Summary The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2,868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2-hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast-to-hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analyzed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24-ceramides in membranes of RsAFP2-treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.

We investigated the cellular mechanisms responsible for the occurrence of miconazole-tolerant persisters in Candida albicans biofilms. Miconazole induced about 30% killing of sessile C. albicans cells at 75 M. The fraction of miconazole-tolerant persisters, i.e., cells that can survive high doses of miconazole (0.6 to 2.4 mM), in these biofilms was 1 to 2%. Since miconazole induces reactive oxygen species (ROS) in sessile C. albicans cells, we focused on a role for superoxide dismutases (Sods) in persistence and found the expression of Sod-encoding genes in sessile C. albicans cells induced by miconazole compared to the expression levels in untreated sessile C. albicans cells. Moreover, addition of the superoxide dismutase inhibitor N,N-diethyldithiocarbamate (DDC) to C. albicans biofilms resulted in an 18-fold reduction of the miconazole-tolerant persister fraction and in increased endogenous ROS levels in these cells. Treatment of biofilms of C. albicans clinical isolates with DDC resulted in an 18-fold to more than 200-fold reduction of their miconazole-tolerant persister fraction. To further confirm the important role for Sods in C. albicans biofilm persistence, we used a ⌬sod4 ⌬sod5 mutant lacking Sods 4 and 5. Biofilms of the ⌬sod4 ⌬sod5 mutant contained at least 3-fold less of the miconazole-tolerant persisters and had increased ROS levels compared to biofilms of the isogenic wild type (WT). In conclusion, the occurrence of miconazole-tolerant persisters in C. albicans biofilms is linked to the ROS-detoxifying activity of Sods. Moreover, Sod inhibitors can be used to potentiate the activity of miconazole against C. albicans biofilms.

ROS levels increased in all Candida biofilms upon addition of miconazole. However, ROS induction was not the only factor that underlies its fungicidal activity, as quenching of ROS did not lead to an enhanced survival of biofilm cells. ROS-induced apoptosis was not observed in C. albicans cells after miconazole treatment.

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14␣-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genomewide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.The class of azole antimycotics constitutes the largest group of synthetic antifungal therapeutics currently in clinical use. The generally accepted mode of antifungal action of azoles is the inhibition of ergosterol biosynthesis arising from a multimechanistic process initiated by the inhibition of two cytochrome P450 enzymes involved in ergosterol biosynthesis, namely the P450 enzyme that catalyzes the lanosterol 14␣-demethylation step and the P450 enzyme that catalyzes ⌬22 desaturation (1). Azole treatment results in predominance of 14␣-methylated sterols and inhibition of subsequent reactions of the ergosterol biosynthesis pathway (1). Apart from inhibition of ergosterol biosynthesis, miconazole induces accumulation of reactive oxygen species (ROS) 3 in susceptible fungi, leading to fungal cell death (2, 3). Moreover, we have demonstrated that miconazole induces actin stabilization prior to this ROS accumulation (4). These data point to an ancillary mode of action for this azole, as was already suggested in the 1970s (5).To obtain further mechanistic insight in the mode of antifungal action of miconazole, we screened in this study the complete haploid collection of 4853 Saccharomyces cerevisiae deletion mutants, individually deleted for nonessential genes, for resistance to miconazole on solid medium. Using this strategy, we demonstrate that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis are resistant to miconazole, suggesting a possible involvement of membrane rafts in the mode of antifungal action of miconazole. These rafts are membrane patches that are enriched in sphingolipids and ergosterol and that are thought to compartmentalize the plasma membrane and to have an important role in cell signaling (6). We investigated the effect of membrane raft-disturbing compounds on (i) miconazole antifungal activity, (ii) miconazole-induced actin cytoskeleton stabilizati...

30Biofilm formation is often associated with persistent Candida albicans infections.