Journal: Antimicrobial Agents and Chemotherapy

Article Title: Genetic interaction analysis of Candida glabrata transcription factors CST6 and UPC2A in the regulation of respiration and fluconazole susceptibility

doi: 10.1128/aac.01294-24

Figure Lengend Snippet: Deletion of CST6 reduces fluconazole hyper-susceptibility of upc2A ∆ mutants. (A) Location of CST6 mutations in evolved strains isolated reported in reference and schematic of Cst6 protein showing position relative to the DNA-binding domain (DBD). (B) A dilution series of the indicated strains were spotted on YPD and YPD with the indicated amount of fluconazole. The plates were incubated for 48 h prior to imaging. The phenotypes are representative of three biological replicates. (C) The indicated strains were plated on YPD medium and incubated in ambient air (normoxia) or in a GAS PAK (hypoxia). (D) Biofilms were generated in RPMI buffered with 0.165 M morpholino-propanesulfonic acid (MOPS) for 72 h before treatment with sham or 1000 µg/mL fluconazole. The wells were incubated for an additional 24 h, and biofilm formation was assayed by metabolic activity as described in Materials and Methods. Bars indicate mean of absorption of the XTT assay, with error bars indicating standard deviation. Asterisks indicate statistically significant differences by one-way analysis of variance followed by Tukey’s correction for multiple comparisons; *<0.05; **<0.01; ***<0.001.

Article Snippet: The decreased susceptibility of the cst6 ∆ mutant on spot dilution assays is consistent with our previously reported observation that its fluconazole minimum inhibitory concentration (MIC) is fourfold increased relative to WT under Clinical and Laboratory Standards Institute (CLSI) conditions ( ).

Techniques: Isolation, Binding Assay, Incubation, Imaging, Generated, Activity Assay, XTT Assay, Standard Deviation

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Genetic interaction analysis of Candida glabrata transcription factors CST6 and UPC2A in the regulation of respiration and fluconazole susceptibility

doi: 10.1128/aac.01294-24

Figure Lengend Snippet: Deletion of CST6 does not increase ergosterol levels, ERG gene expression or CDR1 efflux pump expression in the presence of fluconazole. (A) The ergosterol content of the indicated strains in the presence of fluconazole (16 µg/mL) was determined as described in Materials and Methods. Bars indicate mean and error bars indicate standard deviation for three biological replicates. * indicates statistically significant difference ( P < 0.05) from WT by one-way analysis of variance (ANOVA) and Tukey’s correction for multiple comparisons. The percentage of (B) lanosterol and (C) 14 methyl ergosta-8,24 (28)-dien-3–6-diol (14-MEDD) relative to total sterols in the indicated strains was determined as described in Materials and Methods in the presence of fluconazole (µg/mL). Full data set provided in . (D) The expression of the indicated genes relative for the upc2 A∆, cst6 ∆, and upc2A ∆ cst6 ∆ mutants relative to WT were determined in the presence of fluconazole (16 µg/mL) by qRT-PCR. The fold change is relative WT, and * indicates statistically significant ( P < 0.05) difference by one-way ANOVA and Tukey’s correction for multiple comparisons. Bars indicate mean of three biological replicates performed in technical triplicate, with error bars indicating standard deviation.

Article Snippet: The decreased susceptibility of the cst6 ∆ mutant on spot dilution assays is consistent with our previously reported observation that its fluconazole minimum inhibitory concentration (MIC) is fourfold increased relative to WT under Clinical and Laboratory Standards Institute (CLSI) conditions ( ).

