BACKGROUND
all organisms have evolved several defence systems in order to protect themselves against bacteria, fungi and viruses.
higher organisms have developed a complex network of humoral and cellular responses, called adaptive immunity.
a second defence system, the innate immunity, consists of many components, including small peptides with a broad antimicrobial spectrum  <cit> .
the production of such proteins with antimicrobial activity is not limited to higher eukaryotes, but also found in microorganisms, including fungi.
the diversity of these proteins is reflected in their mode of action and their species-specificity.
some of them form pores in the membrane, others are known to inhibit cell wall synthesis or interfere with nucleic acids and their synthesis  <cit> .
they can be involved in the inhibition of protein synthesis or interfere with cell cycle control  <cit> .
a relatively new group of antimicrobial proteins secreted by filamentous ascomycetes includes small, cationic and cysteine-rich proteins.
so far, only few antifungal proteins have been characterized, namely afp from aspergillus giganteus, anafp from aspergillus niger, paf from penicillium chrysogenum and naf from penicillium nalgiovense  <cit> .
the mode of action of these proteins is not fully understood.
nevertheless, there is evidence, that their toxicity is mediated by interaction with distinct molecules or receptors at the outer layers of the cell, e.g.
cell wall or plasma membrane.
deleterious effects can then be induced either by transmitting signals from the outer layers into the cell, or by internalization of the protein and interaction with internal molecules  <cit> .
similar to substances that perturb the cell wall, such as caspofungin, congo red or calcofluor white   <cit> , the a. giganteus antifungal protein afp was found to modulate the cell wall composition by enhancing the expression of the α- <dig> -glucan synthase a gene , possibly by the activation of the cell wall integrity pathway , and inhibiting chitin synthesis in sensitive fungi  <cit> .
this, however, stands in contrast to the mode of action of the p. chrysogenum antifungal protein paf which fails to activate the cwip  <cit> .
however, the central players that trigger cell wall remodelling in afp-sensitive fungi have not been investigated so far.
another mechanistic function of antifungal proteins is the interference with ion, especially ca2+ ion homeostasis and signalling  <cit> .
we could recently show that the p. chrysogenum antifungal protein paf severely perturbed the ca2+ homeostasis of neurospora crassa by rapidly elevating the cytoplasmic ca2+ c resting level  <cit> .
numerous reports indicate that the activity of antifungal proteins can be antagonized by the external addition of ca2+ ions to the test medium  <cit>  pointing towards the induction of adaptive responses which may be triggered by ca2+ signalling  <cit> .
the aim of this study was to characterize in more detail the mode of action of the a. giganteus afp variant protein afpnn <dig> and to investigate the pathways that might be affected/modulated by this antifungal protein.
therefore, we focussed our interest on the involvement of the cwip and the ca2+ signalling in the toxicity of afpnn <dig>  to address these questions, we used the highly afpnn <dig> sensitive model organisms a. nidulans and a. niger for which appropriate mutant strains were available.
RESULTS
in silico analysis of afpnn5353
clustalw amino acid  sequence analysis of afpnn <dig> with other known antifungal proteins revealed that afpnn <dig> from a. giganteus strain a <dig> is a protein homologous to afp from a. giganteus strain mdh  <dig>  <cit> .
afpnn <dig> exhibits > 90% identity with afp, but only 42% identity with the p. chrysogenum paf and 27% identity with the a. niger anafp.
in fact, the secreted mature form of afpnn <dig> consists of  <dig> aa and differs only in  <dig> aa from afp .
three aa exchanges belong to structurally related aa, one aa exhibits weak similarity and one aa is different .
these aa exchanges do not influence the theoretical isoelectric point  of afpnn <dig>  which is the same as for afp .
most importantly, afpnn <dig> still contains the putative chitin-binding domain ckykaq present in afp but not in paf or anafp and also harbors all conserved cysteine residues important for protein stabilization  <cit> .
antifungal activity of the protein afpnn5353
to investigate the antifungal specificity of afpnn <dig>  fifteen filamentous fungi were tested for their susceptibility to the protein.
since antifungal proteins might be useful for biotechnological applications, filamentous human and plant pathogenic fungi were selected as test organisms  in addition to the model organisms a. nidulans and a. niger.
as shown in table  <dig>  thirteen out of fifteen tested moulds were found to be sensitive against afpnn <dig>  a. nidulans wild type, n. crassa wild type and a. niger wild type were the most sensitive strains to afpnn <dig>  the minimal inhibitory concentration  of afp  was  <dig>  μg/ml for a. nidulans,  <dig>  μg/ml for n. crassa and  <dig> μg/ml for a. niger.
two strains were unaffected at the protein concentrations tested: m. circenelloides and m. genevensis were insensitive against afpnn <dig> when concentrations up to  <dig> μg/ml were used.
aup to  <dig> μg/ml afpnn <dig> was tested
<dig> ×  <dig> conidia/ml were incubated in  <dig> μl cm medium in the presence of various concentrations of afpnn <dig> at 30°c for  <dig> h. growth was determined by measuring the od <dig> nm.
afpnn <dig> interferes with the cell wall integrity of a. nidulans
it is known that antifungal compounds such as congo red, caffeine, cfw or caspofungin interfere with cell wall biosynthesis and weaken the cell wall in fungi .
