Journal: Communications Biology

Article Title: Drug-dependent growth curve reshaping reveals mechanisms of antifungal resistance in Saccharomyces cerevisiae

doi: 10.1038/s42003-022-03228-9

Figure Lengend Snippet: a – d Drug-dependent shrinkage of the shaded area under growth curves (OD 600 ) upon exposure to ( a ) hydrogen peroxide, H 2 O 2 ; ( b ) amphotericin B, AmB; ( c ) caspofungin, CASP; and ( d ) fluconazole, FLC. Here, the representative replicates are shown. For all replicates, see Supplementary Fig. . e Area under each growth curve (AUC) above starting population size, approximated by numerical integration via the trapezoid method with equally spaced 1-h intervals. Red circles represent individual data points. Error bars represent means and standard deviations calculated from AUC of three biological replicates. f Growth curve analysis by piecewise linear fits to ln(OD 600 ) versus time is exemplified by TBR1Δa in normal (N) and 0.8 μg/ml AmB drug-containing (D) medium. The circles and letters next to them (B1, B2, B3) indicate breakpoints identified by the piecewise linear fitting within each growth curve. The breakpoints divide the N curve into 2, and the D curve – into 4 phases: pregrowth, adaptation, regrowth, and stationary phase. To characterize growth curve reshaping, the slope (S) and duration (T) of each growth phase (Supplementary Fig. ) were calculated for all drug concentrations.

Article Snippet: Fluconazole (R&D Systems 3764) was diluted in distilled water and added to the growth media to final concentrations 50, 75, 100, 125, and 150 μg/ml, approximating the concentrations survived by candidiasis-inducing biofilms in clinical samples .

Techniques:

Journal: mBio

Article Title: Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

doi: 10.1128/mBio.02529-18

Figure Lengend Snippet: Perturbation of HSP90 results in a loss of viability of C. auris but does not affect azole resistance. (A) Spotting of C. auris wild-type and tetO-HSP90 strains on YPD or YPD agar plus doxycycline (DOX) plates (right panel). C. albicans wild-type and tetO-HSP90 / tetO-HSP90 strains were included for comparison (left panel). A high concentration (50 μg/ml) of DOX was used to ensure strong repression of HSP90 expression. Overnight cultures were diluted 1,000-fold, at which point 5 μl was spotted in 100-fold dilutions. Plates were incubated at 30°C for 48 h. (B) Fluconazole Etest strip in the presence and absence of DOX. A total of 1 × 10 6 cells of wild-type and tetO- repressible HSP90 strains were plated on YPD agar plates with fluconazole Etest strips in the absence and presence of DOX (0.1 μg/ml or 10 μg/ml). The plates were incubated at 30°C for 48 h. (C) Checkerboard assays with geldanamycin and fluconazole. C. albicans clinical isolate CaCi2 and C. auris isolate Ci6684 were inoculated with a 2-fold gradient of geldanamycin in combination with a 2-fold gradient of fluconazole. Cultures were incubated at 30°C for 48 h. Heat maps represent relative growth levels determined from averages of results of technical replicates normalized to the data from a no-drug well.

Article Snippet: Approximately 1 × 10 3 cells were inoculated with a 2-fold gradient matrix of fluconazole (Carbosynth) or of geldanamycin (LC Laboratories) or of a combination of the two, as indicated, in 96-well microtiter plates to reach the final volume of 200 μl in YPD.

Techniques: Comparison, Concentration Assay, Expressing, Incubation, Stripping Membranes

Journal: mBio

Article Title: Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

doi: 10.1128/mBio.02529-18

Figure Lengend Snippet: Hsp90 mediates tolerance of fluconazole in C. auris . (A) MIC assay for fluconazole in a panel of C. auris clinical isolates. Data were analyzed as described for Fig. 1C . (B) Checkerboard assays with geldanamycin and fluconazole. C. auris strains Ci6684, CDC-382, CDC-387, and CDC-388 were inoculated with a 2-fold gradient of geldanamycin in combination with a 2-fold gradient of fluconazole. Data were analyzed as described for Fig. 1C . After measurement of the growth, cultures were spotted onto drug-free YPD agar plates and allowed to grow at 30°C for 24 h to check for cidality.

Article Snippet: Approximately 1 × 10 3 cells were inoculated with a 2-fold gradient matrix of fluconazole (Carbosynth) or of geldanamycin (LC Laboratories) or of a combination of the two, as indicated, in 96-well microtiter plates to reach the final volume of 200 μl in YPD.

Techniques:

Journal: mBio

Article Title: Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

doi: 10.1128/mBio.02529-18

Figure Lengend Snippet: Fluconazole resistance is mediated by drug efflux in C. auris . (A) Plot of relative CDR1 expression versus MIC 50 in the panel of C. auris isolates. CDR1 transcript levels were normalized against ACT1 and GPD1 transcript levels. MIC 50 values were derived from the MIC assay results presented in Fig. 2 . The Pearson correlation coefficient (r) was calculated for the two values. (B) MIC assay for fluconazole. Ci6684 (wild type [WT]) and cdr1Δ strains were inoculated with a 2-fold gradient of fluconazole. The plates were incubated at 30°C for 48 h. Data were analyzed as described for Fig. 1C .

Article Snippet: Approximately 1 × 10 3 cells were inoculated with a 2-fold gradient matrix of fluconazole (Carbosynth) or of geldanamycin (LC Laboratories) or of a combination of the two, as indicated, in 96-well microtiter plates to reach the final volume of 200 μl in YPD.

Techniques: Expressing, Derivative Assay, Incubation