Calcium signaling mediates antifungal activity of triazole drugs in the Aspergilli

Azoles are widely applied and largely effective as antifungals; however, the increasing prevalence of clinically resistant isolates has yet to be matched by approaches to improve the efficacy of antimicrobial therapy. In this study, using the model fungus Aspergillus nidulans and one of the most common human pathogen Aspergillus fumigatus as research materials, we present the evidence that calcium signaling is involved in the azole-antifungals-induced stress-response reactions. In normal media, antifungal-itraconazole (ITZ) is able to induce the [Ca2+]c increased sharply but the addition of calcium chelator-EGTA or BAPTA almost blocks the calcium influx responses, resulted in the dramatically decreasing of [Ca2+]c transient. Real-time PCR analysis verified that six-tested Ca2+-inducible genes-two calcium channels (cchA/midA), a calmodulin-dependent phosphatase-calcineurin (cnaA), a transcription factor-crzA, and two calcium transporters (pmrA/pmcA)-could be transiently up-regulated by adding ITZ, indicating these components are involved in the azole stress-response reaction. Defect of cnaA or crzA caused more susceptibility to azole antifungals than did single mutants or double deletions of midA and cchA. Notably, EGTA may influence Rh123 accumulation as an azole-mimicking substrate through the process of the drug absorption. In vivo studies of a Galleria mellonella model identified that the calcium chelator works as an adjunct antifungal agent with azoles for invasive aspergillosis. Most importantly, combination of ITZ and EGTA or ITZ with calcium signaling inhibitor-FK506 greatly enhances the ITZ efficacy. Thus, our study provides potential clues that specific inhibitors of calcium signaling could be clinically useful adjuncts to conventional azole antifungals in the Aspergilli.