Fighting pathogenic yeasts with plant defensins and anti-fungal proteins from fungi.

Fungal infections represent a significant health risk worldwide. Opportunistic infections caused by yeasts, particularly by Candida spp. and their virulent emerging isolates, have become a major threat to humans, with an increase in fatal cases of infections attributed to the lack of effective anti-yeast therapies and the emergence of fungal resistance to the currently applied drugs. In this regard, the need for novel anti-fungal agents with modes of action different from those currently available is undeniable. Anti-microbial peptides (AMPs) are promising candidates for the development of novel anti-fungal biomolecules to be applied in clinic. A class of AMPs that is of particular interest is the small cysteine-rich proteins (CRPs). Among CRPs, plant defensins and anti-fungal proteins (AFPs) of fungal origin constitute two of the largest and most promising groups of CRPs showing anti-fungal properties, including activity against multi-resistant pathogenic yeasts. In this review, we update and compare the sequence, structure, and properties of plant defensins and AFPs with anti-yeast activity, along with their in vitro and in vivo potency. We focus on the current knowledge about their mechanism of action that may lead the way to new anti-fungals, as well as on the developments for their effective biotechnological production. KEY POINTS: • Plant defensins and fungal AFPs are alternative anti-yeast agents • Their multi-faceted mode of action makes occurrence of resistance rather improbable • Safe and cost-effective biofactories remain crucial for clinical application.

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

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.

Transcriptomic Profile of Penicillium digitatum Reveals Novel Aspects of the Mode of Action of the Antifungal Protein AfpB.

Antifungal proteins (AFPs) from filamentous fungi are promising biomolecules to control fungal pathogens. Understanding their biological role and mode of action is essential for their future application. AfpB from the citrus fruit pathogen Penicillium digitatum is highly active against fungal phytopathogens, including its native fungus. Our previous data showed that AfpB acts through a multitargeted three-stage process: interaction with the outer mannosylated cell wall, energy-dependent cell internalization, and intracellular actions that result in cell death. Here, we extend these findings by characterizing the functional role of AfpB and its interaction with P. digitatum through transcriptomic studies. For this, we compared the transcriptomic response of AfpB-treated P. digitatum wild type, a ΔafpB mutant, and an AfpB-overproducing strain. Transcriptomic data suggest a multifaceted role for AfpB. Data from the ΔafpB mutant suggested that the afpB gene contributes to the overall homeostasis of the cell. Additionally, these data showed that AfpB represses toxin-encoding genes, and they suggest a link to apoptotic processes. Gene expression and knockout mutants confirmed that genes coding for acetolactate synthase (ALS) and acetolactate decarboxylase (ALD), which belong to the acetoin biosynthetic pathway, contribute to the inhibitory activity of AfpB. Moreover, a gene encoding a previously uncharacterized extracellular tandem repeat peptide (TRP) protein showed high induction in the presence of AfpB, whereas its TRP monomer enhanced AfpB activity. Overall, our study offers a rich source of information to further advance in the characterization of the multifaceted mode of action of AFPs. IMPORTANCE Fungal infections threaten human health worldwide and have a negative impact on food security, damaging crop production and causing animal diseases. At present, only a few classes of fungicides are available due to the complexity of targeting fungi without affecting plant, animal, or human hosts. Moreover, the intensive use of fungicides in agriculture has led to the development of resistance. Therefore, there is an urgent need to develop antifungal biomolecules with new modes of action to fight human-, animal-, and plant-pathogenic fungi. Fungal antifungal proteins (AFPs) offer great potential as new biofungicides to control deleterious fungi. However, current knowledge about their killing mechanism is still limited, which hampers their potential applicability. AfpB from P. digitatum is a promising molecule with potent and specific fungicidal activity. This study further characterizes its mode of action, opening avenues for the development of new antifungals.

Rationally designed antifungal protein chimeras reveal new insights into structure-activity relationship.

Antifungal proteins (AFPs) are promising antimicrobial compounds that represent a feasible alternative to fungicides. Penicillium expansum encodes three phylogenetically distinct AFPs (PeAfpA, PeAfpB and PeAfpC) which show different antifungal profiles and fruit protection effects. To gain knowledge about the structural determinants governing their activity, we solved the crystal structure of PeAfpB and rationally designed five PeAfpA::PeAfpB chimeras (chPeAFPV1-V5). Chimeras showed significant differences in their antifungal activity. chPeAFPV1 and chPeAFPV2 improved the parental PeAfpB potency, and it was very similar to that of PeAfpA. chPeAFPV4 and chPeAFPV5 showed an intermediate profile of activity compared to the parental proteins while chPeAFPV3 was inactive towards most of the fungi tested. Structural analysis of the chimeras evidenced an identical scaffold to PeAfpB, suggesting that the differences in activity are due to the contributions of specific residues and not to induced conformational changes or structural rearrangements. Results suggest that mannoproteins determine protein interaction with the cell wall and its antifungal activity while there is not a direct correlation between binding to membrane phospholipids and activity. This work provides new insights about the relevance of sequence motifs and the feasibility of modifying protein specificity, opening the door to the rational design of chimeras with biotechnological applicability.

