In this study we provide important insights into the mechanistic basis of AFPNN5353, a AFP homologous protein.
Species specificity tests revealed that AFPNN5353 is active against a broad range of filamentous fungi, including human and plant pathogens. Although the proteins AFPNN5353 and AFP are almost identical and show a similar toxicity, MICs for AFPNN5353 differed slightly from those reported for AFP . We attribute this discrepancy to differences in the experimental setups, e.g. fungal strains, medium composition, conidial inoculum, incubation times, cultivation temperature etc., rather than to the differences in the primary sequence of both proteins.
It has been reported that the closely related AFP protein interfered with cell wall synthesis  and our finding that the osmotic stabilizer sorbitol neutralized AFPNN5353 toxicity further corroborated this assumption. Two A. nidulans mutants, the conditional alcA-PkcA and the mpkA deletion mutant showed a hypersensitive phenotype when exposed to AFPNN5353. This is in agreement to the reported function of cell wall stressing agents, such as CFW or caffeine in S. cerevisiae and A. nidulans [[9, 16, 24, 26, 38, 39]] and to the Penicillium antifungal protein PAF . Importantly, Mpk function is essential for CWIP activation in both, unicellular and filamentous fungi [[10, 16, 40]] and triggers the activation of the transcription factors Rlm1p and SBF which regulate the expression of cell cycle regulated genes and genes involved in the synthesis and remodelling of the fungal cell wall in S. cerevisiae [41, 42]. Similarly, RlmA dependent induction of the expression of the ags gene was also reported for aspergilli . Importantly, the activation of the CWIP can occur in a RhoA-dependent, e.g. with CFW [9, 43], or RhoA-independent way, the latter proved for PAF and caffeine [9, 16] and for AFPNN5353 (this study). As proposed by  the dominant rhoA
allele suffers from a perturbation of its GAP binding domain and downstream effectors of Rho-GAP might be disturbed. Therefore, we hypothesize that Rho-GAP targets might be involved in the toxicity of AFPNN5353 similarly to the mode of action of the P. chrysogenum PAF . Our assumption of the activation of the CWIP by AFPNN5353 was further strengthened by the fact, that AFPNN5353 treatment induced agsA expression in the A. niger reporter strain. This result was consistent with the activity of AFP and caspofungin , but differed to the function of PAF, where no CWIP activation and no induction of cell wall biosynthesis genes occurred .
Therefore, we conclude that AFPNN5353 triggers cell wall remodeling via Pkc/Mpk signalling. We further deduce from our data that similarities and differences exist in the molecular targets and the mode of action of antifungal proteins from filamentous fungi, e.g. AFPNN5353 and PAF - despite their homology. This phenomenon was also reported for other closely related antifungal proteins, such as the plant defensins MsDef1 and MtDef4 from Medicago spp. .
Apart from the activation of the CWIP, the perturbation of the Ca2+ homeostasis represents a major mechanistic function of antifungal proteins in sensitive fungi [17, 18]. The intracellular Ca2+ response to AFPNN5353 in A. niger reflected that of the Penicillium antifungal protein PAF in N. crassa . The rapid and sustained increase of the [Ca2+]c resting level depended on a sustained influx of Ca2+ ions from the external medium. Moreover, the AFPNN5353 induced changes in the Ca2+ signature of mechanically perturbed A. niger cells further underlines the disruption of the Ca2+ response and homeostasis by AFPNN5353. The addition of CaCl2 to the growth medium reduced the susceptibility of A. niger towards the antifungal protein and decreased the AFPNN5353 specific rise in the [Ca2+]c resting level. Both observations point towards an adaptive response which is mediated most probably via Ca2+ signalling. First, high extracellular Ca2+ concentrations trigger chitin synthesis in A. niger and thereby confer increased protection against antifungal proteins as shown for AFP . Second, it primes the Ca2+ homeostatic machinery to better maintain a low [Ca2+]c resting level when challenged with the antifungal protein, e.g. by (i) the increase of the activity of existing Ca2+ pumps/transporters to counteract the AFPNN5353-specific intracellular Ca2+ perturbation, or (ii) the modulation of the expression of Ca2+ channels/pumps/exchangers . The former hypothesis (i) might be supported by the observation that the addition of CaCl2 only 10 min before A. niger was challenged with AFPNN5353 restored the low [Ca2+]c resting level. However, the perturbation of the Ca2+ homeostasis by a sustained elevation of the [Ca2+]c resting level indicates that A. niger is not able to restore the low [Ca2+]c resting level after exposure to AFPNN5353 and this might trigger programmed cell death (PCD) on the long term as it was shown to occur in A. nidulans in response to the P. chrysogenum PAF .
Since AFP was shown to cause membrane permeabilization , the influx of Ca2+ might be due to changes in membrane permeability for this ion, if not the formation of pores. However, our staining experiments with CMFDA and PI exclude this possibility at least in the first 10 min of exposure to AFPNN5353 when the [Ca2+]c resting level reaches its maximum. This result is further corroborated by the fact that higher external concentrations of Ca2+ reduced the AFPNN5353 specific rise in [Ca2+]c resting level which - in our opinion - would not occur with leaky membranes. However, we do not exclude changes in membrane permeability at longer exposure times to this antifungal protein and more studies are needed to answer this question.
Finally, we observed that the internalization of AFPNN5353 is characteristic for sensitive but not resistant moulds. A lack of binding of AFPNN5353 to insensitive fungi might point towards the absence or inaccessibility of a putative interacting molecule at the cell surface. AFPNN5353 localized to the cytoplasm of target fungi only when actin filaments were formed. This is in agreement with the endocytotic uptake and intracellular localization of the P. chrysogenum antifungal protein PAF in sensitive filamentous fungi [14, 45]. Importantly, we observed that AFPNN5353 was internalized by hyphae even under sub-inhibitory concentrations (0.2 μg/ml for A. nidulans) which suggests that a threshold concentration is required to cause severe growth defects in target fungi.
The presence of high concentrations of extracellular Ca2+ counteracted AFPNN5353 uptake. This finding parallels well with the report of  that the presence of cations, such as Ca2+, interfered with the binding of AFP to the surface of F. oxysporum and with our observations made with the Penicillium PAF (unpublished data). One possible explanation might be that extracellular Ca2+ ions compete with AFPNN5353 for the same molecular target on the fungal surface which might represent a first binding receptor or even a "gate" for protein uptake [20, 21] or, alternatively, that the interacting target is repressed under these conditions . An additional explanation might be that the primary cell-surface localized AFPNN5353 target might be masked due to a Ca2+-dependent stimulation of chitin synthesis and cell wall remodeling as recently observed for AFP in A. niger . This further suggests that the activation of the CWIP and the agsA induction does not mediate sufficient resistance to survive the toxic effects of AFPNN5353. Instead, according to the "damage-response framework of AFP-fungal interactions" , the chitin response might represent the better strategy for fungi to survive the antifungal attack.