The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a\nphenomenon known as the ââ?¬Å?paradoxical effect,ââ?¬Â which is not consistently observed with other echinocandins (micafungin and\nanidulafungin). Previous studies of A. fumigatus revealed the loss of the paradoxical effect following pharmacological or genetic\ninhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent\nCa2 reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic\nfree Ca2 ([Ca2]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin\nsignaling. While the increase in [Ca2]c was also observed upon treatment with micafungin, another echinocandin without\nthe paradoxical effect, a higher [Ca2]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments\nwith a Ca2-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N=,N=-tetraacetic acid], or the L-type Ca2\nchannel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandinresistant\n(EMFR-S678P) strains. Concomitant with increased [Ca2]c levels at higher concentrations of caspofungin, calmodulin\nand calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation\nat its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth,\nonly at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased\n[Ca2]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational\nlevel and ultimately leads to paradoxical fungal growth.
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