the remodeling of the cell wall by these antifungal compounds is mediated by the activation of the cwip.
in fungi, extracellular signals are transmitted via the membrane bound small gtpase rhoa to the central regulators pkc and mpk, which are regulated by phosphorylation/dephosphorylation.
the signal transduction cascade eventually enforces transcription of cell wall synthesis genes, partly via the transcription factor rlma  <cit> .
respective loss-of-function or conditional mutants show hypersensitive phenotypes in the presence of cell wall perturbing agents  <cit> .
similar to substances that weaken the cell wall, the a. giganteus antifungal protein afp modulates the cell wall composition by inhibiting chitin synthesis in sensitive fungi  and inducing the expression of agsa most likely by the activation of the cwip  <cit> .
to study the involvement of the cwip in afpnn <dig> toxicity, we first tested whether the osmotic stabilizer sorbitol counteracts the toxicity of afpnn <dig>  in the absence of afpnn <dig> a. nidulans proliferated less well in the presence of  <dig> m sorbitol and reached only 30% growth compared to the growth in standard medium .
nevertheless, the addition of  <dig> m sorbitol to the growth medium strongly reduced the activity of afpnn <dig> on a. nidulans wild type.
the osmotic stabilizer ameliorated growth in the presence of  <dig>  μg/ml afpnn <dig> by 80% compared to a 10% growth rate in the absence of sorbitol .
this was even more accentuated when  <dig>  and  <dig>  μg/ml afpnn <dig> were applied, suggesting that afpnn <dig> indeed weakens the cell wall of a. nidulans.
<dig> ×  <dig> conidia/ml were incubated in cm with 0- <dig>  μg/ml afpnn <dig> for  <dig> h. percent values were calculated from percent changes in od <dig> of afpnn <dig> treated a. nidulans compared to untreated controls .
results are expressed as mean ± sd .
to investigate whether afpnn <dig> induces agsa gene transcription similar to afp via the pkc/mpk signalling pathway, we tested the effect of the antifungal protein on the transgenic a. niger strain rd <dig>  which expresses a nuclear-targeted gfp protein fused to the a. niger agsa promoter.
rd <dig>  germlings were treated with afpnn <dig>  for  <dig> h and analyzed microscopically.
as shown in additional file  <dig>  a nuclear signal was clearly detectable in germlings of rd <dig>  treated with ≥  <dig> μg/ml afpnn <dig>  similar to that when exposed to  <dig> μg/ml caspofungin.
in untreated germlings, however, no signal could be observed.
these observations perfectly match with the data obtained for afp  <cit> .
it has to be noted here that antifungal protein concentrations higher than the mic determined for conidia  are needed to inhibit the growth of germlings or hyphae of sensitive fungi  <cit>  .
next, we tested several a. nidulans mutant strains affected in central players of the cwip for their susceptibility to afpnn <dig> by determining their radial growth in the presence or absence of the antifungal protein.
since rhoa is an essential protein in a. nidulans, two strains with ectopic copies of the constitutively active rhoag14v allele and the dominant rhoae40i allele  <cit>  were tested in comparison to the wild type strain .
the rhoag14v mutation prevents the hydrolysis of gtp and therefore renders rhoa constantly active  <cit> .
similarly, the gtp hydrolysis is inhibited in the rhoae40i strain, but this mutation also perturbs the binding of the gtpase activating protein  to rhoa and possibly disturbs downstream effectors of rhoa-gap  <cit> .
the constitutively active rhoag14v and the dominant rhoae40i strain exhibited the same sensitivity towards afpnn <dig> as the wild type strain at low protein concentrations  .
interestingly, the dominant rhoae40i strain was more resistant to afpnn <dig> than the wild type strain or the rhoag14v strain at higher protein concentrations  .
therefore, we suggest that the toxicity of afpnn <dig> is transmitted by rhoa-gap targets and not by rhoa itself.
these mutants performed similarly when exposed to the orthologous p. chrysogenum antifungal protein paf  <cit> .
in addition, mutants defective in pkca and mpka activity were tested for their afpnn <dig> susceptibility.
as pkca is an essential gene in a. nidulans, a conditional alca-pkc mutant strain was used, where the pkca gene was put under the control of the alca promoter, which is repressed by glucose but derepressed by glycerol  <cit> .
both the conditional alca-pkc mutant  and a Δmpka mutant were hypersensitive to afpnn <dig> compared to their recipient strains r <dig> and gr <dig>  respectively, indicating that the activity of pkca and mpka confers a certain resistance to afpnn <dig> .
the hypersensitive phenotype of the Δmpka mutant was also confirmed by liquid growth inhibitory assays.
in unchallenged liquid condition, the gr <dig> and the Δmpka mutant showed a comparable proliferation rate .
in the presence of  <dig>  μg/ml afpnn <dig>  however, the mpka deletion strain did not germinate whereas the gr <dig> strain still exhibited 11% growth.
note that growth inhibition in liquid conditions requires less antifungal protein to monitor its toxicity than on solid media probably due to less diffusion in the latter case .
