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Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus

  • Applied microbial and cell physiology
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Abstract

Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a β subunit of G protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G protein subunit β CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study.

Abstract

Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G -protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a β subunit of G -protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G -protein subunit β CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study.

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Acknowledgments

This work was supported by the Spanish Ministry of Education and Science, Ministry of Economy and Competitiveness, and FEDER (AGL2010-21623, AGL2013-45729-P). Josué Delgado was a recipient of a FPI grant from the Spanish Ministry of Education and Science (BES-2011–043422 y EEBB-I-13–06900). Rebecca A. Owens was funded by a 3U Partnership Award (http://www.3UPartnership.ie/). Mass spectrometry facilities were funded by Science Foundation Ireland (Q-Exactive; 12/RI/2346(3) and PI/11/1188) and the Irish Higher Education Authority (Agilent Ion Trap 6340).

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Authors and Affiliations

  1. Food Hygiene and Safety, Institute of Meat Products, University of Extremadura, Cáceres, Spain

    Josué Delgado, Miguel A. Asensio & Félix Núñez

  2. Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland

    Rebecca A. Owens & Sean Doyle

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  1. Josué Delgado
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  2. Rebecca A. Owens
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  3. Sean Doyle
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Correspondence to Miguel A. Asensio.

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Delgado, J., Owens, R.A., Doyle, S. et al. Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus . Appl Microbiol Biotechnol 99, 8701–8715 (2015). https://doi.org/10.1007/s00253-015-6731-x

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