The Antifungal Activity of the Penicillium chrysogenum Protein PAF Disrupts Calcium Homeostasis in Neurospora crassa

Abstract

ABSTRACT The antifungal protein PAF from Penicillium chrysogenum exhibits growth-inhibitory activity against a broad range of filamentous fungi. Evidence from this study suggests that disruption of Ca 2+ signaling/homeostasis plays an important role in the mechanistic basis of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca 2+ concentrations counteracted PAF toxicity toward PAF-sensitive molds. By using a transgenic Neurospora crassa strain expressing codon-optimized aequorin, PAF was found to cause a significant increase in the resting level of cytosolic free Ca 2+ ([Ca 2+ ] c ). The Ca 2+ signatures in response to stimulation by mechanical perturbation or hypo-osmotic shock were significantly changed in the presence of PAF. BAPTA [bis-(aminophenoxy)-ethane- N , N , N ′, N ′-tetraacetic acid], a Ca 2+ selective chelator, ameliorated the PAF toxicity in growth inhibition assays and counteracted PAF induced perturbation of Ca 2+ homeostasis. These results indicate that extracellular Ca 2+ was the major source of these PAF-induced effects. The L-type Ca 2+ channel blocker diltiazem disrupted Ca 2+ homeostasis in a similar manner to PAF. Diltiazem in combination with PAF acted additively in enhancing growth inhibition and accentuating the change in Ca 2+ signatures in response to external stimuli. Notably, both PAF and diltiazem increased the [Ca 2+ ] c resting level. However, experiments with an aequorin-expressing Δ cch-1 deletion strain of N. crassa indicated that the L-type Ca 2+ channel CCH-1 was not responsible for the observed PAF-induced elevation of the [Ca 2+ ] c resting level. This study is the first demonstration of the perturbation of fungal Ca 2+ homeostasis by an antifungal protein from a filamentous ascomycete and provides important new insights into the mode of action of PAF.