from these data we conclude that afpnn <dig> interferes with the cell wall homeostasis of a. nidulans and that this interaction is mediated by pkca/mpka signalling, although independently from rhoa.
afpnn <dig> disrupts calcium homeostasis in a. niger
supplements other than osmotic stabilizers can also antagonize the activity of antifungal proteins from plants and ascomycetes.
for example, the addition of cations such as ca2+ ions to the growth medium reversed the antifungal activity of the p. chrysogenum paf  <cit> , the a. giganteus afp  <cit>  and of plant defensins  <cit>  which are usually positively charged due to their high pi.
a cation-sensitive antifungal mode of action can for example be associated with the perturbation of the intracellular ca2+ homeostasis by antifungal peptides  <cit>  but might also result from the interference of cations with antifungal-target interaction.
therefore, we tested to which extend these effects also account for the antifungal activity of afpnn <dig>  to this end, we selected a. niger as model organism because this mould was highly sensitive to afpnn <dig> and a transgenic strain was available that expressed the recombinant codon optimized ca2+-sensitive photoprotein aequorin for measuring the c resting level in response to afpnn <dig>  <cit> .
first, we tested the activity of afpnn <dig> in vogels* medium supplemented with 5- <dig> mm cacl <dig> or without cacl <dig> as a control .
addition of cacl <dig> did not influence the growth of a. niger up to a concentration of  <dig> mm.
the growth of a. niger exposed to afpnn <dig>  however, ameliorated in the presence of increasing concentrations of cacl <dig>   <dig> mm cacl <dig> neutralized the toxicity of  <dig> - <dig>  μg/ml afpnn <dig> and the treated samples resumed growth to 100% .
od <dig> was measured after  <dig> h of incubation.
the growth of untreated controls was normalized to 100% to evaluate the percent growth of samples in the presence of afpnn <dig>  vogels* medium without cacl <dig> supplementation contains  <dig>  mm ca2+.
results are expressed as mean ± sd .
next, we determined the influence of afpnn <dig> on the intracellular ca2+ signature.
before afpnn <dig> addition, the resting level of the intracellular ca2+ was  <dig>  μm.
we could show, however, that the c resting level was significantly increased in twelve h old a. niger cultures that were treated with  <dig> μg/ml afpnn <dig>  the c resting level rose to a maximum of  <dig>  μm within the first  <dig> min and stayed elevated throughout the time of measurement , whereas the ca2+ level of the untreated control remained at  <dig>  μm .
this indicated that afpnn <dig> indeed disrupts ca2+ homeostasis in a. niger.
to exclude the possibility that the afpnn <dig> induced rise in the c resting level is due to membrane permeabilization and/or pore formation, we studied the effects of afpnn <dig> on germlings in the presence of cmfda, a membrane permeant dye that is metabolized by viable cells, and the membrane impermeant dye propidium iodide .
additional file  <dig> shows that samples treated with  <dig> μg/ml afpnn <dig> for  <dig> min metabolized cmfda but did not take up pi, resulting in green but no red fluorescence, similar to untreated controls.
this indicated that the plasma membrane was still intact after  <dig> min of protein treatment.
samples exposed to ethanol did not metabolize cmfda but appeared bright red due to pi internalization, indicating that here the membrane was permeabilized.
we therefore conclude that the rapid increase in c within the first  <dig> min of protein treatment is not the result of uncontrolled ca2+ influx due to plasma membrane permeabilization.
the calcium chelator bapta abrogates the afpnn5353-induced calcium signature
the increased c in response to afpnn <dig> treatment could originate from extracellular and/or from intracellular ca2+ stores, such as mitochondria, vacuoles, endoplasmic reticulum or the golgi apparatus.
to discriminate between the extracellular and intracellular source of the c increase, we tested the influence of the ca2+-selective membrane impermeable chelator bapta.
on its own, bapta did not influence the resting level of c in twelve h old a. niger cultures .
however, a pretreatment of the samples with  <dig> mm bapta before the addition of afpnn <dig> inhibited the protein-specific increase in c resting level .
interestingly, the elevated c in response to a  <dig> min afpnn5353-treatment dropped to the resting level immediately after the addition of  <dig> mm bapta , indicating that the afpnn5353-induced elevation of the c resting level requires the continuous influx of extracellular ca2+ and eventually results in loss of c homeostasis.
extracellular calcium ameliorates the afpnn5353-induced rise in c
to decipher the observation that high external cacl <dig> concentrations counteracted afpnn <dig> toxicity , we monitored the effect of externally added ca2+ on the afpnn5353-induced ca2+ signature.
to this end, a. niger germlings were preincubated with  <dig> mm cacl <dig> for  <dig> min before  <dig> μg/ml afpnn <dig> was added and the changes in the c resting level were monitored over a time course of  <dig> min.
this treatment resulted in a less pronounced rise of the c resting level compared to samples without preincubation with cacl <dig>  in contrast, the presence of  <dig> mm cacl <dig> alone had no major effect on the intracellular c resting level which resembled that of the control without afpnn <dig> .
