Cyclic AMP signaling pathway modulates susceptibility of Candida species and Saccharomyces cerevisiae to antifungal azoles and other sterol biosynthesis inhibitors

Azoles are widely used antifungals; however, their efficacy is compromised by fungistatic activity and selection of resistant strains during treatment. Recent studies demonstrated roles for the protein kinase C and calcium signaling pathways in modulating azole activity. Here we explored a role for the signaling pathway mediated by cyclic AMP (cAMP), which is synthesized by the regulated action of adenylate cyclase (encoded by CDC35 in Candida albicans and CYR1 in Saccharomyces cerevisiae) and cyclase-associated protein (encoded by CAP1 and SRV2, respectively). Relative to wild-type strains, C. albicans and S. cerevisiae strains mutated in these genes were hypersusceptible to fluconazole (>4- to >16-fold-decreased 48-h MIC), itraconazole (>8- to >64-fold), or miconazole (16- to >64-fold). Similarly, they were hypersusceptible to terbinafine and fenpropimorph (2- to > 16-fold), which, like azoles, inhibit sterol biosynthesis. Addition of cAMP to the medium at least partially reversed the hypersusceptibility of Ca-cdc35 and Sc-cyr1-2 mutants. An inhibitor of mammalian adenylate cyclase, MDL-12330A, was tested in combination with azoles; a synergistic effect was observed against azole-susceptible and -resistant strains of C. albicans and five of six non-C. albicans Candida species. Analysis of cAMP levels after glucose induction in the presence and absence of MDL-12330A confirmed that it acts by inhibiting cAMP synthesis in yeast. RNA analysis suggested that a defect in azole-dependent upregulation of the multidrug transporter gene CDR1 contributes to the hypersusceptibility of the Ca-cdc35 mutant. Our results implicate cAMP signaling in the yeast azole response; compounds similar to MDL-12330A may be useful adjuvants in azole therapy.