The antifungal protein PAF interferes with PKC/MPK and cAMP/PKA signalling of Aspergillus nidulans

Abstract

The Penicillium chrysogenum antifungal protein PAF inhibits polar growth and induces apoptosis in Aspergillus nidulans. We report here that two signalling cascades are implicated in its antifungal activity. PAF activates the cAMP/protein kinase A (Pka) signalling cascade. A pkaA deletion mutant exhibited reduced sensitivity towards PAF. This was substantiated by the use of pharmacological modulators: PAF aggravated the effect of the activator 8-Br-cAMP and partially relieved the repressive activity of caffeine. Furthermore, the Pkc/mitogen-activated protein kinase (Mpk) signalling cascade mediated basal resistance to PAF, which was independent of the small GTPase RhoA. Non-functional mutations of both genes resulted in hypersensitivity towards PAF. PAF did not increase MpkA phosphorylation or induce enzymes involved in the remodelling of the cell wall, which normally occurs in response to activators of the cell wall integrity pathway. Notably, PAF exposure resulted in actin gene repression and a deregulation of the chitin deposition at hyphal tips of A. nidulans, which offers an explanation for the morphological effects evoked by PAF and which could be attributed to the interconnection of the two signalling pathways. Thus, PAF represents an excellent tool to study signalling pathways in this model organism and to define potential fungal targets to develop new antifungals.

Fig. 1

Growth inhibiting effect of 0–100 μg ml⁻¹ PAF on the A. nidulans RhoA mutant strains, RhoA^G14V and RhoA^E40I, compared with the recipient strain GR5. Controls were left untreated. A total of 2 × 10³ conidia were point inoculated on CM agar plates containing appropriate supplements and were incubated for 48 h at 37°C.

Fig. 2

Growth inhibitory effect of PAF on the A. nidulans mutants ΔmpkA and alcA-PkcA compared with the recipient strains GR5 and R153 respectively. A total of 2 × 10³ conidia were point inoculated on agar plates (CM for GR5 and ΔmpkA, repressive MM containing 1% glucose according to Ronen et al., 2007; for R153 and alcA-PkcA) containing the appropriate supplements and 0–100 μg ml⁻¹ PAF. The plates were incubated at 37°C for 48 h (GR5 and ΔmpkA) or 72 h (R153 and alcA-PkcA mutant).

Fig. 5

Detection of cAMP-dependent PKA phosphorylation activity. Two micrograms of PepTaq A1 peptide were incubated with 50 μg of crude protein extract in a final volume of 25 μl for 30 min as described in the manufacturer's instructions. Samples were separated on a 0.8% agarose gel at 100 V for 20 min. Lanes 1–4: (1) phosphorylated PepTag A1 peptide (positive control) (2) unphosphorylated PepTag A1 peptide (negative control) (3) PepTag A1 peptide exposed to crude cell extract from A. nidulans FGSC4A treated with 100 μg ml⁻¹ PAF for 90 min and (4) PepTag A1 peptide exposed to crude cell extract from untreated A. nidulans. The arrow indicates the loading wells of the agarose gel, and the asterisks indicate the phosphorylated PepTag A1 peptide, which migrated towards the cathode.

Fig. 4

Growth inhibiting effect of PAF on the A. nidulansΔpkaA mutant in comparison to the respective recipient strain RKIS1. A total of 2 × 10³ conidia were point inoculated on CM agar plates containing the respective supplements and incubated for 72 h at 37°C.

Fig. 3

Effect of the cAMP/PkaA signalling pathway modulating substances (A) 8-Br-cAMP and (B) caffeine in combination with PAF on the growth of A. nidulans (FGSC4A). A total of 2 × 10³ conidia were point inoculated on CM agar plates containing 100 μg ml⁻¹ PAF together with 5 mM 8-Br-cAMP and 20 mM caffeine. Plates were incubated at 37°C for 48 h in (A) and 72 h in (B).

Fig. 7

Fluorescence micrographs showing actin and chitin distribution in A. nidulans hyphae in response to PAF treatment (+PAF) (B, D and F–H) compared with untreated controls (Co) (A, C and E). (A–D) shows the transgenic A. nidulans strain expressing actin-GFP (Taheri-Talesh et al., 2008). (A) In the untreated control, characteristic actin patches at the subapical region of the hyphal tip are visible (white arrow heads). (B) When exposed to 50 μg ml⁻¹ PAF, no actin patches were detectable and transition from polar to apolar growth became evident (white arrows). (C and D) Light micrographs of (A) and (B) respectively. (E–H) shows CFW staining of A. nidulans FGSC4A. (E) The untreated control sample exhibits a characteristic cap-like CFW fluorescence at the hyphal tip which corresponds to the site of chitin assembly. (F–H) Incubation with 50 μg ml⁻¹ PAF induces hyperbranching (F) a reduced chitin content and (G and H) a delocalized chitin deposition at the hyphal tips. Scale bar, 10 μm.

Fig. 6

Northern blot analysis of actin (acnA) gene expression. Sixteen-hour-old A. nidulans cultures were exposed to 0 (−) and 50 μg ml⁻¹ PAF (+) for 1.5 and 3 h respectively. Ten micrograms of total RNA was detected for actin gene expression by hybridizing the blotted samples with an actin-specific DIG-dUTP probe (upper panel). The lower panel shows the 26S and 18S rRNA stained with ethidium bromide as loading control. Note that expression of the A. nidulans acnA gene results in two transcripts due to the presence of different 3′ termini (Fidel et al., 1988).

Fig. 8

Tentative model for the mode of action of the antifungal protein PAF in A. nidulans. PAF activates the cAMP/PkaA signalling cascade via heterotrimeric G-protein signalling (Leiter et al., 2005), which leads to apoptosis and to defective actin polymerization and apolar growth (this work and Leiter et al., 2005). Note that the secondary catalytic subunit PkaB could be involved in signal transmission of PAF and caffeine due to partly redundant functions in PkaA signalling in A. nidulans (Ni et al., 2005). Actin assembly and polar growth might also be disturbed via so far unidentified RhoA-GAP effectors. PAF further fails to activate MpkA and probably also PkcA (this work), disrupting basal resistance of A. nidulans towards antifungal activity. In contrast, the cell wall interfering drugs CFW and caffeine induce CWI signalling and cell wall remodelling in a RhoA-dependent and RhoA-independent way respectively (this work and Kuranda et al., 2006; Fujioka et al., 2007). The cell wall remodelling by MpkA-independent pathways, as documented for A. nidulans (Fujioka et al., 2007), were not considered in this diagram. Dotted lines and question marks represent so far unidentified connections. AC, adenylate cyclase; CFW, Calcofluor white; GAP, GTPase activation protein; GEF, guanine nucleotide exchange factor; PAF, Penicillium chrysogenum antifungal protein; TFs, transcription factors.