Journal: mSphere

Article Title: Membrane Integrity Contributes to Resistance of Cryptococcus neoformans to the Cell Wall Inhibitor Caspofungin

doi: 10.1128/msphere.00134-22

Figure Lengend Snippet: Schematic of the caspofungin sensitivity screening assay. Steps and conditions: (a) frozen cells were transferred to YPD agar using a pronged replicator and incubated for 48 h at 25°C; (b) cells were transferred to 600 μL YPD in a deep-well plate using a multichannel pipettor and incubated for 48 h at 25°C; (c) 30 μL was transferred to 600 μL YPD in a deep-well plate and incubated for 48 h at 25°C; (d) cells were centrifuged, washed 2 times, and resuspended in 200 μL PBS; (e) cells were transferred to a clear, flat-bottom plate and optical densities measured at 650 nm; (f) cells were normalized to an OD 650 of 2 in a final volume of 120 μL PBS; (g) cells were diluted 1:20 in 200 μL YNB; (h) cells were diluted 1:10 in 100 μL YNB; (i) cells were diluted 1:10 in 100 μL YNB in 4 replicate plates; (j) caspofungin was added at 10 μg/mL to 3 replicates, PBS was added to the control plate, and the plates were incubated for 48 h at 25°C; (k) XTT reagent was added to all plates, the plates incubated for 3 h at 37°C and centrifuged, and 100 μL transferred to fresh plates; and (l) absorbance was read at OD 490 . Plates incubated in the 2nd, 3rd, and 10th steps were covered with a Breathe-Easy membrane. Image created with BioRender.com.

Article Snippet: Each stress plate was composed of YPD medium supplemented with the indicated concentrations of the following compounds: 0.3% sodium dodecyl sulfate (SDS; Sigma) and 32 μg/mL or 40 μg/mL caspofungin acetate (Biosynth Carbosynth), to investigate cell integrity stress; 2 mM tert-butyl hydroperoxide (tBOOH; Spectrum Chemical) and 0.75 mM NaNO 2 (Sigma), to impose nitrosative and oxidative stress; and 0.5 mg/mL calcofluor white (CFW; MP Biomedicals), 1 mg/mL caffeine (Sigma), and 2.5 mg/mL Congo red (Santa Cruz Biotechnology) to induce cell wall stress.

Techniques: Screening Assay, Incubation, Control, Membrane

Journal: mSphere

Article Title: Membrane Integrity Contributes to Resistance of Cryptococcus neoformans to the Cell Wall Inhibitor Caspofungin

doi: 10.1128/msphere.00134-22

Figure Lengend Snippet: Caspofungin sensitivity screening data for all deletion strains. Normalized growth of each strain on a given plate relative to the untreated cells is shown for the 2015 library (A) and the 2016 library (B) strains. Each dot represents the average percentage of growth relative to the growth of untreated cells after normalization across all strains in the same plate. Error bars show standard deviations. Orange circles represent strains with percent inhibition values of ±2 standard deviations from the plate average. Blue circles are strains with percent inhibition values of ±1 standard deviation from the plate average.

Article Snippet: Each stress plate was composed of YPD medium supplemented with the indicated concentrations of the following compounds: 0.3% sodium dodecyl sulfate (SDS; Sigma) and 32 μg/mL or 40 μg/mL caspofungin acetate (Biosynth Carbosynth), to investigate cell integrity stress; 2 mM tert-butyl hydroperoxide (tBOOH; Spectrum Chemical) and 0.75 mM NaNO 2 (Sigma), to impose nitrosative and oxidative stress; and 0.5 mg/mL calcofluor white (CFW; MP Biomedicals), 1 mg/mL caffeine (Sigma), and 2.5 mg/mL Congo red (Santa Cruz Biotechnology) to induce cell wall stress.

Techniques: Inhibition, Standard Deviation

Journal: mSphere

Article Title: Membrane Integrity Contributes to Resistance of Cryptococcus neoformans to the Cell Wall Inhibitor Caspofungin

doi: 10.1128/msphere.00134-22

Figure Lengend Snippet: Five deletion strains with confirmed caspofungin sensitivity

Article Snippet: Each stress plate was composed of YPD medium supplemented with the indicated concentrations of the following compounds: 0.3% sodium dodecyl sulfate (SDS; Sigma) and 32 μg/mL or 40 μg/mL caspofungin acetate (Biosynth Carbosynth), to investigate cell integrity stress; 2 mM tert-butyl hydroperoxide (tBOOH; Spectrum Chemical) and 0.75 mM NaNO 2 (Sigma), to impose nitrosative and oxidative stress; and 0.5 mg/mL calcofluor white (CFW; MP Biomedicals), 1 mg/mL caffeine (Sigma), and 2.5 mg/mL Congo red (Santa Cruz Biotechnology) to induce cell wall stress.

