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

Praveen R Juvvadi,Alberto Muñoz,William J Steinbach,M Arthur Moseley,Erik J Soderblom,Nick D Read,Frédéric Lamoth

doi:10.1128/aac.00263-15

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 Ca2+ reporter aequorin expression system in A. fumigatus and 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+]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 [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+]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 [Ca2+]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.

Highlights

Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, USAa; Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, United Kingdomb; Infectious Diseases Service, Department of Medicine, Lausanne University Hospital, Lausanne, Switzerlandc; Institute of Microbiology, Lausanne University Hospital, Lausanne, Switzerlandd; Duke Proteomics Facility, Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, USAe; Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USAf
We have previously shown that pharmacologic inhibition of A. fumigatus calcineurin with FK506, deletion of the gene encoding the catalytic subunit of calcineurin (⌬cnaA), or deletion of the gene encoding the major transcription factor in the calcineurin pathway (⌬crzA) abolished the caspofungin-mediated paradoxical growth [3]
We investigated the mechanism for paradoxical growth observed during treatment with higher concentrations of caspofungin by analyzing [Ca2ϩ]c changes and calcineurin activation following treatment of A. fumigatus with the two different echinocandin antifungals, caspofungin and micafungin

Summary

Introduction

Our results indicate that as opposed to micafungin, the increased [Ca2؉]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and leads to paradoxical fungal growth. Based on our previous results demonstrating the involvement of the calcineurin pathway in the paradoxical effect, we hypothesized that a mechanism for the paradoxical effect may involve a transient increase in cytosolic free Ca2ϩ ([Ca2ϩ]c) in the fungal cell following treatment with high concentrations of caspofungin. This Ca2ϩ increase results in the activation of calmodulin-calcineurin signaling, which

Methods

Results

Conclusion