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

Abstract

Antifungal proteins from molds have been proposed as a valuable tool against unwanted molds, but the resistance of some fungi limits their use. Resistance to antimicrobial peptides has been suggested to be due to lack of interaction with the mold or to a successful response. The antifungal protein PgAFP produced by Penicillium chrysogenum inhibits the growth of various ascomycetes, but not Penicillium polonicum. To study the basis for resistance to this antifungal protein, localization of PgAFP and metabolic, structural, and morphological changes were investigated in P. polonicum. PgAFP bound the outer layer of P. polonicum but not regenerated chitin, suggesting an interaction with specific molecules. Comparative two-dimensional gel electrophoresis (2D-PAGE) and comparative quantitative proteomics revealed changes in the relative abundance of several proteins from ribosome, spliceosome, metabolic, and biosynthesis of secondary metabolite pathways. The proteome changes and an altered permeability reveal an active reaction of P. polonicum to PgAFP. The successful response of the resistant mold seems to be based on the higher abundance of protein Rho GTPase Rho1 that would lead to the increased chitin deposition via cell wall integrity (CWI) signaling pathway. Thus, combined treatment with chitinases could provide a complementary means to combat resistance to antifungal proteins.

Effect of potassium sorbate (E‐202) and the antifungal PgAFP protein on Aspergillus carbonarius growth and ochratoxin A production in raisin simulating media

“…59 Reductions and increments in the level of this protein were linked to the sensitivity and resistance of A. flavus and Penicillium polonicum, respectively. 59,60 In general, it appears that PgAFP represses the Rho1 gene expression at the end of the linear phase (first sampling day), except for the strain A171 at 0.95 a w . It means that PgAFP may use this mechanism to control OTA synthesis on sensitive A. carbonarius.…”

“…Finally, the expression of the Rho1 gene was also evaluated because the Rho protein had been demonstrated to be involved in the PgAFP mechanism of action in A. flavus . Reductions and increments in the level of this protein were linked to the sensitivity and resistance of A. flavus and Penicillium polonicum , respectively . In general, it appears that PgAFP represses the Rho1 gene expression at the end of the linear phase (first sampling day), except for the strain A171 at 0.95 a w .…”

Manuscript title: antifungal proteins from moulds: analytical tools and potential application to dry-ripened foods

“…Combining subcellular fractionation with comparative proteomics extends the limits of detection (Komatsu et al 2011;Cagas et al 2011;Moloney et al 2016), while including genomics or alternative proteomic analyses (e.g. gel-free methods) serves to increase sensitivity and validate results (Gautam et al 2008;Cagas et al 2011;Gautam et al 2011;Ene et al 2012;Delgado et al 2015bDelgado et al , 2016.…”

“…Advances in mass spectrometry (MS)-based proteomic methods have enabled in-depth analysis of the protein levels altered in response to an antifungal challenge. Label-free methods have been utilized to profile the AFPs response within both resistant and sensitive fungi, enabling the identification of resistance mechanisms as well as susceptible targets (Delgado et al 2015b;Prado et al 2015;Delgado et al 2016).…”

“…Stress signals caused by echinocandins can also be transmitted in S. cerevisiae from the cell surface integrity sensor Wsc1 to the protein kinase C (PKC) cell integrity signalling pathway, which is controlled by Rho1 protein (Reinoso- Martín et al 2003;Levin 2005). Rho1 binds and activates Pkc, which through Mpk signalling, activates genes involved in cell wall synthesis, resulting in an elevated chitin content (Munro et al 2007;Delgado et al 2016).…”