Development of a FungalBraid Penicillium expansum-based expression system for the production of antifungal proteins in fungal biofactories.

Fungal antifungal proteins (AFPs) have attracted attention as novel biofungicides. Their exploitation requires safe and cost-effective producing biofactories. Previously, Penicillium chrysogenum and Penicillium digitatum produced recombinant AFPs with the use of a P. chrysogenum-based expression system that consisted of the paf gene promoter, signal peptide (SP)-pro sequence and terminator. Here, the regulatory elements of the afpA gene encoding the highly produced PeAfpA from Penicillium expansum were developed as an expression system for AFP production through the FungalBraid platform. The afpA cassette was tested to produce PeAfpA and P. digitatum PdAfpB in P. chrysogenum and P. digitatum, and its efficiency was compared to that of the paf cassette. Recombinant PeAfpA production was only achieved using the afpA cassette, being P. chrysogenum a more efficient biofactory than P. digitatum. Conversely, P. chrysogenum only produced PdAfpB under the control of the paf cassette. In P. digitatum, both expression systems allowed PdAfpB production, with the paf cassette resulting in higher protein yields. Interestingly, these results did not correlate with the performance of both promoters in a luciferase reporter system. In conclusion, AFP production is a complex outcome that depends on the regulatory sequences driving afp expression, the fungal biofactory and the AFP sequence.

Potential of Antifungal Proteins (AFPs) to Control Penicillium Postharvest Fruit Decay.

Penicillium phytopathogenic species provoke severe postharvest disease and economic losses. Penicillium expansum is the main pome fruit phytopathogen while Penicillium digitatum and Penicillium italicum cause citrus green and blue mold, respectively. Control strategies rely on the use of synthetic fungicides, but the appearance of resistant strains and safety concerns have led to the search for new antifungals. Here, the potential application of different antifungal proteins (AFPs) including the three Penicillium chrysogenum proteins (PAF, PAFB and PAFC), as well as the Neosartorya fischeri NFAP2 protein to control Penicillium decay, has been evaluated. PAFB was the most potent AFP against P. digitatum, P. italicum and P. expansum, PAFC and NFAP2 showed moderate antifungal activity, whereas PAF was the least active protein. In fruit protection assays, PAFB provoked a reduction of the incidence of infections caused by P. digitatum and P. italicum in oranges and by P. expansum in apples. A combination of AFPs did not result in an increase in the efficacy of disease control. In conclusion, this study expands the antifungal inhibition spectrum of the AFPs evaluated, and demonstrates that AFPs act in a species-specific manner. PAFB is a promising alternative compound to control Penicillium postharvest fruit decay.

Novel insights in the production, activity and protective effect of Penicillium expansum antifungal proteins.

Antifungal proteins (AFPs) offer a great potential as new biofungicides to control deleterious fungi. The phytopathogenic fungus Penicillium expansum encodes three phylogenetically distinct AFPs, PeAfpA, PeAfpB and PeAfpC. Here, PeAfpA, a potent in vitro self-inhibitory protein, was demonstrated to control the infection caused by P. expansum in Golden apple fruits. We determined the production of the three proteins in different growth media. PeAfpA and PeAfpC were simultaneously produced by P. expansum in three out of the eight media tested as detected by Western blot, whereas PeAfpB was not detected even in those described for class B AFP production. Regardless of the culture medium, the carbon source affected Peafp expression. Notably, the production of PeAfpA was strain-dependent, but analyses of PeafpA regulatory sequences in the three strains studied could not explain differences in protein production. None of the PeAFPs was produced during apple infection, suggesting no relevant role in pathogenesis. PeAfpA together with PeAfpB and also with Penicillium digitatum PdAfpB showed synergistic interaction. The highly active antifungal PeAfpA also showed moderate antibacterial activity. We conclude that there is not a general pattern for Peafp gene expression, protein production or antimicrobial activity and confirm PeAfpA as a promising compound for postharvest conservation.