the values of the c resting levels of the last  <dig> min  measurement of afpnn <dig> treatment in the presence or absence of high ca2+ concentration  are summarized in table  <dig>  the average of the c of the controls which were not exposed to afpnn <dig> was  <dig>  μm in the presence of  <dig>  μm cacl <dig>  and  <dig>  μm in the presence of  <dig> mm cacl <dig>  when afpnn <dig> was added, there was no significant elevation of the c in high-ca2+ medium   whereas the c rised to  <dig>  μm at standard cacl <dig> concentration .
these results suggest that ca2+ externally added prior to the addition of afpnn <dig> counteracts the afpnn <dig> induced perturbation of the c and growth inhibitory effect, at least partly, by controlling the c resting level.
twelve h old germlings were preincubated with  <dig> mm cacl <dig> for  <dig> min before exposure to afpnn <dig>  values represent the average μm concentration of c within the last  <dig> min  of measurement.
afpnn <dig> decreases the amplitude of the c response to mechanical perturbation in a. niger
it is known that a range of external stimuli transiently increase c levels in aspergilli and other fungi  <cit> .
one of these physiological stimuli is mechanical perturbation, which is achieved by the rapid injection of isotonic medium into the test system.
this stimulus results in a unique ca2+ signature, likely involving different components of the ca2+-signalling and ca2+ homeostatic machinery.
changes in this specific ca2+ signature in the presence of compounds, such as afpnn <dig>  can give insights into the mode of action of these compounds.
in our study, twelve h old cultures of a. niger were pre-incubated with afpnn <dig> for  <dig> min and thereafter subjected to mechanical perturbation .
the resulting ca2+ signature, including c resting level, kinetics and amplitude, were determined and compared with controls that were not exposed to the protein but also subjected to mechanical perturbation.
as shown in figure  <dig>  afpnn <dig> provoked a less pronounced c amplitude; however, the c level remained elevated even after the stimulus specific response had stopped.
afpnn <dig> binding and uptake are essential for protein toxicity in a. nidulans
to understand the function of antifungal proteins, the identification of the site of action in target organisms is crucial.
so far, controversial reports exist of the localization of the homologous a. giganteus afp protein.
afp has been detected to bind to outer layers, e.g.
the cell wall or the plasma membrane of sensitive fungi  <cit>  and a time- and concentration-dependent intracellular localization was reported  <cit> .
in another study, alexa-labelled afp was shown to be internalized by the fungal cell and to localize to the nucleus  <cit> .
to dissect the uptake and localization of afpnn <dig>  we performed indirect immunofluorescence staining with a. nidulans wild type exposed to a sublethal concentration of afpnn <dig> .
we applied a protein amount below the toxic concentration for hyphae to maintain the cellular structure and to avoid apoptotic cell disruption  <cit> .
our study revealed that the protein was internalized after  <dig> min of incubation, mostly in hyphal tips, but also within hyphal segments .
the protein seemed not to localize to cell compartments, but was distributed in the cytoplasm.
similar results were obtained with a. niger wild type .
control experiments proved the specificity of the intracellular immunofluorescent signals: no intracellular fluorescent signals were detected in samples where either afpnn <dig>  or the primary antibody or the secondary antibody was omitted .
to analyse the afpnn <dig> localization in more detail, a. nidulans was incubated with afpnn <dig> in the presence of latrunculin b, a potent inhibitor of actin polymerization and endocytosis  <cit> .
at low latrunculin b concentrations , protein uptake was severely reduced compared to the positive control without latrunculin b , whereas  <dig> μg latrunculin b/ml completely inhibited the uptake of  <dig>  μg/ml afpnn <dig>  the solvent of latrunculin b, dmso, had no adverse effect on protein uptake .
this indicates that afpnn <dig> enters the a. nidulans cells by an endocytotic mechanism .
based on our observation that ca2+ ions antagonize the growth inhibitory activity of afpnn <dig>  we questioned whether ca2+ prevents actin-mediated internalisation of the antifungal protein.
indeed, the presence of  <dig> mm cacl <dig> inhibited protein uptake .
most interestingly, no specific fluorescent signals were detectable in m. circinelloides when treated with up to  <dig> μg/ml of antifungal protein , indicating that afpnn <dig> does not bind to insensitive strains.
discussion
in this study we provide important insights into the mechanistic basis of afpnn <dig>  a afp homologous protein.
species specificity tests revealed that afpnn <dig> is active against a broad range of filamentous fungi, including human and plant pathogens.
although the proteins afpnn <dig> and afp are almost identical and show a similar toxicity, mics for afpnn <dig> differed slightly from those reported for afp  <cit> .
