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. Serious fungal infections have increased in recent years as a consequence of the growing number of people who are immunocompromised in association with AIDS, aggressive therapies for cancer and autoimmune disease, or organ and tissue transplantation. The primary pathogen is Candida albicans, normally a commensal of the oral cavity and gastrointestinal tract of humans. This opportunistic yeast causes mucosal infection or less commonly invasive disease that is life threatening unless treated. There are presently three major classes of antifungal agents in clinical use: azoles, allylamines, and polyenes (15). Azoles such as fluconazole, itraconazole, and miconazole inhibit biosynthesis of the membrane component ergosterol by blocking the action of lanosterol demethylase (encoded by ERG11). Allylamines such as terbinafine and morpholines such as fenpropimorph are also sterol biosynthesis inhibitors (SBIs), while polyenes such as amphotericin B directly interact with the cell membrane, causing electrolyte leakage and subsequent cell death. Echinocandins such as caspofungin represent a new class of antifungals that target cell wall biosynthesis. Of all antifungals, azoles are the most commonly used, but their efficacy is limited by lack of fungicidal activity as well as development of clinical resistance. Several mechanisms have been described for the azole resistance of C. albicans, including increased expression of ERG11 and of genes encoding multidrug transporters (encoded by CDR1, CDR2, and MDR1) (14,