FIG 3.

Supplementation with exogenous fatty acids rescues MMV688766-mediated growth inhibition in Candida. Two-fold dilutions of MMV688766 (0 to 100 μM), the fatty acid synthase inhibitor cerulenin (0 to 20 μg/mL), and the sphingolipid biosynthesis inhibitor myriocin (0 to 1 μg/mL) were performed against C. auris (CDC0387) (A) and C. albicans (CaSS1) (B) in YPD medium, in the presence and absence of exogenous fatty acids of various chain length (myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), oleic acid (C18:1), and behenic acid (C22:0)), as well as the early sphingolipid intermediates, dihydrosphingosine, and phytosphingosine. Growth was measured as OD₆₀₀ following a 48-h incubation at 30°C and normalized to the growth of drug-free control wells. Data are displayed using Java TreeView3; see color bar. MIC₈₀ values for each experimental condition are indicated by white numbers on the heat map. (C) C. auris (CDC0387) was grown in the presence and absence of 32 μM MMV688766, 1 μg/mL myriocin, and 5 μg/mL cerulenin for 4 h at 30°C in YPD following a 2-h outgrowth. Cells were washed and stained with 1 μg/mL BODIPY 493/503 to visualize neutral lipid droplets using differential interference contrast (DIC) and an EGFP fluorescence filter to visualize BODIPY staining using a Zeiss Axio Imager.MI at ×100 magnification. Flow cytometry was performed to quantify relative neutral lipid levels in cells exposed to each treatment. Cells were analyzed for incorporation of BODIPY 493/503 measuring relative fluorescence intensity (RFI) and intact cells were gated away from debris. Distributions of fluorescence intensity across each population are plotted in the histogram (no drug: black, cerulenin: green, myriocin: magenta, 32 μg/mL MMV688766: blue). Values describing the mean RFI across each treatment, are included above each peak.