HACIA LA APLICACION DE LAS PROTEINAS ANTIFUNGICAS DE HONGOS (AFPS): DESCIFRANDO SU ACCION ANTIFUNGICA Y PAPEL BIOLOGICO MEDIANTE EDICION GENETICA Y BIOLOGIA SINTETICA

PID2021-125858OB-I00

Nombre agencia financiadora Agencia Estatal de Investigación
Acrónimo agencia financiadora AEI
Programa Programa Estatal para Impulsar la Investigación Científico-Técnica y su Transferencia
Subprograma Subprograma Estatal de Generación de Conocimiento
Convocatoria Proyectos de I+D+I (Generación de Conocimiento y Retos Investigación)
Año convocatoria 2021
Unidad de gestión Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023
Centro beneficiario AGENCIA ESTATAL CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS (CSIC)
Identificador persistente http://dx.doi.org/10.13039/501100011033

Publicaciones

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Studies on the biological role of the antifungal protein PeAfpA from Penicillium expansum by functional gene characterization and transcriptomic profiling

RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia
  • Ropero-Pérez, Carolina
  • Moreno-Giménez, Elena
  • Marcos, Jose F.
  • Manzanares, Paloma
  • Gandía, Mónica
[EN] Antifungal proteins (AFPs) from filamentous fungi have enormous potential as novel biomolecules for the control of fungal diseases. However, little is known about the biological roles of AFPs beyond their antifungal action. Penicillium expansum encodes three phylogenetically different AFPs (PeAfpA, PeAfpB and PeAfpC) with diverse profiles of antifungal activity. PeAfpA stands out as a highly active AFP that is naturally produced at high yields. Here, we provide new data about the function of PeAfpA in P . expansum through phenotypical characterization and transcriptomic studies of null mutants of the corresponding afpA gene. Mutation of afpA did not affect axenic growth, conidiation, virulence, stress responses or sensitivity towards P . expansum AFPs. However, RNA sequencing evidenced a massive transcriptomic change linked to the onset of PeAfpA production. We identified two large gene expression clusters putatively involved in PeAfpA function, which correspond to genes induced or repressed with the production of PeAfpA. Functional enrichment analysis unveiled significant changes in genes related to fungal cell wall remodeling, mobilization of carbohydrates and plasma membrane transporters. This study also shows a putative co-regulation between the three afp genes. Overall, our transcriptomic analyses provide valuable insights for further understanding the biological functions of AFPs., This work was supported by grant PID2021-125858OB-100 and the Severo Ochoa Excellence Program CEX2021-001189-S, funded by MCIN/AEI/10.13039/501100011033 and by "ERDF, a way of making Europe." C.R.P. and EM -G were recipients of predoctoral grants ACIF/2021/364 funded by Conselleria d'Educaci o (Generalitat Valenciana, Comunitat Valenciana, Spain) and FPU18/02019 funded by MCIN/AEI/10.13039/501100011033 and by "ESF Investing in your future", respectively.




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

RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia
  • Giner-Llorca, Moisés
  • Locascio, Antonella|||0000-0001-8026-1937
  • Alonso del Real, Javier
  • Marcos, Jose F.
  • Manzanares, Paloma
[EN] 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., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by grants RTI2018-101115-B-C21, PID2021-125858OB-100 and the Severo Ochoa Excellence Program CEX2021-001189-S funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe". MGL was a recipient of a predoctoral grant FPU19/02066 funded by MCIN/AEI/10.13039/501100011033 and by "ESF Investing in your future".




