The penta-EF-hand protein Pef1 of Candida albicans functions at sites of membrane perturbation to support polarized growth and membrane integrity

The fungal plasma membrane is the target of fungicidal compounds, such as polyenes and saponins, that directly interact with fungus-specific ergosterol to cause deleterious membrane disruption. To counter membrane attack, diverse eukaryotic cells employ Ca2+-binding penta-EF (PEF)-hand proteins, including the human ortholog, ALG-2, to maintain membrane integrity. Candida albicans is a major fungal pathogen in humans, where increasing resistance to current antifungal drugs that target the plasma membrane is a serious cause of concern. Combinatorial treatments that additionally compromise the plasma membrane offer a way forward, but our mechanistic understanding of how fungi respond to direct membrane disruption remains limited. Here, we characterized the PEF-hand ortholog, Pef1, in this polymorphic species. GFP-tagged Pef1 localized at sites of polarized growth in yeast and hyphal cells of C. albicans. On treatment of cells with the polyene drug, amphotericin B, or the saponin, tomatine, GFP-Pef1 appeared as punctate spots at the membrane. In a similar manner, loss of calcineurin, but not of its transcription factor, Crz1, caused a punctate localization pattern of GFP-Pef1, which correlated with the serum sensitivity of the cna1{Delta} mutant. While deletion of PEF1 impaired yeast cell separation, filamentation was not affected. Strikingly, pef1{Delta} hyphae could not maintain plasma membrane integrity in serum. Consistent with this, the mutant exhibited attenuated virulence in an insect larvae infection model. Taken together, these observations suggest that Pef1 localizes to sites of membrane perturbation in order to maintain cell integrity, including sites of dynamic polarized growth and fungicide-induced membrane disruption.