Calcium-Mediated Induction of Paradoxical Growth following Caspofungin Treatment Is Associated with Calcineurin Activation and Phosphorylation in Aspergillus fumigatus

Abstract

ABSTRACTThe echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition ofAspergillus fumigatus, a phenomenon known as the “paradoxical effect,” which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies ofA. fumigatusrevealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca2+reporter aequorin expression system inA. fumigatusand showed that caspofungin elicits a transient increase in cytosolic free Ca2+([Ca2+]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca2+]cwas also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca2+]cincrease was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca2+-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid], or the L-type Ca2+channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca2+]clevels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca2+]cat high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.