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

RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia
  • Giner-Llorca, Moisés
  • Gallego del Sol, Francisca
  • Marcos, Jose F.
  • Marina, Alberto
  • Manzanares, Paloma
[EN] 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., We would like to thank the IBV-CSIC Crystallogenesis Facility for protein crystallization screenings. The structural results reported in this article derive from measurements made at the synchrotron DLS (Didcot, UK), ALBA (Cerdanyola del Valles, Spain) and ESRF (Grenoble, France). Data collection experiments for the best crystals were carried at XALOC, I24 and ID23-2 beamlines at ALBA, DLS and ESRF Synchrotrons, respectively. X-ray diffraction data collection was supported by block allocation group (BAG) DLS Proposal MX28394, ALBA Proposal 2020074406 and ESRF proposal MX-2351. We acknowledge the ESRF, ALBA and DLS synchrotrons for provision of beam time and we would like to thank beamline staff for assistance. This work was supported by grants RTI2018-101115-B-C21, PID2021-125858OB-100 (to PM and JFM) and PID2019-108541GB-I00 (to AM) funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe", and PROMETEO/2018/066 (to PM and JFM) and PROMETEO/2020/012 (to AM) by Valencian Government (Conselleria de Educacion, Investigacion, Cultura y Deporte). MGL was recipient of a predoctoral grant FPU19/02066 funded by MCIN/AEI/10.13039/501100011033 and by "ESF Investing in your future".




Data of the manuscript Studies on the biological role of the antifungal protein PeAfpA from Penicillium expansum by functional gene characterization and transcriptomic profiling"

Digital.CSIC. Repositorio Institucional del CSIC
  • Ropero Pérez, Carolina
  • Moreno Giménez, Elena
  • Marcos López, José Francisco
  • Manzanares, Paloma
  • Gandía, Mónica
This work was supported by grant PID2021-125858OB-100 and the Severo Ochoa Excellence Program CEX2021-001189-S, funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF, a way of making Europe.” C.R.P. and EM-G were recipients of predoctoral grants ACIF/2021/364 funded by Conselleria d’Educació (Generalitat Valenciana, Comunitat Valenciana, Spain) and FPU18/02019 funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”, respectively., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 2021-001189-S), Data for Figure 1; -- Data for Figure 2; -- Data for Figure 3; -- Data for Figure 6; -- Data for Figure 7; -- Data for Supplementary Figure 1; -- Data for Supplementary Figure 2; -- Data for Supplementary Figure 3; -- Data for Supplementary Figure 4, Peer reviewed




Studies on the biological role of the antifungal protein PeAfpA from Penicillium expansum by functional gene characterization and transcriptomic profiling

Digital.CSIC. Repositorio Institucional del CSIC
  • Ropero Pérez, Carolina
  • Moreno Giménez, Elena
  • Marcos López, José Francisco
  • Manzanares, Paloma
  • Gandía, Mónica
Antifungal proteins (AFPs) from filamentous fungi have enormous potential as novel biomolecules for the control of fungal diseases. However, little is known about the biological roles of AFPs beyond their antifungal action. Penicillium expansum encodes three phylogenetically different AFPs (PeAfpA, PeAfpB and PeAfpC) with diverse profiles of antifungal activity. PeAfpA stands out as a highly active AFP that is naturally produced at high yields. Here, we provide new data about the function of PeAfpA in P. expansum through phenotypical characterization and transcriptomic studies of null mutants of the corresponding afpA gene. Mutation of afpA did not affect axenic growth, conidiation, virulence, stress responses or sensitivity towards P. expansum AFPs. However, RNA sequencing evidenced a massive transcriptomic change linked to the onset of PeAfpA production. We identified two large gene expression clusters putatively involved in PeAfpA function, which correspond to genes induced or repressed with the production of PeAfpA. Functional enrichment analysis unveiled significant changes in genes related to fungal cell wall remodeling, mobilization of carbohydrates and plasma membrane transporters. This study also shows a putative co-regulation between the three afp genes. Overall, our transcriptomic analyses provide valuable insights for further understanding the biological functions of AFPs., This work was supported by grant PID2021-125858OB-100 and the Severo Ochoa Excellence Program CEX2021-001189-S, funded by MCIN/AEI/10.13039/501100011033 and by “ERDF, a way of making Europe.” C.R.P. and EM-G were recipients of predoctoral grants ACIF/2021/364 funded by Conselleria d'Educació (Generalitat Valenciana, Comunitat Valenciana, Spain) and FPU18/02019 funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”, respectively., Peer reviewed