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  <cit>  and our finding that the osmotic stabilizer sorbitol neutralized afpnn <dig> 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 afpnn <dig>  this is in agreement to the reported function of cell wall stressing agents, such as cfw or caffeine in s. cerevisiae and a. nidulans  <cit>  and to the penicillium antifungal protein paf  <cit> .
importantly, mpk function is essential for cwip activation in both, unicellular and filamentous fungi  <cit>  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  <cit> .
similarly, rlma dependent induction of the expression of the ags gene was also reported for aspergilli  <cit> .
importantly, the activation of the cwip can occur in a rhoa-dependent, e.g.
with cfw  <cit> , or rhoa-independent way, the latter proved for paf and caffeine  <cit>  and for afpnn <dig> .
as proposed by  <cit>  the dominant rhoae40i 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 afpnn <dig> similarly to the mode of action of the p. chrysogenum paf  <cit> .
our assumption of the activation of the cwip by afpnn <dig> was further strengthened by the fact, that afpnn <dig> treatment induced agsa expression in the a. niger reporter strain.
this result was consistent with the activity of afp and caspofungin  <cit> , but differed to the function of paf, where no cwip activation and no induction of cell wall biosynthesis genes occurred  <cit> .
therefore, we conclude that afpnn <dig> 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.
afpnn <dig> and paf - despite their homology.
this phenomenon was also reported for other closely related antifungal proteins, such as the plant defensins msdef <dig> and mtdef <dig> from medicago spp.
<cit> .
apart from the activation of the cwip, the perturbation of the ca2+ homeostasis represents a major mechanistic function of antifungal proteins in sensitive fungi  <cit> .
the intracellular ca2+ response to afpnn <dig> in a. niger reflected that of the penicillium antifungal protein paf in n. crassa  <cit> .
the rapid and sustained increase of the c resting level depended on a sustained influx of ca2+ ions from the external medium.
moreover, the afpnn <dig> induced changes in the ca2+ signature of mechanically perturbed a. niger cells further underlines the disruption of the ca2+ response and homeostasis by afpnn <dig>  the addition of cacl <dig> to the growth medium reduced the susceptibility of a. niger towards the antifungal protein and decreased the afpnn <dig> specific rise in the 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  <cit> .
second, it primes the ca2+ homeostatic machinery to better maintain a low c resting level when challenged with the antifungal protein, e.g.
by  the increase of the activity of existing ca2+ pumps/transporters to counteract the afpnn5353-specific intracellular ca2+ perturbation, or  the modulation of the expression of ca2+ channels/pumps/exchangers  <cit> .
the former hypothesis  might be supported by the observation that the addition of cacl <dig> only  <dig> min before a. niger was challenged with afpnn <dig> restored the low c resting level.
however, the perturbation of the ca2+ homeostasis by a sustained elevation of the c resting level indicates that a. niger is not able to restore the low c resting level after exposure to afpnn <dig> and this might trigger programmed cell death  on the long term as it was shown to occur in a. nidulans in response to the p. chrysogenum paf  <cit> .
since afp was shown to cause membrane permeabilization  <cit> , 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  <dig> min of exposure to afpnn <dig> when the c resting level reaches its maximum.
this result is further corroborated by the fact that higher external concentrations of ca2+ reduced the afpnn <dig> specific rise in 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 afpnn <dig> is characteristic for sensitive but not resistant moulds.
a lack of binding of afpnn <dig> to insensitive fungi might point towards the absence or inaccessibility of a putative interacting molecule at the cell surface.
afpnn <dig> 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  <cit> .
importantly, we observed that afpnn <dig> was internalized by hyphae even under sub-inhibitory concentrations  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 afpnn <dig> uptake.
this finding parallels well with the report of  <cit>  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 .
one possible explanation might be that extracellular ca2+ ions compete with afpnn <dig> for the same molecular target on the fungal surface which might represent a first binding receptor or even a "gate" for protein uptake  <cit>  or, alternatively, that the interacting target is repressed under these conditions  <cit> .
an additional explanation might be that the primary cell-surface localized afpnn <dig> 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  <cit> .
this further suggests that the activation of the cwip and the agsa induction does not mediate sufficient resistance to survive the toxic effects of afpnn <dig>  instead, according to the "damage-response framework of afp-fungal interactions"  <cit> , the chitin response might represent the better strategy for fungi to survive the antifungal attack.
CONCLUSIONS
based on the growth inhibitory activity, antifungal proteins like afpnn <dig> can be well considered as promising candidates for future antimycotic drug developments.
however, for biotechnological exploitation, the detailed knowledge on the mode of action is demanded.
our study shows that the detrimental effects caused by the a. giganteus antifungal protein afpnn <dig> in sensitive target aspergilli are based on the interaction of this protein with more than one signalling pathway.
in figure  <dig>  we present a tentative working model.
the toxicity of afpnn <dig> is mediated via pkca/mpka signalling which occurs independently from rhoa.
instead, so far unidentified rhoa-gap effector molecules might contribute to afpnn <dig> toxicity.
the activation of the cwip by afpnn <dig> induces the agsa gene expression which is, however, insufficient to counteract toxicity of the protein.
furthermore, afpnn <dig> leads to an immediate and significant increase of the c resting level in the cell.
this sustained perturbation of the ca2+ homeostasis could lead to pcd  <cit> .
the presence of extracellular ca2+ neutralizes the toxic effects of afpnn <dig> and improves the resistance of the target organism possibly by decreasing the elevated c resting level and stimulating the fortification of the cell wall by the induction of chsd expression as shown for afp  <cit> .
further investigations are in progress to clarify how these pathways are interconnected and interfere with each other on the molecular level.