Techniques: Ubiquitin Proteomics

Journal: mSphere

Article Title: Membrane Integrity Contributes to Resistance of Cryptococcus neoformans to the Cell Wall Inhibitor Caspofungin

doi: 10.1128/msphere.00134-22

Figure Lengend Snippet: The caspofungin sensitivity was replicated in the new deletion strains. We tested KN99 and the cft1 Δ, erg4 Δ, myo1 Δ, and ysp2 Δ deletion strains for sensitivity to caspofungin by measuring the MICs of caspofungin in YNB using a microdilution assay (A) and on agar plates (B). Cells at an OD of 0.001 were incubated with caspofungin at concentrations of 2 to 32 μg/mL for 48 h at 30°C. Cell growth was measured at OD 650 and plotted as the percentage of the growth of the untreated control. Error bars represent the standard deviation of the average of three replicates.

Article Snippet: Each stress plate was composed of YPD medium supplemented with the indicated concentrations of the following compounds: 0.3% sodium dodecyl sulfate (SDS; Sigma) and 32 μg/mL or 40 μg/mL caspofungin acetate (Biosynth Carbosynth), to investigate cell integrity stress; 2 mM tert-butyl hydroperoxide (tBOOH; Spectrum Chemical) and 0.75 mM NaNO 2 (Sigma), to impose nitrosative and oxidative stress; and 0.5 mg/mL calcofluor white (CFW; MP Biomedicals), 1 mg/mL caffeine (Sigma), and 2.5 mg/mL Congo red (Santa Cruz Biotechnology) to induce cell wall stress.

Techniques: Microdilution Assay, Incubation, Control, Standard Deviation

Journal: mSphere

Article Title: Membrane Integrity Contributes to Resistance of Cryptococcus neoformans to the Cell Wall Inhibitor Caspofungin

doi: 10.1128/msphere.00134-22

Figure Lengend Snippet: Caspofungin-sensitive deletion strains also showed sensitivity to membrane stress. Cell wall stress phenotypes of cft1 Δ, erg4 Δ, myo1 Δ, and ysp2 Δ strains were compared to those of the parental KN99 strain. Overnight cultures grown in YPD were diluted to an OD 650 of 1 and serially diluted 1:10 four times before being spotted on agar plates containing the indicated stressor. The plates were incubated at 30°C for 3 days, except for a YPD plate whose stressor was growth at 37°C (YPD 37).

Article Snippet: Each stress plate was composed of YPD medium supplemented with the indicated concentrations of the following compounds: 0.3% sodium dodecyl sulfate (SDS; Sigma) and 32 μg/mL or 40 μg/mL caspofungin acetate (Biosynth Carbosynth), to investigate cell integrity stress; 2 mM tert-butyl hydroperoxide (tBOOH; Spectrum Chemical) and 0.75 mM NaNO 2 (Sigma), to impose nitrosative and oxidative stress; and 0.5 mg/mL calcofluor white (CFW; MP Biomedicals), 1 mg/mL caffeine (Sigma), and 2.5 mg/mL Congo red (Santa Cruz Biotechnology) to induce cell wall stress.

Techniques: Membrane, Incubation

Journal: mSphere

Article Title: Membrane Integrity Contributes to Resistance of Cryptococcus neoformans to the Cell Wall Inhibitor Caspofungin

doi: 10.1128/msphere.00134-22

Figure Lengend Snippet: High temperature increases the sensitivity of deletion strains to caspofungin. The KN99 and cft1 Δ, erg4 Δ, myo1 Δ, and ysp2 Δ strains were grown overnight in YPD and diluted to an OD 650 of 1 before being spotted in 4-step serial dilutions on YPD agar plates containing 32 or 40 μg/mL caspofungin. The plates were incubated for 3 days at 37°C (A) or 39°C (B).

Article Snippet: Each stress plate was composed of YPD medium supplemented with the indicated concentrations of the following compounds: 0.3% sodium dodecyl sulfate (SDS; Sigma) and 32 μg/mL or 40 μg/mL caspofungin acetate (Biosynth Carbosynth), to investigate cell integrity stress; 2 mM tert-butyl hydroperoxide (tBOOH; Spectrum Chemical) and 0.75 mM NaNO 2 (Sigma), to impose nitrosative and oxidative stress; and 0.5 mg/mL calcofluor white (CFW; MP Biomedicals), 1 mg/mL caffeine (Sigma), and 2.5 mg/mL Congo red (Santa Cruz Biotechnology) to induce cell wall stress.

Techniques: Incubation