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

Digital.CSIC. Repositorio Institucional del CSIC
  • Manzanares, Paloma
  • Giner-Llorca, Moisés
  • Marcos López, José Francisco
  • Garrigues, Sandra
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., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by grant PID2021-125858OB-100 and the Severo Ochoa Excellence Program CEX2021-001189-S funded by MCIN/AEI/ https://doi.org/10.13039/501100011033 and by “ERDF: A way of making Europe.” This study forms part of the AGROALNEXT program and was supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana. S. G. holds a Juan de la Cierva Incorporación grant (IJC2020-042749-I) funded by MCIN/AEI/ https://doi.org/10.13039/501100011033, “ERDF: A way of making Europe” and ‘NextGenerationEU/PRTR.’ M. G. L. was a recipient of a predoctoral grant FPU19/02066 funded by MCIN/AEI/ https://doi.org/10.13039/501100011033 and by “ESF: Investing in your future.”, With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 2021-001189-S), Peer reviewed




Increasing the efficiency of CRISPR/Cas9-mediated genome editing in the citrus postharvest pathogen Penicillium digitatum [DATASET]

Digital.CSIC. Repositorio Institucional del CSIC
  • Ropero Pérez, Carolina
  • Marcos López, José Francisco
  • Manzanares, Paloma
  • Garrigues, Sandra
Compressed Folder content: Figure 1, Figure 2, Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3, N/A, Grant PID2021-125858OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ‘ERDF A way of making Europe’. Juan de la Cierva Incorporación grant (IJC2020-042749-I) funded by MCIN/AEI/ 10.13039/501100011033, “ERDF A way of making Europe” and ‘NextGenerationEU’/PRTR’. Predoctoral grant (ACIF/2021/364) funded by Conselleria d’Educació (Generalitat Valenciana, Comunitat Valenciana, Spain)., Figure 1.--Figure 2.--Supplementary Figure 1.--Supplementary Figure 2.--Supplementary Figure 3, Peer reviewed




Increasing the efficiency of CRISPR/Cas9-mediated genome editing in the citrus postharvest pathogen Penicillium digitatum

Digital.CSIC. Repositorio Institucional del CSIC
  • Ropero Pérez, Carolina
  • Marcos López, José Francisco
  • Manzanares, Paloma
  • Garrigues, Sandra
Background: Penicillium digitatum is a fungal plant pathogen that causes the green mold disease in harvested citrus fruits. Due to its economical relevance, many efforts have focused on the development of genetic engineering tools for this fungus. Adaptation of the CRISPR/Cas9 technology was previously accomplished with self-replicative AMA1-based plasmids for marker-free gene editing, but the resulting efficiency (10%) limited its practical implementation. In this study, we aimed to enhance the efficiency of the CRISPR/Cas9-mediated gene editing in P. digitatum to facilitate its practical use.
Results: Increasing the culture time by performing additional culture streaks under selection conditions in a medium that promotes slower growth rates significantly improved the gene editing efficiency in P. digitatum up to 54–83%. To prove this, we disrupted five candidate genes that were chosen based on our previous high-throughput gene expression studies aimed at elucidating the transcriptomic response of P. digitatum to the antifungal protein PdAfpB. Two of these genes lead to visual phenotypic changes (PDIG_53730/pksP, and PDIG_54100/arp2) and allowed to start the protocol optimization. The other three candidates (PDIG_56860, PDIG_33760/rodA and PDIG_68680/dfg5) had no visually associated phenotype and were targeted to confirm the high efficiency of the protocol.
Conclusion: Genome editing efficiency of P. digitatum was significantly increased from 10% to up to 83% through the modification of the selection methodology, which demonstrates the feasibility of the CRISPR/Cas9 system for gene disruption in this phytopathogenic fungus. Moreover, the approach described in this study might help increase CRISPR/Cas9 gene editing efficiencies in other economically relevant fungal species for which editing efficiency via CRISPR/Cas9 is still low., This work was supported by the Severo Ochoa Excellence Program CEX2021-001189-S, and grant PID2021-125858OB-100 funded by MCIN/AEI/https://doi.org/10.13039/501100011033 and by ‘ERDF A way of making Europe’. SG holds a Juan de la Cierva Incorporación grant (IJC2020-042749-I) funded by MCIN/AEI/ https://doi.org/10.13039/501100011033, “ERDF A way of making Europe” and ‘NextGenerationEU’/PRTR’. CR-P holds a predoctoral grant (ACIF/2021/364) funded by Conselleria d’Educació (Generalitat Valenciana, Comunitat Valenciana, Spain).
Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2021-001189-S), Peer reviewed




