The antifungal activity of the Penicillium chrysogenum protein PAF disrupts calcium homeostasis in Neurospora crassa

Eukaryot Cell. 2010 Sep;9(9):1374-82. doi: 10.1128/EC.00050-10. Epub 2010 Jul 9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antifungal Agents / metabolism
  • Antifungal Agents / pharmacology*
  • Calcium / metabolism*
  • Fungal Proteins / metabolism
  • Fungal Proteins / pharmacology*
  • Neurospora crassa / drug effects*
  • Neurospora crassa / genetics
  • Neurospora crassa / growth & development
  • Neurospora crassa / metabolism*
  • Penicillium chrysogenum / chemistry
  • Penicillium chrysogenum / metabolism
  • Signal Transduction / drug effects

Substances

  • Antifungal Agents
  • Fungal Proteins
  • PAF protein, Penicillium chrysogenum
  • Calcium