The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Florentine Marx - One of the best experts on this subject based on the ideXlab platform.

  • potential of Antifungal Proteins afps to control penicillium postharvest fruit decay
    Journal of Fungi, 2021
    Co-Authors: Monica Gandia, Florentine Marx, László Galgóczy, Anant Kakar, Moises Ginerllorca, Jeanett Holzknecht, Pedro Martinezculebras, Jose F Marcos, Paloma Manzanares
    Abstract:

    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.

  • cysteine rich Antifungal Proteins from filamentous fungi are promising bioactive natural compounds in anti candida therapy
    Israel Journal of Chemistry, 2019
    Co-Authors: Annie Yap, László Galgóczy, Florentine Marx
    Abstract:

    The emerging number of life-threatening invasive fungal infections caused by drug-resistant Candida strains urges the need for the development and application of fundamentally new and safe Antifungal strategies in the clinical treatment. Recent studies demonstrated that the extracellular cysteine-rich and cationic Antifungal Proteins (crAFPs) originating from filamentous fungi, and de novo designed synthetic peptide derivatives of these crAFPs provide a feasible basis for this approach. This mini-review focuses on the global challenges of the anti-Canidia therapy and on the crAFPs as potential drug candidates to overcome existing problems. The advantages and limitations in the use of crAFPs and peptide derivatives compared to those of conventional Antifungal drugs will also be critically discussed.

  • Solution structure and novel insights into phylogeny and mode of action of the Neosartorya (Aspergillus) fischeri Antifungal protein (NFAP).
    International journal of biological macromolecules, 2019
    Co-Authors: Dorottya Hajdu, Florentine Marx, Anna Huber, András Czajlik, Liliána Tóth, Zoltán Kele, Sándor Kocsubé, Ádám Fizil, László Galgóczy, Gyula Batta
    Abstract:

    Small, cysteine-rich and cationic Antifungal Proteins from natural sources are promising candidates for the development of novel treatment strategies to prevent and combat infections caused by drug-resistant fungi. However, limited information about their structure and Antifungal mechanism hampers their future applications. In the present study, we determined the solution structure, dynamics and associated solvent areas of the Neosartorya (Aspergillus) fischeri Antifungal protein NFAP. Genome mining within the genus revealed the presence of orthologous genes in N. fischeri and Neosartorya spathulata, and genes encoding closely related Proteins can be found in Penicillium brasiliensis and Penicillium oxalicum. We show that the tertiary structure of these putative Proteins can be resolved using the structure of NFAP as reliable template for in silico prediction. Localization studies with fluorescence-labelled protein pointed at an energy-dependent uptake mechanism of NFAP in the sensitive model fungus Neurospora crassa and subsequent cytoplasmic localization coincided with cell-death induction. The presented results contribute to a better understanding of the structure/function relationship of NFAP and related Proteins and pave the way towards future Antifungal drug development.

  • Three Antifungal Proteins From Penicillium expansum: Different Patterns of Production and Antifungal Activity.
    Frontiers in microbiology, 2018
    Co-Authors: Sandra Garrigues, Florentine Marx, Monica Gandia, Jose F Marcos, Maria Coca, Laia Castillo, Paloma Manzanares
    Abstract:

    Antifungal Proteins of fungal origin (AFPs) are small, secreted, cationic, and cysteine-rich Proteins. Filamentous fungi encode a wide repertoire of AFPs belonging to different phylogenetic classes, which offer a great potential to develop new Antifungals for the control of pathogenic fungi. The fungus Penicillium expansum is one of the few reported to encode three AFPs each belonging to a different phylogenetic class (A, B, and C). In this work, the production of the putative AFPs from P. expansum was evaluated, but only the representative of class A, PeAfpA, was identified in culture supernatants of the native fungus. The biotechnological production of PeAfpB and PeAfpC was achieved in Penicillium chrysogenum with the P. chrysogenum-based expression cassette, which had been proved to work efficiently for the production of other related AFPs in filamentous fungi. Western blot analyses confirmed that P. expansum only produces PeAfpA naturally, whereas PeAfpB and PeAfpC could not be detected. From the three AFPs from P. expansum, PeAfpA showed the highest Antifungal activity against all fungi tested, including plant and human pathogens. P. expansum was also sensitive to its self-AFPs PeAfpA and PeAfpB. PeAfpB showed moderate Antifungal activity against filamentous fungi, whereas no activity could be attributed to PeAfpC at the conditions tested. Importantly, none of the PeAFPs showed hemolytic activity. Finally, PeAfpA was demonstrated to efficiently protect against fungal infections caused by Botrytis cinerea in tomato leaves and Penicillium digitatum in oranges. The strong Antifungal potency of PeAfpA, together with the lack of cytotoxicity, and significant in vivo protection against phytopathogenic fungi that cause postharvest decay and plant diseases, make PeAfpA a promising alternative compound for application in agriculture, but also in medicine or food preservation.

