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

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.

Increased chitin biosynthesis contributes to the resistance of Penicillium polonicum against the antifungal protein PgAFP

“…Gels obtained from 3 biological replicates and 2 technical replicates per treatment were stained and analyzed using Progenesis™ SameSpot software (TotalLab, Newcastle, UK) as previously described Collins et al 2013;Owens et al 2014). Spot intensities were normalized in Progenesis SameSpots software (Delgado et al 2015b). Protein spots showing differences (p < 0.05, fold change ≥1.5) were excised, destained, and in-gel trypsin digested (Shevchenko et al 2007).…”

“…Mycelial lysates were centrifuged to remove cell debris and the subsequent supernatant precipitated with TCA/acetone (Carpentier et al 2005). The following two proteomic analyses were carried out from these precipitated lysates, similar to the procedure described by Delgado et al (2015b).…”

“…Mechanisms of action of antifungal proteins from molds have been described as multifactorial, where membrane permeabilization, changes in actin distribution, chitin biosynthesis inhibition, destabilization of cell wall, Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-7020-4) contains supplementary material, which is available to authorized users. and oxidative stress lead to apoptosis (Leiter et al 2005;Moreno et al 2006;Hagen et al 2007;Binder et al 2010;Virágh et al 2015;Delgado et al 2015b). AFP binds chitin, inhibits chitin biosynthesis, permeabilizes the cell membrane, and penetrates into the cell and binds nucleic acids (Liu et al 2002;Moreno et al 2006;Hagen et al 2007), and AcAFP also binds chitin altering cell wall (Skouri-Gargouri et al 2009), whereas PAF, NFAP, and PgAFP lead to apoptosis mediated by G-protein signaling (Binder et al 2010(Binder et al , 2015Virágh et al 2015;Delgado et al 2015b).…”

“…On the other hand, comparative quantitative proteomics is able to identify proteins not detectable by 2D-PAGE. These two techniques have been used to complementarily evaluate the effect of PgAFP on the proteins involved in signaling pathways and selecting adequate tests to study the metabolic response in molds (Delgado et al 2015b). In addition, localization of the antifungal protein in non-sensitive molds can give valuable information on the possible interaction at the surface or inside the cell.…”

“…and oxidative stress lead to apoptosis (Leiter et al 2005;Moreno et al 2006;Hagen et al 2007;Binder et al 2010;Virágh et al 2015;Delgado et al 2015b). AFP binds chitin, inhibits chitin biosynthesis, permeabilizes the cell membrane, and penetrates into the cell and binds nucleic acids (Liu et al 2002;Moreno et al 2006;Hagen et al 2007), and AcAFP also binds chitin altering cell wall (Skouri-Gargouri et al 2009), whereas PAF, NFAP, and PgAFP lead to apoptosis mediated by G-protein signaling (Binder et al 2010(Binder et al , 2015Virágh et al 2015;Delgado et al 2015b). Both PAF and NFAP activate the cAMP/protein kinase A pathway via G-protein signaling (Leiter et al 2005;Virágh et al 2015) and PgAFP provoked a lower amount G-protein subunit β CpcB (Delgado et al 2015b).…”