The interaction with fungal cell wall polysaccharides determines the salt tolerance of antifungal plant defensins

Abstract

The fungal cell wall is the first point of contact between fungal pathogens and host organisms. It serves as a protective barrier against biotic and abiotic stresses and as a signal to the host that a fungal pathogen is present. The fungal cell wall is made predominantly of carbohydrates and glycoproteins, many of which serve as binding receptors for host defence molecules or activate host immune responses through interactions with membrane-bound receptors. Plant defensins are a large family of cationic antifungal peptides that protect plants against fungal disease. Binding of the plant defensin NaD1 to the fungal cell wall has been described but the specific component of the cell wall with which this interaction occurred was unknown. The effect of binding was also unclear, that is whether the plant defensin used fungal cell wall components as a recognition motif for the plant to identify potential pathogens or if the cell wall acted to protect the fungus against the defensin. Here we describe the interaction between the fungal cell wall polysaccharides chitin and β-glucan with NaD1 and other plant defensins. We discovered that the β-glucan layer protects the fungus against plant defensins and the loss of activity experienced by many cationic antifungal peptides at elevated salt concentrations is due to sequestration by fungal cell wall polysaccharides. This has limited the development of cationic antifungal peptides for the treatment of systemic fungal diseases in humans as the level of salt in serum is enough to inactivate most cationic peptides.

Membrane-Interacting Antifungal Peptides

“…The initial association of cationic AMPs with anionic bilayers is mostly directed by electrostatic interactions due to their net charge being opposed to that of the target membrane (von Deuster and Knecht, 2011;Alvares et al, 2017). Therefore, the presence of cations can significantly reduce the antifungal activity of AMPs, as demonstrated in case of e.g., AFP, coprisin (dung beetle), DmAMP1, HNP-1, HsAFP1, indolicidin (cattle), NaD1, OefDef1.1, P113 (a 12-amino-acid fragment of histatin 5), RsAFP2, and hBD-1,-2 (Figures 2C-E; Osborn et al, 1995;Goldman et al, 1997;Bals et al, 1998;Edgerton et al, 2000;Lee et al, 2003Lee et al, , 2012Lee et al, , 2014Theis et al, 2003;Bleackley et al, 2019;Li et al, 2019;Cheng et al, 2020). Similarly, in yeast mutants lacking Agp2p, a plasma membrane regulator of polyamine and carnitine transport, the antifungal activity of NaD1 is significantly reduced.…”

“…Recently, the interaction of both chitin and β-glucan with the tobacco plant defensin NaD1 was described (Figures 1, 2C). Herein, the yeast cell wall protects target cells against the antifungal activity of NaD1, as the killing of spheroplasts occurred at lower concentrations as compared to cells with intact walls (Bleackley et al, 2019). Note that AMPs can have multiple fungal targets, as NaD1 also interacts with PA and PIP 2 (Figure 2C) (cfr.…”

“…Deletion of AGP2 was suggested to result in an accumulation of cationic molecules at the cell surface, thereby repelling cationic AMPs (Bleackley et al, 2014). Since the hydrolysis of the cell wall by zymolyase treatment restored the activity of NaD1 in high salt conditions (100 mM NaCl), the loss of antifungal activity at elevated salt concentrations was recently attributed to the sequestration of cations by fungal cell wall polysaccharides (Bleackley et al, 2019). Note that Bleackley et al (2019) only evaluated the effect of monovalent cations, being Na + , thereby reporting the plant defensin DmAMP1 to be salt tolerant.…”

“…Since the hydrolysis of the cell wall by zymolyase treatment restored the activity of NaD1 in high salt conditions (100 mM NaCl), the loss of antifungal activity at elevated salt concentrations was recently attributed to the sequestration of cations by fungal cell wall polysaccharides (Bleackley et al, 2019). Note that Bleackley et al (2019) only evaluated the effect of monovalent cations, being Na + , thereby reporting the plant defensin DmAMP1 to be salt tolerant. In contrast, Osborn et al (1995) earlier reported that the antifungal activity of DmAMP1 against both N. crassa and Fusarium culmorum is significantly reduced in the presence of both 1 and 5 mM Ca 2+ or Mg 2+ .…”

Antifungal Peptides and Proteins to Control Toxigenic Fungi and Mycotoxin Biosynthesis

“…The activity of antifungal AMPs could be compromised of different characteristics of food matrices such as high concentration of salts [37,128]. Nevertheless, several natural antifungal AMPs are active in the presence of high salt concentrations and divalent cations [74,222]. Moreover, the efficacy of antifungal AMPs can be improved by means of structure stabilization, peptide concatemerization, and/or generation of peptide hybrid fusions [37,74].…”