 all organisms have evolved several defence systems in order to protect themselves against bacteria, fungi and viruses. higher organisms have developed a complex network of humoral and cellular responses, called adaptive immunity. a second defence system, the innate immunity, consists of many components, including small peptides with a broad antimicrobial spectrum. the production of such proteins with antimicrobial activity is not limited to higher eukaryotes, but also found in microorganisms, including fungi. the diversity of these proteins is reflected in their mode of action and their species-specificity. some of them form pores in the membrane, others are known to inhibit cell wall synthesis or interfere with nucleic acids and their synthesis. they can be involved in the inhibition of protein synthesis or interfere with cell cycle control. a relatively new group of antimicrobial proteins secreted by filamentous ascomycetes includes small, cationic and cysteine-rich proteins. so far, only few antifungal proteins have been characterized, namely afp from aspergillus giganteus, anafp from aspergillus niger, paf from penicillium chrysogenum and naf from penicillium nalgiovense. the mode of action of these proteins is not fully understood. nevertheless, there is evidence, that their toxicity is mediated by interaction with distinct molecules or receptors at the outer layers of the cell, e.g. cell wall or plasma membrane. deleterious effects can then be induced either by transmitting signals from the outer layers into the cell, or by internalization of the protein and interaction with internal molecules. similar to substances that perturb the cell wall, such as caspofungin, congo red or calcofluor white, the a. giganteus antifungal protein afp was found to modulate the cell wall composition by enhancing the expression of the α- -glucan synthase a gene, possibly by the activation of the cell wall integrity pathway, and inhibiting chitin synthesis in sensitive fungi. this, however, stands in contrast to the mode of action of the p. chrysogenum antifungal protein paf which fails to activate the cwip. however, the central players that trigger cell wall remodelling in afp-sensitive fungi have not been investigated so far. another mechanistic function of antifungal proteins is the interference with ion, especially ca2+ ion homeostasis and signalling. we could recently show that the p. chrysogenum antifungal protein paf severely perturbed the ca2+ homeostasis of neurospora crassa by rapidly elevating the cytoplasmic ca2+ c resting level. numerous reports indicate that the activity of antifungal proteins can be antagonized by the external addition of ca2+ ions to the test medium pointing towards the induction of adaptive responses which may be triggered by ca2+ signalling. the aim of this study was to characterize in more detail the mode of action of the a. giganteus afp variant protein afpnn and to investigate the pathways that might be affected/modulated by this antifungal protein. therefore, we focussed our interest on the involvement of the cwip and the ca2+ signalling in the toxicity of afpnn to address these questions, we used the highly afpnn sensitive model organisms a. nidulans and a. niger for which appropriate mutant strains were available.  in silico analysis of afpnn5353 clustalw amino acid sequence analysis of afpnn with other known antifungal proteins revealed that afpnn from a. giganteus strain a is a protein homologous to afp from a. giganteus strain mdh. afpnn exhibits > 90% identity with afp, but only 42% identity with the p. chrysogenum paf and 27% identity with the a. niger anafp. in fact, the secreted mature form of afpnn consists of aa and differs only in aa from afp. three aa exchanges belong to structurally related aa, one aa exhibits weak similarity and one aa is different. these aa exchanges do not influence the theoretical isoelectric point of afpnn which is the same as for afp. most importantly, afpnn still contains the putative chitin-binding domain ckykaq present in afp but not in paf or anafp and also harbors all conserved cysteine residues important for protein stabilization. antifungal activity of the protein afpnn5353 to investigate the antifungal specificity of afpnn fifteen filamentous fungi were tested for their susceptibility to the protein. since antifungal proteins might be useful for biotechnological applications, filamentous human and plant pathogenic fungi were selected as test organisms in addition to the model organisms a. nidulans and a. niger. as shown in table thirteen out of fifteen tested moulds were found to be sensitive against afpnn a. nidulans wild type, n. crassa wild type and a. niger wild type were the most sensitive strains to afpnn the minimal inhibitory concentration of afp was μg/ml for a. nidulans, μg/ml for n. crassa and μg/ml for a. niger. two strains were unaffected at the protein concentrations tested: m. circenelloides and m. genevensis were insensitive against afpnn when concentrations up to μg/ml were used. aup to μg/ml afpnn was tested × conidia/ml were incubated in μl cm medium in the presence of various concentrations of afpnn at 30°c for h. growth was determined by measuring the od nm. afpnn interferes with the cell wall integrity of a. nidulans it is known that antifungal compounds such as congo red, caffeine, cfw or caspofungin interfere with cell wall biosynthesis and weaken the cell wall in fungi. the remodeling of the cell wall by these antifungal compounds is mediated by the activation of the cwip. in fungi, extracellular signals are transmitted via the membrane bound small gtpase rhoa to the central regulators pkc and mpk, which are regulated by phosphorylation/dephosphorylation. the signal transduction cascade eventually enforces transcription of cell wall synthesis genes, partly via the transcription factor rlma. respective loss-of-function or conditional mutants show hypersensitive phenotypes in the presence of cell wall perturbing agents. similar to substances that weaken the cell wall, the a. giganteus antifungal protein afp modulates the cell wall composition by inhibiting chitin synthesis in sensitive fungi and inducing the expression