Effect of antifungal proteins (AFPs) on the viability of heat-resistant fungi (HRFs) and the preservation of fruit juices

Digital.CSIC. Repositorio Institucional del CSIC
  • Hernández García, Laura
  • Manzanares, Paloma
  • Marcos López, José Francisco
  • Martínez Culebras, Pedro V
The control of heat-resistant fungi (HRFs), which cause spoilage of heat-treated fruit products, is considered a challenge for the fruit juice and beverage industry and requires new strategies for the development of antifungal compounds. In this study, four antifungal proteins (AFPs) from Penicillium digitatum (PdAfpB) and Penicillium expansum (PeAfpA, PeAfpB and PeAfpC), were evaluated against conidia from a representative collection of HRFs. A total of 19 strains from 16 different species belonging to the genera Aspergillus, Hamigera, Paecilomyces, Rasamsonia, Sarocladium, Talaromyces and Thermoascus were included in the study. PeAfpA and PdAfpB exhibited potent antifungal activity in synthetic media, completely inhibiting the growth of most of the fungi evaluated in the range of 0.5-32 μg/mL. The efficacy of the four AFPs was also tested in fruit juices against ascospores of five HRFs relevant to the food industry, including P. fulvus, P. niveus, P. variotii, A. fischeri and T. flavus. PdAfpB was the most effective protein in fruit juices, since it completely inhibited the growth of the five species tested in at least one of the fruit juices evaluated. This is the first study to demonstrate the activity of AFPs against fungal ascospores. Finally, a challenge test study showed that PdAfpB, at a concentration of 32 μg/mL, protected apple fruit juice artificially inoculated with ascospores of P. variotii for 17 days, highlighting the potential of the protein as a preservative in the fruit juice industry., This work was supported by grant PID2021-125858OB-100 and the Severo Ochoa Excellence Program CEX2021–001189-S funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”., Peer reviewed




Dynamics of interaction and internalisation of the antifungal protein PeAfpA into Penicillium digitatum morphotypes [DATASET]