  • Efficient production and characterization of the novel and highly active Antifungal protein AfpB from Penicillium digitatum
    Scientific Reports, 2017
    Co-Authors: Sandra Garrigues, Florentine Marx, Monica Gandia, Jose F Marcos, Maria Coca, Attila Borics, Crina Popa, Paloma Manzanares
    Abstract:

    Filamentous fungi encode distinct Antifungal Proteins (AFPs) that offer great potential to develop new Antifungals. Fungi are considered immune to their own AFPs as occurs in Penicillium chrysogenum , the producer of the well-known PAF. The Penicillium digitatum genome encodes only one afp gene ( afpB ), and the corresponding protein (AfpB) belongs to the class B phylogenetic cluster. Previous attempts to detect AfpB were not successful. In this work, immunodetection confirmed the absence of AfpB accumulation in wild type and previous recombinant constitutive P . digitatum strains. Biotechnological production and secretion of AfpB were achieved in P . digitatum with the use of a P . chrysogenum -based expression cassette and in the yeast Pichia pastoris with the α-factor signal peptide. Both strategies allowed proper protein folding, efficient production and single-step purification of AfpB from culture supernatants. AfpB showed Antifungal activity higher than the P . chrysogenum PAF against the majority of the fungi tested, especially against Penicillium species and including P . digitatum , which was highly sensitive to the self-AfpB. Spectroscopic data suggest that native folding is not required for activity. AfpB also showed notable ability to withstand protease and thermal degradation and no haemolytic activity, making AfpB a promising candidate for the control of pathogenic fungi.

László Galgóczy - One of the best experts on this subject based on the ideXlab platform.

  • potential of Antifungal Proteins afps to control penicillium postharvest fruit decay
    Journal of Fungi, 2021
    Co-Authors: Monica Gandia, Florentine Marx, László Galgóczy, Anant Kakar, Moises Ginerllorca, Jeanett Holzknecht, Pedro Martinezculebras, Jose F Marcos, Paloma Manzanares
    Abstract:

    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.

  • cysteine rich Antifungal Proteins from filamentous fungi are promising bioactive natural compounds in anti candida therapy
    Israel Journal of Chemistry, 2019
    Co-Authors: Annie Yap, László Galgóczy, Florentine Marx
    Abstract:

    The emerging number of life-threatening invasive fungal infections caused by drug-resistant Candida strains urges the need for the development and application of fundamentally new and safe Antifungal strategies in the clinical treatment. Recent studies demonstrated that the extracellular cysteine-rich and cationic Antifungal Proteins (crAFPs) originating from filamentous fungi, and de novo designed synthetic peptide derivatives of these crAFPs provide a feasible basis for this approach. This mini-review focuses on the global challenges of the anti-Canidia therapy and on the crAFPs as potential drug candidates to overcome existing problems. The advantages and limitations in the use of crAFPs and peptide derivatives compared to those of conventional Antifungal drugs will also be critically discussed.

