Novel findings about the mode of action of the antifungal protein PeAfpA against Saccharomyces cerevisiae

Abstract

Antifungal proteins (AFPs) from filamentous fungi offer the potential to control fungal infections that threaten human health and food safety. AFPs exhibit broad antifungal spectra against harmful fungi, but limited knowledge of their killing mechanism hinders their potential applicability. PeAfpA from Penicillium expansum shows strong antifungal potency against plant and human fungal pathogens and stands above other AFPs for being active against the yeast Saccharomyces cerevisiae. We took advantage of this and used a model laboratory strain of S. cerevisiae to gain insight into the mode of action of PeAfpA by combining (i) transcriptional profiling, (ii) PeAfpA sensitivity analyses of deletion mutants available in the S. cerevisiae genomic deletion collection and (iii) cell biology studies using confocal microscopy. Results highlighted and confirmed the role of the yeast cell wall (CW) in the interaction with PeAfpA, which can be internalized through both energy-dependent and independent mechanisms. The combined results also suggest an active role of the CW integrity (CWI) pathway and the cAMP-PKA signalling in the PeAfpA killing mechanism. Besides, our studies revealed the involvement of phosphatidylinositol metabolism and the participation of ROX3, which codes for the subunit 19 of the RNA polymerase II mediator complex, in the yeast defence strategy. In conclusion, our study provides clues about both the killing mechanism of PeAfpA and the fungus defence strategies against the protein, suggesting also targets for the development of new antifungals. KEY POINTS: • PeAfpA is a cell-penetrating protein with inhibitory activity against S. cerevisiae. • The CW integrity (CWI) pathway is a key player in the PeAfpA killing mechanism. • Phosphatidylinositol metabolism and ROX3 are involved in the yeast defence strategy.

Keywords: Antifungal proteins (AFPs); Cell wall integrity; Cell-penetrating protein; Phosphatidylinositol metabolism; Transcriptomics; Yeast deletion collection.

Fig. 1

Dose–response curve showing the antifungal activity of PeAfpA against S. cerevisiae BY4741. Curve shows mean ± SD of three replicates at each PeAfpA concentration

Fig. 2

A Principal component analysis of the expressed genes across the treatments. B Heat map of the 100 most variable genes across all treatments sorted by transcripts per million (TPM). Hierarchical clustering is included for the treatments (horizontal axis) and genes (vertical axis). Colour scaling was applied to the genes (row Z-score represented). C Venn diagram showing unique and shared differentially expressed genes (DEGs) among the treatments. Condition 1, S. cerevisiae BY4741 grown in absence of PeAfpA; condition 2, S. cerevisiae BY4741 grown in presence of 1 μg/mL PeAfpA; condition 3, S. cerevisiae BY4741 grown in presence of 2 μg/mL PeAfpA; condition 4, S. cerevisiae BY4741 grown in presence of 4 μg/mL PeAfpA

Fig. 3

GO enrichment and KEGG pathways analysis for the upregulated differentially expressed genes (DEGs) of condition 4 (S. cerevisiae BY4741 grown in presence of 4 μg/mL PeAfpA). The chart shows the top 20 GO terms from each category that were significantly enriched (false discovery rate (FDR) < 0.01). Within each category, GO terms or KEGG pathways are sorted in decreasing order according to their fold enrichment value. Dot size correlates with the number of genes identified inside each pathway. Dot colour indicates relative significance by correlating with –log10(FDR)

Fig. 4

GO enrichment and KEGG pathways analysis for the downregulated differentially expressed genes (DEGs) of condition 4 (S. cerevisiae BY4741 grown in presence of 4 μg/mL PeAfpA). The chart shows the top 20 GO terms from each category that were significantly enriched (false discovery rate (FDR) < 0.01). Within each category, GO terms or KEGG pathways are sorted in decreasing order according to their fold enrichment value. Dot size correlates with the number of genes identified inside each pathway. Dot colour indicates relative significance by correlating with –log10(FDR)

Fig. 5

GO enrichment and KEGG pathways analysis for the upregulated differentially expressed genes (DEGs) which are shared between conditions 3 (S. cerevisiae BY4741 grown in presence of 2 μg/mL PeAfpA) and 4 (S. cerevisiae BY4741 grown in presence of 4 μg/mL PeAfpA). The chart shows the top 20 GO terms from each category that were significantly enriched (false discovery rate (FDR) < 0.01). Within each category, GO terms or KEGG pathways are sorted in decreasing order according to their fold enrichment value. Dot size correlates with the number of genes identified inside each pathway. Dot colour indicates relative significance by correlating with –log10(FDR)

Fig. 6

Antifungal activity of PeAfpA at 32 µg/mL against S. cerevisiae BY4741 and 102 deletion mutant strains. Bars show means and standard deviation of triplicate values. Bar corresponding to the parental strain BY4741 is coloured in grey. Mutant strains have been classified in three groups: significantly sensitive (red), significantly tolerant (green) and not significant (blue). Statistical significance was assessed by t-test between each mutant and its corresponding wild type data (p-value < 0.05)

Fig. 7

PeAfpA interaction with S. cerevisiae cells. A Representative confocal laser microscopy images of S. cerevisiae cells incubated with 8 µg/mL of BODIPY-PeAfpA. Cells were incubated for the indicated periods of time with BODIPY-PeAfpA (left and middle images) or pre-treated with NaN₃ (10 mM, 15 min) and then incubated 60 min with BODIPY-PeAfpA (right images) and stained with calcofluor white (CFW; 25 μM) or the cell death marker propidium iodide (PI; 2 μM). Upper panels are bright-field images; middle panels are fluorescent images (BODIPY); lower panels are fluorescent merged images (BODIPY, CFW, PI). No PI signal was observed; in the merged images, the red channel for picture processing was included. Bars correspond to 10 μm. B Representative confocal laser microscopy images of S. cerevisiae cells incubated with 10-μM melittin and stained with PI or CFW. Left panel corresponds to bright field; middle panel corresponds to PI fluorescent images; right panel corresponds to fluorescent merged images (PI and CFW). Bars correspond to 10 μm

Fig. 8

Effect of PeAfpA on S. cerevisiae cells. A Dose–response curves of PeAfpA against S. cerevisiae BY4741 and the selected mutant strains for microscopy analysis. Error bars show standard deviation of three technical replicates. Arrow indicates the protein concentration used in microscopy assays. B Representative confocal laser microscopy images of S. cerevisiae parental and mutant cells incubated with 8 µg/mL of BODIPY-PeAfpA for 60 min and stained with calcofluor white (CFW; 25 μM) or the cell death marker propidium iodide (PI; 2 μM). Bright field, fluorescent BODIPY (green) and merged (BODIPY, CFW and PI) images are shown. Insert in vps34Δ cells corresponds to fluorescent PI images. Bars correspond to 10 μm