The flavodoxin-like protein CgPst2 plays an essential role in menadione and benzoquinone resistance.

A. Serial dilution spotting assay illustrating MD (95 μM) and BQ (4 mM) sensitivity of indicated C . glabrata mutants. The Q-KO strain lacks four flavodoxin-like proteins, CgPst2, CgRfs1, CgPst3 and CgYcp4. B. Total intracellular glutathione content of log-phase cells (10.0 OD 600 ). Data (mean ± SEM; n = 4–5) represent total glutathione concentration normalized to one ml of cell lysate. *, p ≤ 0.05, ***, p ≤ 0.001, ****, p ≤ 0.0001; unpaired two-tailed Student’s t test. C. Ratio of oxidized (GSSG) to reduced (GSH) glutathione. 4-vinylpyridine was used to derivatize/block all free thiols in cell lysates, followed by GSSG estimation using DTNB [5,5-dithio-bis-(2-nitrobenzoic acid); Ellman’s Reagent]. Reduced GSH levels were calculated by subtracting GSSG levels from total glutathione levels. Data represent (mean ± SEM; n = 3–5). D. Survival of indicated C . glabrata strains in the murine model of systemic candidiasis. Six to eight-week-old female BALB/c mice were infected with cells (4X10 7 cells; 100 μl cell suspension) of overnight YPD/CAA-grown C . glabrata strains, wt , Cgpst2Δ , Q-KO and Q-KO expressing CgPST2 , through tail vein injections. At 7 th day post infection, mice were sacrificed, target organs (kidneys, liver and spleen) collected, homogenized in sterile PBS and appropriate homogenate dilutions were plated on YPD medium containing penicillin and streptomycin antibiotics. Total fungal burden in each mouse organ was determined, and plotted. Diamonds and bars indicate CFUs recovered from an individual mouse, and CFU geometric mean (n = 8–12), respectively, for each organ. **, p < 0.01; Mann-Whitney U-test. E. Size exclusion chromatogram of purified CgPst2 protein. 300 μg 6XHIS-FLAG-CgPst2 was applied to a Sephacryl S-200 column, and protein elution profiles were determined using the absorbance values at 280 nm. The peak corresponds to 29 kDa CgPst2 protein. AU, Arbitrary Units. F. NADH:quinone oxidoreductase activity in cell lysates (500 μg) of log-phase wt , Cgpst2 Δ and Q-KO cells, as measured ...