of agsa most likely by the activation of the cwip. to study the involvement of the cwip in afpnn toxicity, we first tested whether the osmotic stabilizer sorbitol counteracts the toxicity of afpnn in the absence of afpnn a. nidulans proliferated less well in the presence of m sorbitol and reached only 30% growth compared to the growth in standard medium. nevertheless, the addition of m sorbitol to the growth medium strongly reduced the activity of afpnn on a. nidulans wild type. the osmotic stabilizer ameliorated growth in the presence of μg/ml afpnn by 80% compared to a 10% growth rate in the absence of sorbitol. this was even more accentuated when and μg/ml afpnn were applied, suggesting that afpnn indeed weakens the cell wall of a. nidulans. × conidia/ml were incubated in cm with 0- μg/ml afpnn for h. percent values were calculated from percent changes in od of afpnn treated a. nidulans compared to untreated controls. results are expressed as mean ± sd. to investigate whether afpnn induces agsa gene transcription similar to afp via the pkc/mpk signalling pathway, we tested the effect of the antifungal protein on the transgenic a. niger strain rd which expresses a nuclear-targeted gfp protein fused to the a. niger agsa promoter. rd germlings were treated with afpnn for h and analyzed microscopically. as shown in additional file a nuclear signal was clearly detectable in germlings of rd treated with ≥ μg/ml afpnn similar to that when exposed to μg/ml caspofungin. in untreated germlings, however, no signal could be observed. these observations perfectly match with the data obtained for afp. it has to be noted here that antifungal protein concentrations higher than the mic determined for conidia are needed to inhibit the growth of germlings or hyphae of sensitive fungi. next, we tested several a. nidulans mutant strains affected in central players of the cwip for their susceptibility to afpnn by determining their radial growth in the presence or absence of the antifungal protein. since rhoa is an essential protein in a. nidulans, two strains with ectopic copies of the constitutively active rhoag14v allele and the dominant rhoae40i allele were tested in comparison to the wild type strain. the rhoag14v mutation prevents the hydrolysis of gtp and therefore renders rhoa constantly active. similarly, the gtp hydrolysis is inhibited in the rhoae40i strain, but this mutation also perturbs the binding of the gtpase activating protein to rhoa and possibly disturbs downstream effectors of rhoa-gap. the constitutively active rhoag14v and the dominant rhoae40i strain exhibited the same sensitivity towards afpnn as the wild type strain at low protein concentrations. interestingly, the dominant rhoae40i strain was more resistant to afpnn than the wild type strain or the rhoag14v strain at higher protein concentrations. therefore, we suggest that the toxicity of afpnn is transmitted by rhoa-gap targets and not by rhoa itself. these mutants performed similarly when exposed to the orthologous p. chrysogenum antifungal protein paf. in addition, mutants defective in pkca and mpka activity were tested for their afpnn susceptibility. as pkca is an essential gene in a. nidulans, a conditional alca-pkc mutant strain was used, where the pkca gene was put under the control of the alca promoter, which is repressed by glucose but derepressed by glycerol. both the conditional alca-pkc mutant and a Δmpka mutant were hypersensitive to afpnn compared to their recipient strains r and gr respectively, indicating that the activity of pkca and mpka confers a certain resistance to afpnn. the hypersensitive phenotype of the Δmpka mutant was also confirmed by liquid growth inhibitory assays. in unchallenged liquid condition, the gr and the Δmpka mutant showed a comparable proliferation rate. in the presence of μg/ml afpnn however, the mpka deletion strain did not germinate whereas the gr strain still exhibited 11% growth. note that growth inhibition in liquid conditions requires less antifungal protein to monitor its toxicity than on solid media probably due to less diffusion in the latter case. from these data we conclude that afpnn interferes with the cell wall homeostasis of a. nidulans and that this interaction is mediated by pkca/mpka signalling, although independently from rhoa. afpnn disrupts calcium homeostasis in a. niger supplements other than osmotic stabilizers can also antagonize the activity of antifungal proteins from plants and ascomycetes. for example, the addition of cations such as ca2+ ions to the growth medium reversed the antifungal activity of the p. chrysogenum paf, the a. giganteus afp and of plant defensins which are usually positively charged due to their high pi. a cation-sensitive antifungal mode of action can for example be associated with the perturbation of the intracellular ca2+ homeostasis by antifungal peptides but might also result from the interference of cations with antifungal-target interaction. therefore, we tested to which extend these effects also account for the antifungal activity of afpnn to this end, we selected a. niger as model organism because this mould was highly sensitive to afpnn and a transgenic strain was available that expressed the recombinant codon optimized ca2+-sensitive photoprotein aequorin for measuring the c resting level in response to afpnn. first, we tested the activity of afpnn in vogels* medium supplemented with 5- mm cacl or without cacl as a control. addition of cacl did not influence the growth of a. niger up to a concentration of mm. the growth of a. niger exposed to afpnn however, ameliorated in the presence of increasing concentrations of cacl mm cacl neutralized the toxicity of - μg/ml afpnn and the treated samples resumed growth to 100%. od was measured after h of incubation. the growth of untreated controls was normalized to 100% to evaluate the percent growth of samples in the presence of afpnn vogels* medium without cacl supplementation contains mm ca2+. results are expressed as mean ± sd. next, we determined the influence of afpnn on the intracellular ca2+ signature. before afpnn addition, the resting level of the intracellular ca2+ was μm. we could show, however, that