Proper folding of the antifungal protein PAF is required for optimal activity

Abstract

The Penicillium chrysogenumantifungal protein PAF is secreted into the supernatant after elimination of a preprosequence. PAF is actively internalized into the hyphae of sensitive molds and provokes growth retardation as well as changes in morphology. Thus far, no information is available on the exact mode of action of PAF, nor on the function of its prosequence in protein activity. Therefore, we sought to investigate the effects of secreted PAF as well as of intracellularly retained pro-PAF and mature PAF on the sensitive ascomycete Aspergillus nidulans, and transformed this model organism by expression vectors containing 5'-sequentially truncated paf-coding sequences under the control of the inducible P. chrysogenum-derived xylanase promoter. Indirect immunofluorescence staining revealed the localization of recombinant PAF predominantly in the hyphal tips of the transformant Xylpaf1 which expressed prepro-PAF, whereas the protein was found to be distributed intracellularly within all segments of hyphae of the transformants Xylpaf2 and Xylpaf3 which expressed pro-PAF and mature PAF, respectively. Growth retardation of Xylpaf1 and Xylpaf3 hyphae was detected by proliferation assays and by light microscopy analysis. Using transmission electron microscopy of ultrathin hyphal sections a marked alteration of the mitochondrial ultrastructure in Xylpaf1 was observed and an elevated amount of carbonylated proteins pointed to severe oxidative stress in this strain. The effects induced by secreted recombinant PAF resembled those evoked by native PAF. The results give evidence that properly folded PAF is a prerequisite for its activity.