  • Solution structure and novel insights into phylogeny and mode of action of the Neosartorya (Aspergillus) fischeri Antifungal protein (NFAP).
    International journal of biological macromolecules, 2019
    Co-Authors: Dorottya Hajdu, Florentine Marx, Anna Huber, András Czajlik, Liliána Tóth, Zoltán Kele, Sándor Kocsubé, Ádám Fizil, László Galgóczy, Gyula Batta
    Abstract:

    Small, cysteine-rich and cationic Antifungal Proteins from natural sources are promising candidates for the development of novel treatment strategies to prevent and combat infections caused by drug-resistant fungi. However, limited information about their structure and Antifungal mechanism hampers their future applications. In the present study, we determined the solution structure, dynamics and associated solvent areas of the Neosartorya (Aspergillus) fischeri Antifungal protein NFAP. Genome mining within the genus revealed the presence of orthologous genes in N. fischeri and Neosartorya spathulata, and genes encoding closely related Proteins can be found in Penicillium brasiliensis and Penicillium oxalicum. We show that the tertiary structure of these putative Proteins can be resolved using the structure of NFAP as reliable template for in silico prediction. Localization studies with fluorescence-labelled protein pointed at an energy-dependent uptake mechanism of NFAP in the sensitive model fungus Neurospora crassa and subsequent cytoplasmic localization coincided with cell-death induction. The presented results contribute to a better understanding of the structure/function relationship of NFAP and related Proteins and pave the way towards future Antifungal drug development.

  • Structural determinants of Neosartorya fischeri Antifungal protein (NFAP) for folding, stability and Antifungal activity.
    Scientific reports, 2017
    Co-Authors: László Galgóczy, Zoltán Kele, Györgyi Váradi, Attila Borics, Mate Viragh, Hargita Ficze, Florentine Marx
    Abstract:

    The recent global challenges to prevent and treat fungal infections strongly demand for the development of new Antifungal strategies. The structurally very similar cysteine-rich Antifungal Proteins from ascomycetes provide a feasible basis for designing new Antifungal molecules. The main structural elements responsible for folding, stability and Antifungal activity are not fully understood, although this is an essential prerequisite for rational protein design. In this study, we used the Neosartorya fischeri Antifungal protein (NFAP) to investigate the role of the disulphide bridges, the hydrophobic core, and the N-terminal amino acids in the formation of a highly stable, folded, and Antifungal active protein. NFAP and its mutants carrying cysteine deletion (NFAPΔC), hydrophobic core deletion (NFAPΔh), and N-terminal amino acids exchanges (NFAPΔN) were produced in Pichia pastoris. The recombinant NFAP showed the same features in structure, folding, stability and activity as the native protein. The data acquired with mass spectrometry, structural analyses and Antifungal activity assays of NFAP and its mutants proved the importance of the disulphide bonding, the hydrophobic core and the correct N-terminus for folding, stability and full Antifungal function. Our findings provide further support to the comprehensive understanding of the structure-function relationship in members of this protein group.

  • a penicillium chrysogenum based expression system for the production of small cysteine rich Antifungal Proteins for structural and functional analyses
    Microbial Cell Factories, 2016
    Co-Authors: Christoph Sonderegger, Anna Huber, Ádám Fizil, László Galgóczy, Jose F Marcos, Paloma Manzanares, Sandra Garrigues, Attila Borics, Nikoletta Hegedus, Gyula Batta
    Abstract:

    Small, cysteine-rich and cationic Antifungal Proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure–function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous Antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant Proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed Proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant Proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and Antifungal activity of the recombinant Proteins were analysed by ESI–MS, ECD and NMR spectroscopy and growth inhibition assays. This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded Proteins with high quality for structure–function analyses.

Jose F Marcos - One of the best experts on this subject based on the ideXlab platform.

  • potential of Antifungal Proteins afps to control penicillium postharvest fruit decay
    Journal of Fungi, 2021
    Co-Authors: Monica Gandia, Florentine Marx, László Galgóczy, Anant Kakar, Moises Ginerllorca, Jeanett Holzknecht, Pedro Martinezculebras, Jose F Marcos, Paloma Manzanares
    Abstract:

    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.