Techniques: Gene Expression, Expressing, Standard Deviation, Quantitative RT-PCR

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Genetic interaction analysis of Candida glabrata transcription factors CST6 and UPC2A in the regulation of respiration and fluconazole susceptibility

doi: 10.1128/aac.01294-24

Figure Lengend Snippet: Effect of the cst6 ∆ upc2A ∆ mutant on gene expression relative to single mutants in the presence of fluconazole. (A) Heatmap comparing the expression (RNA-seq) of the indicated ERG genes and the adhesin EPA3 for the upc2 A∆, cst6 ∆, and upc2A ∆ cst6 ∆ mutants relative to WT (BG2). Biological process GO term analysis of genes downregulated in the upc2A ∆ cst6 ∆ (B) and upc2A ∆ (C) mutants in the presence of fluconazole with the number of genes in each GO term group listed on the x-axis. The FDR was determined by Benjamini-Hochberg analysis. (D) Representative mitochondrial and respiration genes downregulated in the upc2A ∆ mutant is shown.

Article Snippet: The decreased susceptibility of the cst6 ∆ mutant on spot dilution assays is consistent with our previously reported observation that its fluconazole minimum inhibitory concentration (MIC) is fourfold increased relative to WT under Clinical and Laboratory Standards Institute (CLSI) conditions ( ).

Techniques: Mutagenesis, Gene Expression, Expressing, RNA Sequencing

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Genetic interaction analysis of Candida glabrata transcription factors CST6 and UPC2A in the regulation of respiration and fluconazole susceptibility

doi: 10.1128/aac.01294-24

Figure Lengend Snippet: Forced respiration with glycerol medium increases fluconazole susceptibility of cst6 ∆, upc2A ∆, and upc2A ∆ cst6 ∆ mutants. (A) The upc2 A∆, cst6 ∆, and upc2A ∆ cst6 ∆ mutants were plated on YP + 2% glucose and YP + 2% glycerol medium and incubated at 30°C or 37°C for 48–72 h. (B) The minimum inhibitory concentration (MIC) of fluconazole was determined after incubation for 24 h (glucose) or 48 h (glycerol) at 37°C. (C) The effect of loss of Upc2A function on the expression of ERG genes in the cst6 ∆ mutant by RNA-seq. Full data set is in . All changes were statistically significant (adjusted P < 0.05).

Article Snippet: The decreased susceptibility of the cst6 ∆ mutant on spot dilution assays is consistent with our previously reported observation that its fluconazole minimum inhibitory concentration (MIC) is fourfold increased relative to WT under Clinical and Laboratory Standards Institute (CLSI) conditions ( ).

Techniques: Incubation, Concentration Assay, Expressing, Mutagenesis, RNA Sequencing

Journal: Nature Communications

Article Title: Forward and reverse genetic dissection of morphogenesis identifies filament-competent Candida auris strains

doi: 10.1038/s41467-021-27545-5

Figure Lengend Snippet: a Wild-type AR0382 (Clade I), Δelm1 , and Δelm1 + ELM1 strains in the AR0382 background were standardized to an optical density of OD 600 = 1.0 in PBS before inoculating 20 Galleria mellonella larvae per C. auris strain with 50 μL of prepared inoculum. Larvae were maintained at 37 °C and monitored daily for survival for 5 days. Source data are provided as a Source Data file. Statistical differences were determined using a Mantel−Cox log-rank test. WT- Δelm1 , *** p < 0.0001; WT- Δelm1 + ELM1 , ns: p = 0.51; Δelm1-Δelm1 + ELM1 , *** p < 0.0001. b Gradient MIC test strips were used to determine the susceptibility of wild-type AR0382, morphogenic mutants, and complemented strains in the AR0382 background to fluconazole, caspofungin, and amphotericin B. The color and intensity of each point corresponds to the Log 2 fold change in MIC for each strain relative to the wild type. A complete list of MICs is available in Supplementary Table . c MIC test strips with zones of inhibition for mutants with substantially altered antifungal susceptibilities. Δtao3 exhibited reduced susceptibility to fluconazole and amphotericin B compared to the wild type. Δelm1 exhibited increased susceptibility to caspofungin.

Article Snippet: MIC test strips containing 0.016–256 mg/L Fluconazole (Liofilchem REF 921471), 0.002–32 mg/L Caspofungin (Liofilchem REF 921541), or 0.002–32 mg/L Amphotericin B (Liofilchem REF 921531) were placed onto the surface of the agar.

Techniques: Inhibition