Figure 4. ML316 renders respiration toxic and increases citrate levels.

(a) C. albicans strain CaCi2 with genetic deletion of mitochondrial COX1 are resistant to ML316 but are unable to respire or grow in the presence of fluconazole. Serial dilutions of CaCi-2 or CaCi-2 cox1 were spotted on media with carbon sources supporting fermentation (glucose) or enforcing respiration (glycerol) in presence or absence of ML316 (5 µM) or Flu (16 µM) as indicated. Plates were imaged after 2 days at 30 °C. (b) Citrate levels increase dramatically upon treatment with ML316 but not with other mitochondrial poisons. LC/MS was used to measure levels of citrate in C. albicans grown in synthetic defined media (2% glucose) following 30 or 60 min exposure to ML316 (5 µM), Antimycin (1 µM), or 2,4-dinitrophenol (25 µg/mL). All values were normalized to the mean for DMSO-treated samples. Values represent the mean and s.e.m. of determinations from 2 independent experiments, each consisting of 3 independent cultures. (c) ML316 increases citrate levels in wild-type Candida but not in ML316-resistant mutant strains expressing either mir1R or carrying a deletion of COX4. Citrate levels were determined by LC/MS in strains growing in glucose after 60 min exposure to ML316 (5 µM). In the genotypes indicated on the x-axis, “V” denotes empty vector control. Citrate levels were normalized to the mean of wild type, DMSO-treated samples. Mean and s.e.m. from 2 independent experiments each consisting of 3 independent cultures are shown.