Digital.CSIC. Repositorio Institucional del CSIC
  • Giner Llorca, Moisés
  • Ropero Pérez, Carolina
  • Garrigues, Sandra
  • Thomson, Darren D.
  • Bignell, Elaine M.
  • Manzanares, Paloma
  • Marcos López, José Francisco
Raw data of manuscript "Dynamics of interaction and internalisation of the antifungal protein PeAfpA into Penicillium digitatum morphotypes", This dataset includes the raw data from the manuscript “Dynamics of interaction and internalisation of the antifungal protein PeAfpA into Penicillium digitatum morphotypes”. This data comprises Excel sheets used to generate all the plots and graphs of the article (.xlsx files), 16-bit, 8-bit and RGB confocal microscopy images (.tif files), statistical analyses (.pzfx and .txt files) and “Readme” files (.txt files) to specificy experimental conditions for data collection and software-specific requirements to visualise each file., This work was supported by grant PID2021-125858OB-I00, and the Severo Ochoa Excellence Program CEX2021-001189-S, funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF, a way of making Europe”. M.G.L. and C.R.P. were recipients of predoctoral grants FPU19/02066 and ACIF/2021/364 funded by MCIN/AEI/10.13039/501100011033 and by ESF-Investing in Your future and by ”Conselleria d’Educació (Generalitat Valenciana, Comunitat Valenciana, Spain), respectively. M.G.L. international exchange was supported by EST23/00233 grant (funded by MCIN/AEI/10.13039/ 501100011033 and by ESF-Investing in Your future) and SEG10314 EMBO Scientific Exchange Grant. S.G. holds a Juan de la Cierva Incorporación grant (IJC2020-042749-I) from the Spanish Ministerio de Ciencia e Innovación, funded by the European Union, NextGenerationEU PRTR. Work undertaken with EMB and DDT was funded by the Medical Research Council Centre for Medical Mycology at the University of Exeter (MR/N006364/2 and MR/V033417/1), that forms part of the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC), and the BBSRC project grant BB/V017004/1 to EMB., dor (dc.descripction.sponsorship): With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2021-001189-S), Figure 1_data: Antifungal activity of the antifungal proteins PeAfpA and PdAfpB against Penicillium digitatum conidia. -- Figure 2_data ROS production in Penicilliumn digitatum germlings, induced by the treatment with antifungal proteins (PeAfpA or PdAfpB).-- Figure 3_data Evaluation of the interaction of PeAfpA and PdAfpB with P. digitaum conidia. Panels show confocal microscopy images of P. digitatum conidia after 15 min or 90 min of treatment with 8 µg/mL PeAfpA (A, B), or after 90 min of treatment with 8 µg/mL PdAfpB (C).-- Figure 4_data Comparison of the PeAfpA interaction with conidia of P. digitatum wild-type strain CECT 20796 (WT) and the KO mutant strains ΔpksP and Δarp2. -- Figure 5_data BODIPY-PeAfpA internalisation dynamics in P. digitatum hyphae in low-dose conditions (2 µg/mL).-- Figure 6_data PeAfpA internalises through hyphal tips and provokes vacuolisation and hyphal burst. Raw images used for each panel are provided.-- Figure 7_data: PeAfpA co-localises with CFW in the fungal cell wall. Raw images used for each panel are provided.-- Figure 8_data: PeAfpA internalisation in fungal hyphae is energy-dependent but not an endocytic process. Raw images used for each panel are provided.-- Supplementary Figure 1_data: Antifungal activity of BODIPY-labelled and -unlabelled antifungal proteins (PeAfpA and PdAfpB) against Penicillium digitatum conidia. Raw OD600 measurements are provided in two separated Excel files. -- Supplementary Figure 2_data: Raw data for the amino acid alignment of the Aspergillus fumigatus RodA protein and the orthologous protein from Penicillium digitatum. Raw pictures for panel B are provided.-- Supplementary Figure 3_data: Raw OD measurements for the dose response curves of P. digitatum CECT 20796 and deletion mutant strains (ΔpksP and Δarp2) treated with PeAfpA and PdAfpB. -- Supplementary Figure 4_data: Evaluation of the colocalization of PeAfpA and CFW in P. digitatum hyphae. Rae images for each panel are provided, as well as raw pixel intensity data.-- Supplementary Figure 5_data: Raw images from insets shown in Figure 5., Peer reviewed




Comparing the activity and interactions of the antifungal protein PeAfpA with conventional fungicides and food preservatives against mycotoxigenic fungi

