title: 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 of 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 Ca(2+) reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic free Ca(2+) ([Ca(2+)]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca(2+)]c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca(2+)]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca(2+)-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid], or the L-type Ca(2+) channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca(2+)]c levels 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 [Ca(2+)]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.
authors.affiliation: Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Praveen R
authors.initials: PR
authors.lastname: Juvvadi
authors.affiliation: Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, United Kingdom.
authors.forename: Alberto
authors.initials: A
authors.lastname: Muñoz
authors.affiliation: Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, USA Infectious Diseases Service, Department of Medicine, Lausanne University Hospital, Lausanne, Switzerland Institute of Microbiology, Lausanne University Hospital, Lausanne, Switzerland.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Lamoth
authors.affiliation: Duke Proteomics Facility, Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, USA.
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Soderblom
authors.affiliation: Duke Proteomics Facility, Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, USA.
authors.forename: M Arthur
authors.initials: MA
authors.lastname: Moseley
authors.affiliation: Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, United Kingdom.
authors.forename: Nick D
authors.initials: ND
authors.lastname: Read
authors.affiliation: Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, USA Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA bill.steinbach@duke.edu.
authors.forename: William J
authors.initials: WJ
authors.lastname: Steinbach
mesh_terms: D000077612:Anidulafungin; D000935:Antifungal Agents / Q000494:pharmacology; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000378:metabolism*; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D000077336:Caspofungin; D054714:Echinocandins / Q000494:pharmacology*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism; D055666:Lipopeptides / Q000494:pharmacology; D000077551:Micafungin; D010766:Phosphorylation / Q000502:physiology*
chemical_list: D000935:Antifungal Agents; D054714:Echinocandins; D005656:Fungal Proteins; D055666:Lipopeptides; D004533:Egtazic Acid; D000077612:Anidulafungin; D019703:Calcineurin; D000077336:Caspofungin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D000077551:Micafungin; D002118:Calcium