (A) represent averages of triplicate counts and error bars represent standard deviations. Scale bar in (B) is 10 microns long and applies to all images.

    Article Snippet: Alternatively, 0.1 ml of a 0.5 or 5 mg/ml solution of fluconazole (Sequoia Research Products, United Kingdom) in PBS was injected per mouse i.p. just prior to tail vein infection, corresponding to doses of 2.5 or 25 mg/kg.

    Techniques: Mouse Assay, Injection, Infection, Staining, Labeling

    Whole genome sequencing identifies aneuploidies that are associated with the restoration of azole resistance to a C . albicans clinical isolate lacking RGD1 . A) RGD1 was deleted from a clinical isolate obtained late in treatment from a patient undergoing fluconazole therapy (CaCi-17). Resistant mutants were obtained by plating 2x10 8 cells on YPD plates containing a high concentration of miconazole. Spontaneous mutants were selected after 5 days at 30°C. Azole resistance of the mutants was verified by MIC assay. Cells were grown as described in Fig 1 . Growth was measured after 48 hours. B) Spontaneous azole resistant mutants show fitness defect in the absence of the selective pressure but show enhanced growth in the presence of fluconazole. Strains were grown in the absence or presence of 128 μg/mL fluconazole. Growth was assessed by OD 600 measurements every 15 minutes for 48 hours in a Tecan plate reader. C) Copy number variation was analyzed using the Y MAP pipeline. Amplification of chromosome 7 as well as a portion of chromosome 3 occurred in all independent spontaneous resistant mutants. D) Chromosome 3 haplotype map analyzed using Y MAP . The monosomic region of Chr3L was generated from different haplotypes in the four azole-resistant isolates: haplotype A (Cyan) in isolates R1 and R2, and haplotype B (magenta) in isolates R3 and R4.

    Journal: PLoS Genetics

    Article Title: Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs

    doi: 10.1371/journal.pgen.1007319

    Figure Lengend Snippet: Whole genome sequencing identifies aneuploidies that are associated with the restoration of azole resistance to a C . albicans clinical isolate lacking RGD1 . A) RGD1 was deleted from a clinical isolate obtained late in treatment from a patient undergoing fluconazole therapy (CaCi-17). Resistant mutants were obtained by plating 2x10 8 cells on YPD plates containing a high concentration of miconazole. Spontaneous mutants were selected after 5 days at 30°C. Azole resistance of the mutants was verified by MIC assay. Cells were grown as described in Fig 1 . Growth was measured after 48 hours. B) Spontaneous azole resistant mutants show fitness defect in the absence of the selective pressure but show enhanced growth in the presence of fluconazole. Strains were grown in the absence or presence of 128 μg/mL fluconazole. Growth was assessed by OD 600 measurements every 15 minutes for 48 hours in a Tecan plate reader. C) Copy number variation was analyzed using the Y MAP pipeline. Amplification of chromosome 7 as well as a portion of chromosome 3 occurred in all independent spontaneous resistant mutants. D) Chromosome 3 haplotype map analyzed using Y MAP . The monosomic region of Chr3L was generated from different haplotypes in the four azole-resistant isolates: haplotype A (Cyan) in isolates R1 and R2, and haplotype B (magenta) in isolates R3 and R4.

    Article Snippet: In brief, minimum MIC assays were set up in 2-fold serial dilutions of fluconazole (Sequoia Research Products Ltd.), miconazole (Sigma-Aldrich), FK506 (A.G. Scientific, Inc.), geldanamycin (A.G. Scientific, Inc.), terbinafine (Sigma-Aldrich), SDS (Bioshop), amphotericin B (Sigma-Aldrich), or caspofungin (generously provided by Terry Roemer from Merck Research Laboratories) in a final volume of 200 μL per well.

