Harnessing the calcineurin pathway for the development of novel antifungal drug interventions against Candida species.

摘要

Candida albicans is a commensal fungal organism found in the microflora of the gastrointestinal tract, vaginal mucosa, and on the skin of healthy individuals. However, this organism is an opportunistic pathogen that can over-proliferate in immunocompromised hosts and cause disease manifestations ranging from skin infections to disseminated candidiasis. Azole drugs such as fluconazole are fungistatic agents that block the production of ergosterol, a key fungal cell membrane component that maintains membrane integrity. Previous studies established that calcineurin (a serine/threonine protein phosphatase) is essential for virulence and survival of C. albicans during membrane perturbation by azoles, and the calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are dramatically synergistic with azoles against C. albicans. Using in vitro susceptibility assays, we demonstrated that non-azole drugs (terbinafine and fenpropimorph) also act synergistically with CsA, FK506, or L-685,818 (an FK506 analog) against C. albicans and fluconazole-resistant strains of C. glabrata and C. krusei. We tested the therapeutic utility of fluconazole or terbinafine combined with FK506 in a murine model of disseminated candidiasis, and found that this treatment model was not optimal for demonstrating in vivo drug synergy. We subsequently established a murine model of C. albicans keratitis and conducted treatment studies in which topical forms of fluconazole and CsA were administered alone and in combination. Calcineurin contributed to pathogenicity in this corneal infection model, and combination treatment resolved corneal infections more rapidly than monotherapy with either drug, thus illustrating a successful in vivo application of the azole-calcineurin inhibitor drug combination. To elucidate calcineurin-dependent mechanisms involved in virulence and azole tolerance, we constructed mutants of the zinc-finger transcription factor Crzl, a proposed downstream target of calcineurin. The in vitro phenotypes of crz1/crz1 mutants differed from calcineurin mutants, and Crz1 was not required for virulence. Therefore, Crz1 is not the sole target of calcineurin, and the C. albicans calcineurin-mediated mechanisms that confer azole tolerance are distinct from those that regulate virulence. Taken together, these studies demonstrate the utility of pharmacological targeting of the calcineurin pathway, and outline a useful approach to antifungal drug therapy that combines existing drugs that target separate pathways in order to maximize the therapeutic potential of each drug.