Mapping and Identification of Antifungal Peptides in the Putative Antifungal Protein AfpB from the Filamentous Fungus Penicillium digitatum.

Florentine Marx,Sandra Garrigues,Attila Borics,Jose F Marcos,Mónica Gandía,Paloma Manzanares

doi:10.3389/fmicb.2017.00592

Abstract

Antifungal proteins (AFPs) from Ascomycetes are small cysteine-rich proteins that are abundantly secreted and show antifungal activity against non-producer fungi. A gene coding for a class B AFP (AfpB) was previously identified in the genome of the plant pathogen Penicillium digitatum. However, previous attempts to detect the AfpB protein were not successful despite the high expression of the corresponding afpB gene. In this work, the structure of the putative AfpB was modeled. Based on this model, four synthetic cysteine-containing peptides, PAF109, PAF112, PAF118, and PAF119, were designed and their antimicrobial activity was tested and characterized. PAF109 that corresponds to the γ-core motif present in defensin-like antimicrobial proteins did not show antimicrobial activity. On the contrary, PAF112 and PAF118, which are cationic peptides derived from two surface-exposed loops in AfpB, showed moderate antifungal activity against P. digitatum and other filamentous fungi. It was also confirmed that cyclization through a disulfide bridge prevented peptide degradation. PAF116, which is a peptide analogous to PAF112 but derived from the Penicillium chrysogenum antifungal protein PAF, showed activity against P. digitatum similar to PAF112, but was less active than the native PAF protein. The two AfpB-derived antifungal peptides PAF112 and PAF118 showed positive synergistic interaction when combined against P. digitatum. Furthermore, the synthetic hexapeptide PAF26 previously described in our laboratory also exhibited synergistic interaction with the peptides PAF112, PAF118, and PAF116, as well as with the PAF protein. This study is an important contribution to the mapping of antifungal motifs within the AfpB and other AFPs, and opens up new strategies for the rational design and application of antifungal peptides and proteins.

Highlights

There is an urgent need to develop new antifungal molecules with properties and mechanisms of action different from existing ones (Brown et al, 2012; Fisher et al, 2012)
To identify putative antimicrobial motifs in AfpB, the cysteinecontaining peptides PAF109, PAF112, PAF118, and PAF119 were derived from the AfpB sequence (Table 1)
Most of the studies based on peptides derived from antifungal cysteine-rich proteins (CRPs) have focused on defensins and the highly conserved γ-motif (Sagaram et al, 2011; Avitabile et al, 2013; van der Weerden and Anderson, 2013; Muñoz et al, 2014; Kaewklom et al, 2016), but not on fungal antifungal proteins (AFPs)

Summary

Introduction

There is an urgent need to develop new antifungal molecules with properties and mechanisms of action different from existing ones (Brown et al, 2012; Fisher et al, 2012). Antimicrobial peptides and proteins (AMPs) have been found in a broad variety of species (Hancock and Sahl, 2006) and are candidates for the development of novel therapeutic compounds (Zasloff, 2002; Brogden, 2005; Fjell et al, 2012) including antifungals (Shah and Read, 2013). AFPs belong to the broad class of defensins and are produced by some species of filamentous Ascomycetes, mostly from the genus Aspergillus and Penicillium (Marx et al, 2008; Meyer, 2008; Galgóczy et al, 2010; Silva et al, 2014). One of the most studied AFPs is the Penicillium chrysogenum PAF protein (55 amino acids in length) that can be purified from the culture supernatants of the producer fungus P. chrysogenum (Marx et al, 2008). PAF inhibits the growth of filamentous human and plant pathogenic fungi at μM concentrations and is non-toxic to mammalian cells in vitro (Marx et al, 2008) and in vivo (Palicz et al, 2013)

Methods

Results

Conclusion