Digital.CSIC. Repositorio Institucional del CSIC
  • Hernández-García, Laura
  • Molinos, Xabier
  • MANZANARES MIR, PALOMA MARIA
  • Marcos López, José Francisco
  • Martínez-Culebras, Pedro V.
Prevention of fungal contamination and the occurrence of mycotoxins in food and feeds requires the development of new antifungal approaches. The antifungal proteins (AFPs) produced by some fungi provide great potential for the control of contaminating fungi. In the present study, the antifungal activity of the protein PeAfpA from Penicillium expansum was compared with fungicides used in post-harvest control (imazalil and thiabendazole) and food preservatives (calcium propionate, sodium, sodium benzoate, potassium sorbate, and natamycin), against 23 fungal species belonging to the genera Penicillium, Fusarium, Byssochlamys, Aspergillus and Alternaria. In general, PeAfpA had the lowest minimum inhibitory concentration (MIC) followed by natamycin, the fungicides, and the chemical preservatives. PeAfpA was able to completely inhibit the growth of all tested fungi at concentrations ranging from 0.5 to 8 μg mL−1. In addition, we assessed the effects of PeAfpA in combination with imazalil, thiabendazole, natamycin, or potassium sorbate against four representative fungal species. Our results provide evidence for partial synergistic and additive effects between the protein PeAfpA and the other compounds tested. This study concludes that PeAfpA, alone or in combination with fungicides or food preservatives, has a great potential to prevent fungal contamination and reduce the required dosage of fungicides or chemical preservatives used in food conservation., This work was supported by grant PID2021-125858OB-100 and the Severo Ochoa Excellence Program CEX2021-001189-S funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”., Peer reviewed




Dynamics of interaction and internalisation of the antifungal protein PeAfpA into Penicillium digitatum morphotypes

Digital.CSIC. Repositorio Institucional del CSIC
  • Giner Llorca, Moisés
  • Ropero Pérez, Carolina
  • Garrigues, Sandra
  • Thomson, Darren D.
  • Bignell, Elaine M.
  • Manzanares, Paloma
  • Marcos López, José Francisco
Antifungal proteins (AFPs) as the highly active PeAfpA from Penicillium expansum or PdAfpB from Penicillium digitatum exert promising antifungal activity, but their mode of action is not fully understood. We characterised the interaction of PeAfpA against P. digitatum, comparing it to the less active PdAfpB. Despite similar effect on conidia germination, PeAfpA did not induce a burst of reactive oxygen species as PdAfpB. Live-cell fluorescence microscopy revealed complex dynamics of interaction and internalisation of both proteins with distinct P. digitatum morphotypes (quiescent conidia, swollen conidia, germlings and hyphae). Labelled PeAfpA co-localised at the cell wall of quiescent conidia, where its localisation was punctate and not uniformly distributed. This pattern changed during germination to a uniform distribution with increased intensity. Conidia from mutants of genes involved in melanin biosynthesis (pksP/alb1 or arp2) showed an altered distribution of PeAfpA but later mimicked the wild type trend of changes during germination. In swollen conidia and germlings, PeAfpA remained attached to the cell wall. In hyphae, PeAfpA was internalised through the growing hyphal tip after binding to the cell wall, in a non-endocytic but energy-dependent process that caused vacuolisation, which preceded cell death. These results may help the development of biofungicides based on AFPs., This work was supported by grant PID2021-125858OB-I00, and the Severo Ochoa Excellence Program CEX2021-001189-S, funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF, a way of making Europe”. M.G.L. and C.R.P. were recipients of predoctoral grants FPU19/02066 and ACIF/2021/364 funded by MCIN/AEI/10.13039/501100011033 and by ESF-Investing in Your future and by “Conselleria d'Educació” (Generalitat Valenciana, Comunitat Valenciana, Spain), respectively. M.G.L. international exchange was supported by EST23/00233 grant (funded by MCIN/AEI/10.13039/ 501100011033 and by ESF-Investing in Your future) and SEG10314 EMBO Scientific Exchange Grant. S.G. holds a Juan de la Cierva Incorporación grant (IJC2020-042749-I) from the Spanish Ministerio de Ciencia e Innovación, funded by the European Union, NextGenerationEU PRTR. Work undertaken with EMB and DDT was funded by the Medical Research Council Centre for Medical Mycology at the University of Exeter (MR/N006364/2 and MR/V033417/1), that forms part of the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC), and the BBSRC project grant BB/V017004/1 to EMB., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2021-001189-S), Peer reviewed