Vacuolar iron export alters the synergy between doxycycline and fluconazole by affecting cidal ROS levels in Candida...

Fungal infections are combatted using three main classes of antifungals, of which the azoles, considered to be fungistatic, are the most widely used. Slow growth of Candida albicans at supra-minimal inhibitory concentrations (MIC) of fluconazole (FLC), termed tolerance, is routinely observed. A combination therapy resulting in the eradication of this fungistatic character would be a valid therapeutic strategy, and indeed, the synergistic combination of the antibiotic doxycycline and FLC has such an effect. We hypothesized that iron-requiring mitochondrial functions may be the targets of the synergistic combination. The proteome enriched for mitochondria obtained from FLC + Fe-treated cells hinted that iron alleviated the FLC stress and that intracellular iron homeostasis, more specifically the vacuolar iron exporter Smf3, might be a key factor during FLC treatment, as its expression was induced. Moreover, a ROS assay revealed that a smf3Δ/Δ strain treated with FLC accumulated ROS to a similar extent as that displayed by the WT undergoing a FLC+DOX combination treatment. Thus, deletion of SMF3 mimics the addition of doxycycline in wild-type cells. The ROS accumulation can be attenuated through overexpression of the mitochondrial superoxide dismutase SOD2, and this restored the synergy between DOX and FLC in the smf3Δ/Δ background. ROS accumulation, in part through altered iron availability from the vacuolar storage pool, is thus the molecular mechanism underlying the synergy between doxycycline and FLC. Furthermore, no effect on either cidality or tolerance was observed in the smf3Δ/Δ strain, highlighting that synergy is not necessarily an indication of cidal therapies.IMPORTANCEAzoles are widely used against Candida albicans, yet many cells survive above the minimal inhibitory concentrations (MIC) by growing slowly, which can prolong infection and foster resistance. We show that intracellular iron homeostasis alters the fluconazole characteristics by affecting ROS accumulation in mitochondria, and that this is the molecular mechanism underlying the combination therapy of fluconazole and doxycycline. These results place iron release from the vacuole at the center of azole responses, suggesting novel ways to boost azole efficacy.