  • differential susceptibility of mycotoxin producing fungi to distinct Antifungal Proteins afps
    Food Microbiology, 2021
    Co-Authors: Monica Gandia, Jose F Marcos, P V Martinezculebras, Alicia Boronat, Paloma Manzanares
    Abstract:

    Abstract The global challenge to prevent fungal spoilage and mycotoxin contamination on foods and feeds require the development of new Antifungal strategies. Filamentous fungi encode diverse Antifungal Proteins (AFPs), which offer a great potential for the control of contaminant fungi. In this study, four AFPs from Penicillium digitatum (PdAfpB) and Penicillium expansum (PeAfpA, PeAfpB and PeAfpC) belonging to classes A, B and C, were tested against a representative panel of mycotoxin-producing fungi. They included a total of 38 strains representing 32 different species belonging to the genera Alternaria, Aspergillus, Byssochlamys, Fusarium and Penicillium. PeAfpA exhibited a potent Antifungal activity, since the growth of all tested fungi was completely inhibited by concentrations ranging from 0.5 to 16 μg/mL. PdAfpB and PeAfpB, although less effective than PeAfpA, showed significant activity against most of the mycotoxigenic fungi tested. Importantly, PeAfpC previously described as inactive, showed a powerful inhibition against B. spectabilis strains, which are important spoilage and mycotoxin fungi in pasteurized foods. Although less effective than in liquid media, AFPs affected fungal growth on solid media. This study also underlines the potential of these AFPs, in particular PeAfpA, as future Antifungal agents for applications in foods, on growing crops or during postharvest storage.

  • novel insights in the production activity and protective effect of penicillium expansum Antifungal Proteins
    International Journal of Biological Macromolecules, 2020
    Co-Authors: Monica Gandia, Moises Ginerllorca, Jose F Marcos, Sandra Garrigues, Ana Monge, Helena Orozco, Paloma Manzanares
    Abstract:

    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.

  • the Antifungal protein afpb induces regulated cell death in its parental fungus penicillium digitatum
    mSphere, 2020
    Co-Authors: Adria Bugeda, Monica Gandia, Jose F Marcos, Paloma Manzanares, Sandra Garrigues, Maria Coca
    Abstract:

    ABSTRACT Filamentous fungi produce small cysteine-rich Proteins with potent, specific Antifungal activity, offering the potential to fight fungal infections that severely threaten human health and food safety and security. The genome of the citrus postharvest fungal pathogen Penicillium digitatum encodes one of these Antifungal Proteins, namely AfpB. Biotechnologically produced AfpB inhibited the growth of major pathogenic fungi at minimal concentrations, surprisingly including its parental fungus, and conferred protection to crop plants against fungal infections. This study reports an in-depth characterization of the AfpB mechanism of action, showing that it is a cell-penetrating protein that triggers a regulated cell death program in the target fungus. We prove the importance of AfpB interaction with the fungal cell wall to exert its killing activity, for which protein mannosylation is required. We also show that the potent activity of AfpB correlates with its rapid and efficient uptake by fungal cells through an energy-dependent process. Once internalized, AfpB induces a transcriptional reprogramming signaled by reactive oxygen species that ends in cell death. Our data show that AfpB activates a self-injury program, suggesting that this protein has a biological function in the parental fungus beyond defense against competitors, presumably more related to regulation of the fungal population. Our results demonstrate that this protein is a potent Antifungal that acts through various targets to kill fungal cells through a regulated process, making AfpB a promising compound for the development of novel biofungicides with multiple fields of application in crop and postharvest protection, food preservation, and medical therapies. IMPORTANCE Disease-causing fungi pose a serious threat to human health and food safety and security. The limited number of licensed Antifungals, together with the emergence of pathogenic fungi with multiple resistance to available Antifungals, represents a serious challenge for medicine and agriculture. Therefore, there is an urgent need for new compounds with high fungal specificity and novel Antifungal mechanisms. Antifungal Proteins in general, and AfpB from Penicillium digitatum in particular, are promising molecules for the development of novel Antifungals. This study on AfpB’s mode of action demonstrates its potent, specific fungicidal activity through the interaction with multiple targets, presumably reducing the risk of evolving fungal resistance, and through a regulated cell death process, uncovering this protein as an excellent candidate for a novel biofungicide. The in-depth knowledge on AfpB mechanistic function presented in this work is important to guide its possible future clinical and agricultural applications.