    Techniques: Sequencing, Concentration Assay, Amplification, Generated

    The ERG3 genetic interaction network in S . cerevisiae uncovers species-specific circuitry important for azole resistance. A) Genetic nodes highlighting the negative genetic interactions with an SGA score (ε) ≤ -0.25 in YPD medium. 100 genes were identified as having a genetic interaction with ScERG3 , and they are coloured based on their GO Slim Process category. B) Microbroth dilution minimum inhibitory concentration (MIC) assay for S . cerevisiae ERG3 genetic interactors for which gene deletion abrogates azole resistance of the erg3 mutant. Strains were complemented with their respective wild-type allele to verify the azole sensitivity phenotype was due to the specific gene deletion. MIC assays were conducted in YPD and growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements. Data was quantitatively displayed with colour using Treeview (see colour bar). C) MIC assay for C . albicans erg3 homozygous deletion mutants that also harboured homozygous deletions of genes originally identified in S . cerevisiae as being important for erg3- mediated resistance. MIC assay was performed as in part B. For the bottom panel, fluconazole MIC assays were conducted in YPD medium with 1 μg/mL doxycycline to achieve transcriptional repression of the target gene in the conditional expression strains.

    Journal: PLoS Genetics

    Article Title: Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs

    doi: 10.1371/journal.pgen.1007319

    Figure Lengend Snippet: The ERG3 genetic interaction network in S . cerevisiae uncovers species-specific circuitry important for azole resistance. A) Genetic nodes highlighting the negative genetic interactions with an SGA score (ε) ≤ -0.25 in YPD medium. 100 genes were identified as having a genetic interaction with ScERG3 , and they are coloured based on their GO Slim Process category. B) Microbroth dilution minimum inhibitory concentration (MIC) assay for S . cerevisiae ERG3 genetic interactors for which gene deletion abrogates azole resistance of the erg3 mutant. Strains were complemented with their respective wild-type allele to verify the azole sensitivity phenotype was due to the specific gene deletion. MIC assays were conducted in YPD and growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements. Data was quantitatively displayed with colour using Treeview (see colour bar). C) MIC assay for C . albicans erg3 homozygous deletion mutants that also harboured homozygous deletions of genes originally identified in S . cerevisiae as being important for erg3- mediated resistance. MIC assay was performed as in part B. For the bottom panel, fluconazole MIC assays were conducted in YPD medium with 1 μg/mL doxycycline to achieve transcriptional repression of the target gene in the conditional expression strains.

    Article Snippet: In brief, minimum MIC assays were set up in 2-fold serial dilutions of fluconazole (Sequoia Research Products Ltd.), miconazole (Sigma-Aldrich), FK506 (A.G. Scientific, Inc.), geldanamycin (A.G. Scientific, Inc.), terbinafine (Sigma-Aldrich), SDS (Bioshop), amphotericin B (Sigma-Aldrich), or caspofungin (generously provided by Terry Roemer from Merck Research Laboratories) in a final volume of 200 μL per well.

    Techniques: Concentration Assay, Mutagenesis, Expressing

    Loss of PEP8 results in abnormal vacuolar morphology and overwhelms the functional capacity of calcineurin. A) Strains of C . albicans were grown to log-phase prior to staining with membrane dye FM4-64. Images were captured using differential interference contrast (DIC) microscopy and fluorescence microscopy with a TRITC/DsRED filter set on a Zeiss Axio Observer.Z1 (Carl Zeiss) using 100x magnification. Scale bar represents 10 μm. B) Deletion of PEP8 increases fluconazole-induction of two calcineurin-dependent transcripts, UTR2 and CRH11 . Transcript levels were monitored by qRT-PCR and normalized to ACT1 and GPD1 . Error bars represent standard error of the mean for triplicate samples. Treatment conditions were compared using a one-way ANOVA with Bonferroni post-test. Double asterisk indicates a significant difference in transcript level relative to both wildtype with fluconazole and erg3Δ/erg3Δ strain plus fluconazole. (** P

    Journal: PLoS Genetics

    Article Title: Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs

    doi: 10.1371/journal.pgen.1007319

    Figure Lengend Snippet: Loss of PEP8 results in abnormal vacuolar morphology and overwhelms the functional capacity of calcineurin. A) Strains of C . albicans were grown to log-phase prior to staining with membrane dye FM4-64. Images were captured using differential interference contrast (DIC) microscopy and fluorescence microscopy with a TRITC/DsRED filter set on a Zeiss Axio Observer.Z1 (Carl Zeiss) using 100x magnification. Scale bar represents 10 μm. B) Deletion of PEP8 increases fluconazole-induction of two calcineurin-dependent transcripts, UTR2 and CRH11 . Transcript levels were monitored by qRT-PCR and normalized to ACT1 and GPD1 . Error bars represent standard error of the mean for triplicate samples. Treatment conditions were compared using a one-way ANOVA with Bonferroni post-test. Double asterisk indicates a significant difference in transcript level relative to both wildtype with fluconazole and erg3Δ/erg3Δ strain plus fluconazole. (** P

