Antifungal Peptides of the AFP Family Revisited: Are These Cannibal Toxins?

Abstract

The emergence and spread of pathogenic fungi resistant to currently used antifungal drugs represents a serious challenge for medicine and agriculture. The use of smart antimicrobials, so-called "dirty drugs" which affect multiple cellular targets, is one strategy to prevent resistance. Of special interest is the exploitation of the AFP family of antimicrobial peptides, which include its founding member AFP from Aspergillus giganteus. This latter is a highly potent inhibitor of chitin synthesis and affects plasma membrane integrity in many human and plant pathogenic fungi. A transcriptomic meta-analysis of the afp-encoding genes in A. giganteus and A. niger predicts a role for these proteins during asexual sporulation, autophagy, and nutrient recycling, suggesting that AFPs are molecules important for the survival of A. niger and A. giganteus under nutrient limitation. In this review, we discuss parallels which exist between AFPs and bacterial cannibal toxins and provide arguments that the primary function of AFPs could be to kill genetically identical siblings. We hope that this review inspires computational and experimental biologists studying alternative explanations for the nature and function of antimicrobial peptides beyond the general assumption that they are mere defense molecules to fight competitors.

Keywords: AFP; AnAFP; Aspergillus giganteus; Aspergillus niger; Bacillus; antifungal; antimicrobial peptide; cannibal toxin; mode of action; sporulation.

Figure 1

Electrostatic surface potentials of AFP, PAF, and AnAFP. AFP and PAF were derived from PDB accession codes 1AFP or 1KCN, respectively, whereas the structure of AnAFP was generated by molecular modeling using the structure of AFP as the template. Negatively charged regions are colored red, positively charged ones blue, and uncharged ones white. Graphical representations displaying top, side, and bottom views of the peptides were generated using the program GRASP2 [11]. An alignment of AFP, PAF, and AnAFP is given below. The box depicts residues of the γ-core motif. Arrows on top represent beta-strands.

Figure 2

Expression of genes encoding antifungal peptides in aspergilli are under tight time-dependent control: (A) Oscillating expression of the afp gene as visualized in a 6-day old colony of an A. giganteus reporter strain. Here, the reporter gene β-glucuronidase (uidA) was put under control of the afp promoter. Induction of Pafp::uidA reporter expression results in blue color formation on agar plates in a circadian manner (indicated by arrows). Blue color formation is visible only in the vegetative medium and occurs when A. giganteus vegetative hyphae achieve the competence to form aerial hyphae/conidiophores. Picture is reproduced from [17] with permission from Springer Nature. (B) Luciferase expression under control of the anafp promoter was measured using the reporter strain PK2.9 (Panafp::luc). Reporter activity was measured as luminescent counts per second normalized to culture optical density during 4 days of submerged cultivation of strain PK2.9 in microtiter plates. Picture is taken from [10], licensed under CC-BY 4.0. LCPS, luminescent counts per second.

Figure 3

Morphological differentiation of A. niger during substrate-limited growth in retentostat cultures as visualized by DIC (differential interference contrast) microscopy (upper panel) and fluorescence microscopy (bottom panel). Mycelium of an Panafp::eyfp reporter strain after 1 day (μ < 0.1 h⁻¹), 2 days (μ ~ 0.01 h⁻¹), and 6 days (μ ~ 0.005 h⁻¹). Fluorescence represents the activated anafp promoter and is only visible in individual compartments. Note that after day 6, newly formed spores become visible (arrows). Picture is taken from [10], licensed under CC-BY 4.0. μ: growth rate. Bar = 20 µm.