  • the myosin motor domain containing chitin synthases are involved in cell wall integrity and sensitivity to Antifungal Proteins in penicillium digitatum
    Frontiers in Microbiology, 2019
    Co-Authors: Monica Gandia, Paloma Manzanares, Sandra Garrigues, Begona Bolos, Jose F Marcos
    Abstract:

    Penicillium digitatum is the main postharvest pathogen of citrus fruit and is responsible for important economic losses in spite of the massive use of fungicides. The fungal cell wall (CW) and its specific component chitin are potential targets for the development of new Antifungal molecules. Among these are the Antifungal peptides and Proteins that specifically interact with fungal CW. Chitin is synthesized by a complex family of chitin synthases (Chs), classified into up to eight classes within three divisions. Previously, we obtained and characterized a mutant of P. digitatum in the class VII gene (ΔchsVII), which contains a short myosin motor-like domain (MMD). In this report, we extend our previous studies to the characterization of mutants in chsII and in the gene coding for the other MMD-Chs (chsV), and study the role of chitin synthases in the sensitivity of P. digitatum to the self-Antifungal protein AfpB, and to AfpA obtained from P. expansum. The ΔchsII mutant showed no significant phenotypic and virulence differences with the wild type strain, except in the production and morphology of the conidia. In contrast, mutants in chsV showed a more dramatic phenotype than the previous ΔchsVII, with reduced growth and conidial production, increased chitin content, changes in mycelial morphology and a decrease in virulence to citrus fruit. Mutants in chsVII were specifically more tolerant than the wild type to nikkomycin Z, an Antifungal inhibitor of chitin biosynthesis. Treatment of P. digitatum with its own Antifungal protein AfpB resulted in an overall reduction in the expression of the chitin synthase genes. The mutants corresponding to MMD chitin synthases exhibited differential sensitivity to the Antifungal Proteins AfpA and AfpB, ΔchsVII being more susceptible than its parental strain and ΔchsV being slightly more tolerant despite its reduced growth in liquid broth. Taking these results together, we conclude that the MMD-containing chitin synthases affect cell wall integrity and sensitivity to Antifungal Proteins in P. digitatum.

Paloma Manzanares - One of the best experts on this subject based on the ideXlab platform.

  • potential of Antifungal Proteins afps to control penicillium postharvest fruit decay
    Journal of Fungi, 2021
    Co-Authors: Monica Gandia, Florentine Marx, László Galgóczy, Anant Kakar, Moises Ginerllorca, Jeanett Holzknecht, Pedro Martinezculebras, Jose F Marcos, Paloma Manzanares
    Abstract:

    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.

  • differential susceptibility of mycotoxin producing fungi to distinct Antifungal Proteins afps
    Food Microbiology, 2021
    Co-Authors: Monica Gandia, Jose F Marcos, P V Martinezculebras, Alicia Boronat, Paloma Manzanares
    Abstract:

    Abstract The global challenge to prevent fungal spoilage and mycotoxin contamination on foods and feeds require the development of new Antifungal strategies. Filamentous fungi encode diverse Antifungal Proteins (AFPs), which offer a great potential for the control of contaminant fungi. In this study, four AFPs from Penicillium digitatum (PdAfpB) and Penicillium expansum (PeAfpA, PeAfpB and PeAfpC) belonging to classes A, B and C, were tested against a representative panel of mycotoxin-producing fungi. They included a total of 38 strains representing 32 different species belonging to the genera Alternaria, Aspergillus, Byssochlamys, Fusarium and Penicillium. PeAfpA exhibited a potent Antifungal activity, since the growth of all tested fungi was completely inhibited by concentrations ranging from 0.5 to 16 μg/mL. PdAfpB and PeAfpB, although less effective than PeAfpA, showed significant activity against most of the mycotoxigenic fungi tested. Importantly, PeAfpC previously described as inactive, showed a powerful inhibition against B. spectabilis strains, which are important spoilage and mycotoxin fungi in pasteurized foods. Although less effective than in liquid media, AFPs affected fungal growth on solid media. This study also underlines the potential of these AFPs, in particular PeAfpA, as future Antifungal agents for applications in foods, on growing crops or during postharvest storage.

