The genus Malassezia includes yeasts that are commonly found on the skin or hair of animals and humans as commensals and are associated with a number of skin disorders. We have previously developed an Agrobacterium tumefaciens transformation system effective for both targeted gene deletion and insertional mutagenesis in Malassezia furfur and M. sympodialis In the present study, these molecular resources were applied to characterize the immunophilin FKBP12 as the target of tacrolimus (FK506), ascomycin, and pimecrolimus, which are calcineurin inhibitors that are used as alternatives to corticosteroids in the treatment of inflammatory skin disorders such as those associated with Malassezia species. While M. furfur and M. sympodialis showed in vitro sensitivity to these agents, fkb1? mutants displayed full resistance to all three of them, confirming that FKBP12 is the target of these calcineurin inhibitors and is essential for their activity. We found that calcineurin inhibitors act additively with fluconazole through an FKBP12-dependent mechanism. Spontaneous M. sympodialis isolates resistant to calcineurin inhibitors had mutations in the gene encoding FKBP12 in regions predicted to affect the interactions between FKBP12 and FK506 based on structural modeling. Due to the presence of homopolymer nucleotide repeats in the gene encoding FKBP12, an msh2? hypermutator of M. sympodialis was engineered and exhibited an increase of more than 20-fold in the rate of emergence of resistance to FK506 compared to that of the wild-type strain, with the majority of the mutations found in these repeats.IMPORTANCEMalassezia species are the most abundant fungal components of the mammalian and human skin microbiome. Although they belong to the natural skin commensal flora of humans, they are also associated with a variety of clinical skin disorders. The standard treatment for Malassezia-associated inflammatory skin infections is topical corticosteroids, although their use has adverse side effects and is not recommended for long treatment periods. Calcineurin inhibitors have been proposed as a suitable alternative to treat patients affected by skin lesions caused by Malassezia Although calcineurin inhibitors are well-known as immunosuppressive drugs, they are also characterized by potent antimicrobial activity. In the present study, we investigated the mechanism of action of FK506 (tacrolimus), ascomycin (FK520), and pimecrolimus in M. furfur and M. sympodialis and found that the conserved immunophilin FKBP12 is the target of these drugs with which it forms a complex that directly binds calcineurin and inhibits its signaling activity. We found that FKBP12 is also required for the additive activity of calcineurin inhibitors with fluconazole. Furthermore, the increasing natural occurrence in fungal pathogen populations of mutator strains poses a high risk for the rapid emergence of drug resistance and adaptation to host defense. This led us to generate an engineered hypermutator msh2? mutant strain of M. sympodialis and genetically evaluate mutational events resulting in a substantially increased rate of resistance to FK506 compared to that of the wild type. Our study paves the way for the novel clinical use of calcineurin inhibitors with lower immunosuppressive activity that could be used clinically to treat a broad range of fungal infections, including skin disorders caused by Malassezia. Copyright © 2017 Ianiri et al.