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

ABSTRACT

The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a phenomenon known as the “paradoxical effect”, which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies on A. fumigatus revealed 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 in A. fumigatus and show that caspofungin elicits a transient increase in [Ca²⁺]_c (cytosolic free Ca²⁺) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca²⁺]_c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca²⁺]_c increase was noted with the paradoxical growth concentration of caspofungin. Treatments with a Ca²⁺-selective chelator, BAPTA, 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 to increased [Ca²⁺]_c levels at higher concentration 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⁺]_c at high concentrations of caspofungin activates calmodulin-calcineurin signaling both at a transcriptional and post-translational level and ultimately leads to paradoxical fungal growth.