  • novel insights in the production activity and protective effect of penicillium expansum Antifungal Proteins
    International Journal of Biological Macromolecules, 2020
    Co-Authors: Monica Gandia, Moises Ginerllorca, Jose F Marcos, Sandra Garrigues, Ana Monge, Helena Orozco, Paloma Manzanares
    Abstract:

    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.

  • the Antifungal protein afpb induces regulated cell death in its parental fungus penicillium digitatum
    mSphere, 2020
    Co-Authors: Adria Bugeda, Monica Gandia, Jose F Marcos, Paloma Manzanares, Sandra Garrigues, Maria Coca
    Abstract:

    ABSTRACT Filamentous fungi produce small cysteine-rich Proteins with potent, specific Antifungal activity, offering the potential to fight fungal infections that severely threaten human health and food safety and security. The genome of the citrus postharvest fungal pathogen Penicillium digitatum encodes one of these Antifungal Proteins, namely AfpB. Biotechnologically produced AfpB inhibited the growth of major pathogenic fungi at minimal concentrations, surprisingly including its parental fungus, and conferred protection to crop plants against fungal infections. This study reports an in-depth characterization of the AfpB mechanism of action, showing that it is a cell-penetrating protein that triggers a regulated cell death program in the target fungus. We prove the importance of AfpB interaction with the fungal cell wall to exert its killing activity, for which protein mannosylation is required. We also show that the potent activity of AfpB correlates with its rapid and efficient uptake by fungal cells through an energy-dependent process. Once internalized, AfpB induces a transcriptional reprogramming signaled by reactive oxygen species that ends in cell death. Our data show that AfpB activates a self-injury program, suggesting that this protein has a biological function in the parental fungus beyond defense against competitors, presumably more related to regulation of the fungal population. Our results demonstrate that this protein is a potent Antifungal that acts through various targets to kill fungal cells through a regulated process, making AfpB a promising compound for the development of novel biofungicides with multiple fields of application in crop and postharvest protection, food preservation, and medical therapies. IMPORTANCE Disease-causing fungi pose a serious threat to human health and food safety and security. The limited number of licensed Antifungals, together with the emergence of pathogenic fungi with multiple resistance to available Antifungals, represents a serious challenge for medicine and agriculture. Therefore, there is an urgent need for new compounds with high fungal specificity and novel Antifungal mechanisms. Antifungal Proteins in general, and AfpB from Penicillium digitatum in particular, are promising molecules for the development of novel Antifungals. This study on AfpB’s mode of action demonstrates its potent, specific fungicidal activity through the interaction with multiple targets, presumably reducing the risk of evolving fungal resistance, and through a regulated cell death process, uncovering this protein as an excellent candidate for a novel biofungicide. The in-depth knowledge on AfpB mechanistic function presented in this work is important to guide its possible future clinical and agricultural applications.

  • the myosin motor domain containing chitin synthases are involved in cell wall integrity and sensitivity to Antifungal Proteins in penicillium digitatum
    Frontiers in Microbiology, 2019
    Co-Authors: Monica Gandia, Paloma Manzanares, Sandra Garrigues, Begona Bolos, Jose F Marcos
    Abstract:

    Penicillium digitatum is the main postharvest pathogen of citrus fruit and is responsible for important economic losses in spite of the massive use of fungicides. The fungal cell wall (CW) and its specific component chitin are potential targets for the development of new Antifungal molecules. Among these are the Antifungal peptides and Proteins that specifically interact with fungal CW. Chitin is synthesized by a complex family of chitin synthases (Chs), classified into up to eight classes within three divisions. Previously, we obtained and characterized a mutant of P. digitatum in the class VII gene (ΔchsVII), which contains a short myosin motor-like domain (MMD). In this report, we extend our previous studies to the characterization of mutants in chsII and in the gene coding for the other MMD-Chs (chsV), and study the role of chitin synthases in the sensitivity of P. digitatum to the self-Antifungal protein AfpB, and to AfpA obtained from P. expansum. The ΔchsII mutant showed no significant phenotypic and virulence differences with the wild type strain, except in the production and morphology of the conidia. In contrast, mutants in chsV showed a more dramatic phenotype than the previous ΔchsVII, with reduced growth and conidial production, increased chitin content, changes in mycelial morphology and a decrease in virulence to citrus fruit. Mutants in chsVII were specifically more tolerant than the wild type to nikkomycin Z, an Antifungal inhibitor of chitin biosynthesis. Treatment of P. digitatum with its own Antifungal protein AfpB resulted in an overall reduction in the expression of the chitin synthase genes. The mutants corresponding to MMD chitin synthases exhibited differential sensitivity to the Antifungal Proteins AfpA and AfpB, ΔchsVII being more susceptible than its parental strain and ΔchsV being slightly more tolerant despite its reduced growth in liquid broth. Taking these results together, we conclude that the MMD-containing chitin synthases affect cell wall integrity and sensitivity to Antifungal Proteins in P. digitatum.