    Article Snippet: In brief, minimum MIC assays were set up in 2-fold serial dilutions of fluconazole (Sequoia Research Products Ltd.), miconazole (Sigma-Aldrich), FK506 (A.G. Scientific, Inc.), geldanamycin (A.G. Scientific, Inc.), terbinafine (Sigma-Aldrich), SDS (Bioshop), amphotericin B (Sigma-Aldrich), or caspofungin (generously provided by Terry Roemer from Merck Research Laboratories) in a final volume of 200 μL per well.

    Techniques: Functional Assay, Staining, Microscopy, Fluorescence, Quantitative RT-PCR

    C . albicans functional genomic screen identifies novel genetic circuitry important for azole tolerance and resistance. A) Schematic of functional genomic screen to identify genes important for fluconazole (FL) tolerance. Thirteen genes were identified and prioritized for follow-up analysis based on the robust hypersensitivity of the corresponding deletion mutants. B) The 13 genes that were identified as having important roles in fluconazole tolerance, coloured based on their GO Slim Process category. Note many genes belong to multiple categories. C) MIC assay for C . albicans erg3 homozygous deletion mutants that also harboured homozygous deletions of genes implicated in fluconazole tolerance in part B. Homozygous deletion of either RGD1 or PEP8 abrogates the erg3- mediated azole resistance phenotype. MIC assay was performed as described in Fig 1 . Growth was analyzed after 24 hours (see colour bar). D) Homozygous deletion of either RGD1 or PEP8 reduces azole resistance of two resistant clinical isolates collected from an HIV patient undergoing fluconazole therapy. CaCi-2 represents an isolate collected early in treatment and CaCi-17 represents an isolate collected later in treatment. MIC assay was performed as described in part C.

    Journal: PLoS Genetics

    Article Title: Global analysis of genetic circuitry and adaptive mechanisms enabling resistance to the azole antifungal drugs

    doi: 10.1371/journal.pgen.1007319

    Figure Lengend Snippet: C . albicans functional genomic screen identifies novel genetic circuitry important for azole tolerance and resistance. A) Schematic of functional genomic screen to identify genes important for fluconazole (FL) tolerance. Thirteen genes were identified and prioritized for follow-up analysis based on the robust hypersensitivity of the corresponding deletion mutants. B) The 13 genes that were identified as having important roles in fluconazole tolerance, coloured based on their GO Slim Process category. Note many genes belong to multiple categories. C) MIC assay for C . albicans erg3 homozygous deletion mutants that also harboured homozygous deletions of genes implicated in fluconazole tolerance in part B. Homozygous deletion of either RGD1 or PEP8 abrogates the erg3- mediated azole resistance phenotype. MIC assay was performed as described in Fig 1 . Growth was analyzed after 24 hours (see colour bar). D) Homozygous deletion of either RGD1 or PEP8 reduces azole resistance of two resistant clinical isolates collected from an HIV patient undergoing fluconazole therapy. CaCi-2 represents an isolate collected early in treatment and CaCi-17 represents an isolate collected later in treatment. MIC assay was performed as described in part C.

    Article Snippet: In brief, minimum MIC assays were set up in 2-fold serial dilutions of fluconazole (Sequoia Research Products Ltd.), miconazole (Sigma-Aldrich), FK506 (A.G. Scientific, Inc.), geldanamycin (A.G. Scientific, Inc.), terbinafine (Sigma-Aldrich), SDS (Bioshop), amphotericin B (Sigma-Aldrich), or caspofungin (generously provided by Terry Roemer from Merck Research Laboratories) in a final volume of 200 μL per well.

    Techniques: Functional Assay