title: The antifungal activity of the Penicillium chrysogenum protein PAF disrupts calcium homeostasis in Neurospora crassa.
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 Deltacch-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.
authors.affiliation: Biocenter, Division of Molecular Biology, Innsbruck Medical University, Fritz-Pregl Strasse 3, A-6020 Innsbruck, Austria.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Binder
authors.forename: Meiling
authors.initials: M
authors.lastname: Chu
authors.forename: Nick D
authors.initials: ND
authors.lastname: Read
authors.forename: Florentine
authors.initials: F
authors.lastname: Marx
mesh_terms: D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism / Q000494:pharmacology*; D009492:Neurospora crassa / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010408:Penicillium chrysogenum / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; C097832:PAF protein, Penicillium chrysogenum; D002118:Calcium