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  • in vivo applicability of neosartorya fischeri Antifungal protein 2 nfap2 in treatment of vulvovaginal candidiasis
    Antimicrobial Agents and Chemotherapy, 2018
    Co-Authors: Renato Kovacs, Györgyi Váradi, Jeanett Holzknecht, Attila Borics, G Toth, Zoltan Hargitai, Csaba Papp, Attila Farkas, Lilana Toth, Ilona Kovacs
    Abstract:

    As a consequence of emerging numbers of vulvovaginitis cases caused by azole-resistant and biofilm-forming Candida species, fast and efficient treatment of this infection has become challenging. The problem is further exacerbated by the severe side effects of azoles as long-term-use medications in the recurrent form. There is therefore an increasing demand for novel and safely applicable effective Antifungal therapeutic strategies. The small, cysteine-rich, and cationic Antifungal Proteins from filamentous ascomycetes are potential candidates, as they inhibit the growth of several Candida spp. in vitro; however, no information is available about their in vivo Antifungal potency against yeasts. In the present study, we investigated the possible therapeutic application of one of their representatives in the treatment of vulvovaginal candidiasis, Neosartorya fischeri Antifungal protein 2 (NFAP2). NFAP2 inhibited the growth of a fluconazole (FLC)-resistant Candida albicans strain isolated from a vulvovaginal infection, and it was effective against both planktonic cells and biofilm in vitro We observed that the fungal cell-killing activity of NFAP2 is connected to its pore-forming ability in the cell membrane. NFAP2 did not exert cytotoxic effects on primary human keratinocytes and dermal fibroblasts at the MIC in vitro. In vivo murine vulvovaginitis model experiments showed that NFAP2 significantly decreases the number of FLC-resistant C. albicans cells, and combined application with FLC enhances the efficacy. These results suggest that NFAP2 provides a feasible base for the development of a fundamental new, safely applicable mono- or polytherapeutic topical agent for the treatment of superficial candidiasis.

  • Efficient production and characterization of the novel and highly active Antifungal protein AfpB from Penicillium digitatum
    Scientific Reports, 2017
    Co-Authors: Sandra Garrigues, Florentine Marx, Monica Gandia, Jose F Marcos, Maria Coca, Attila Borics, Crina Popa, Paloma Manzanares
    Abstract:

    Filamentous fungi encode distinct Antifungal Proteins (AFPs) that offer great potential to develop new Antifungals. Fungi are considered immune to their own AFPs as occurs in Penicillium chrysogenum , the producer of the well-known PAF. The Penicillium digitatum genome encodes only one afp gene ( afpB ), and the corresponding protein (AfpB) belongs to the class B phylogenetic cluster. Previous attempts to detect AfpB were not successful. In this work, immunodetection confirmed the absence of AfpB accumulation in wild type and previous recombinant constitutive P . digitatum strains. Biotechnological production and secretion of AfpB were achieved in P . digitatum with the use of a P . chrysogenum -based expression cassette and in the yeast Pichia pastoris with the α-factor signal peptide. Both strategies allowed proper protein folding, efficient production and single-step purification of AfpB from culture supernatants. AfpB showed Antifungal activity higher than the P . chrysogenum PAF against the majority of the fungi tested, especially against Penicillium species and including P . digitatum , which was highly sensitive to the self-AfpB. Spectroscopic data suggest that native folding is not required for activity. AfpB also showed notable ability to withstand protease and thermal degradation and no haemolytic activity, making AfpB a promising candidate for the control of pathogenic fungi.

  • Structural determinants of Neosartorya fischeri Antifungal protein (NFAP) for folding, stability and Antifungal activity.
    Scientific reports, 2017
    Co-Authors: László Galgóczy, Zoltán Kele, Györgyi Váradi, Attila Borics, Mate Viragh, Hargita Ficze, Florentine Marx
    Abstract:

    The recent global challenges to prevent and treat fungal infections strongly demand for the development of new Antifungal strategies. The structurally very similar cysteine-rich Antifungal Proteins from ascomycetes provide a feasible basis for designing new Antifungal molecules. The main structural elements responsible for folding, stability and Antifungal activity are not fully understood, although this is an essential prerequisite for rational protein design. In this study, we used the Neosartorya fischeri Antifungal protein (NFAP) to investigate the role of the disulphide bridges, the hydrophobic core, and the N-terminal amino acids in the formation of a highly stable, folded, and Antifungal active protein. NFAP and its mutants carrying cysteine deletion (NFAPΔC), hydrophobic core deletion (NFAPΔh), and N-terminal amino acids exchanges (NFAPΔN) were produced in Pichia pastoris. The recombinant NFAP showed the same features in structure, folding, stability and activity as the native protein. The data acquired with mass spectrometry, structural analyses and Antifungal activity assays of NFAP and its mutants proved the importance of the disulphide bonding, the hydrophobic core and the correct N-terminus for folding, stability and full Antifungal function. Our findings provide further support to the comprehensive understanding of the structure-function relationship in members of this protein group.

  • a penicillium chrysogenum based expression system for the production of small cysteine rich Antifungal Proteins for structural and functional analyses
    Microbial Cell Factories, 2016
    Co-Authors: Christoph Sonderegger, Anna Huber, Ádám Fizil, László Galgóczy, Jose F Marcos, Paloma Manzanares, Sandra Garrigues, Attila Borics, Nikoletta Hegedus, Gyula Batta
    Abstract:

    Small, cysteine-rich and cationic Antifungal Proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure–function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous Antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant Proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed Proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant Proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and Antifungal activity of the recombinant Proteins were analysed by ESI–MS, ECD and NMR spectroscopy and growth inhibition assays. This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded Proteins with high quality for structure–function analyses.

  • nfap2 a novel cysteine rich anti yeast protein from neosartorya fischeri nrrl 181 isolation and characterization
    AMB Express, 2016
    Co-Authors: Liliána Tóth, Zoltán Kele, László Galgóczy, Györgyi Váradi, Attila Borics, Laszlo Nagy, Mate Viragh, Miklos Tako, Csaba Vagvolgyi
    Abstract:

    The increasing incidence of fungal infections and damages due to drug-resistant fungi urges the development of new Antifungal strategies. The cysteine-rich Antifungal Proteins from filamentous ascomycetes provide a feasible base for protection against molds due to their potent Antifungal activity on them. In contrast to this, they show no or weak activity on yeasts, hence their applicability against this group of fungi is questionable. In the present study a 5.6 kDa anti-yeast protein (NFAP2) is isolated, identified and characterized from the ferment broth of Neosartorya fischeri NRRL 181. Based on a phylogenetic analysis, NFAP2 and its putative homologs represent a new group of ascomycetous cysteine-rich Antifungal Proteins. NFAP2 proved to be highly effective against tested yeasts involving clinically relevant Candida species. NFAP2 did not cause metabolic inactivity and apoptosis induction, but its plasma membrane disruption ability was observed on Saccharomyces cerevisiae. The Antifungal activity was maintained after high temperature treatment presumably due to the in silico predicted stable tertiary structure. The disulfide bond-stabilized, heat-resistant folded structure of NFAP2 was experimentally proved. After further investigations of Antifungal mechanism, structure and toxicity, NFAP2 could be applicable as a potent Antifungal agent against yeasts.