the c resting level was significantly increased in twelve h old a. niger cultures that were treated with μg/ml afpnn the c resting level rose to a maximum of μm within the first min and stayed elevated throughout the time of measurement, whereas the ca2+ level of the untreated control remained at μm. this indicated that afpnn indeed disrupts ca2+ homeostasis in a. niger. to exclude the possibility that the afpnn induced rise in the c resting level is due to membrane permeabilization and/or pore formation, we studied the effects of afpnn on germlings in the presence of cmfda, a membrane permeant dye that is metabolized by viable cells, and the membrane impermeant dye propidium iodide. additional file shows that samples treated with μg/ml afpnn for min metabolized cmfda but did not take up pi, resulting in green but no red fluorescence, similar to untreated controls. this indicated that the plasma membrane was still intact after min of protein treatment. samples exposed to ethanol did not metabolize cmfda but appeared bright red due to pi internalization, indicating that here the membrane was permeabilized. we therefore conclude that the rapid increase in c within the first min of protein treatment is not the result of uncontrolled ca2+ influx due to plasma membrane permeabilization. the calcium chelator bapta abrogates the afpnn5353-induced calcium signature the increased c in response to afpnn treatment could originate from extracellular and/or from intracellular ca2+ stores, such as mitochondria, vacuoles, endoplasmic reticulum or the golgi apparatus. to discriminate between the extracellular and intracellular source of the c increase, we tested the influence of the ca2+-selective membrane impermeable chelator bapta. on its own, bapta did not influence the resting level of c in twelve h old a. niger cultures. however, a pretreatment of the samples with mm bapta before the addition of afpnn inhibited the protein-specific increase in c resting level. interestingly, the elevated c in response to a min afpnn5353-treatment dropped to the resting level immediately after the addition of mm bapta, indicating that the afpnn5353-induced elevation of the c resting level requires the continuous influx of extracellular ca2+ and eventually results in loss of c homeostasis. extracellular calcium ameliorates the afpnn5353-induced rise in c to decipher the observation that high external cacl concentrations counteracted afpnn toxicity, we monitored the effect of externally added ca2+ on the afpnn5353-induced ca2+ signature. to this end, a. niger germlings were preincubated with mm cacl for min before μg/ml afpnn was added and the changes in the c resting level were monitored over a time course of min. this treatment resulted in a less pronounced rise of the c resting level compared to samples without preincubation with cacl in contrast, the presence of mm cacl alone had no major effect on the intracellular c resting level which resembled that of the control without afpnn. the values of the c resting levels of the last min measurement of afpnn treatment in the presence or absence of high ca2+ concentration are summarized in table the average of the c of the controls which were not exposed to afpnn was μm in the presence of μm cacl and μm in the presence of mm cacl when afpnn was added, there was no significant elevation of the c in high-ca2+ medium whereas the c rised to μm at standard cacl concentration. these results suggest that ca2+ externally added prior to the addition of afpnn counteracts the afpnn induced perturbation of the c and growth inhibitory effect, at least partly, by controlling the c resting level. twelve h old germlings were preincubated with mm cacl for min before exposure to afpnn values represent the average μm concentration of c within the last min of measurement. afpnn decreases the amplitude of the c response to mechanical perturbation in a. niger it is known that a range of external stimuli transiently increase c levels in aspergilli and other fungi. one of these physiological stimuli is mechanical perturbation, which is achieved by the rapid injection of isotonic medium into the test system. this stimulus results in a unique ca2+ signature, likely involving different components of the ca2+-signalling and ca2+ homeostatic machinery. changes in this specific ca2+ signature in the presence of compounds, such as afpnn can give insights into the mode of action of these compounds. in our study, twelve h old cultures of a. niger were pre-incubated with afpnn for min and thereafter subjected to mechanical perturbation. the resulting ca2+ signature, including c resting level, kinetics and amplitude, were determined and compared with controls that were not exposed to the protein but also subjected to mechanical perturbation. as shown in figure afpnn provoked a less pronounced c amplitude; however, the c level remained elevated even after the stimulus specific response had stopped. afpnn binding and uptake are essential for protein toxicity in a. nidulans to understand the function of antifungal proteins, the identification of the site of action in target organisms is crucial. so far, controversial reports exist of the localization of the homologous a. giganteus afp protein. afp has been detected to bind to outer layers, e.g. the cell wall or the plasma membrane of sensitive fungi and a time- and concentration-dependent intracellular localization was reported. in another study, alexa-labelled afp was shown to be internalized by the fungal cell and to localize to the nucleus. to dissect the uptake and localization of afpnn we performed indirect immunofluorescence staining with a. nidulans wild type exposed to a sublethal concentration of afpnn. we applied a protein amount below the toxic concentration for hyphae to maintain the cellular structure and to avoid apoptotic cell disruption. our study revealed that the protein was internalized after min of incubation, mostly in hyphal tips, but also within hyphal segments. the protein seemed not to localize to cell compartments, but was distributed in the cytoplasm. similar results were obtained with a. niger wild type. control experiments proved the specificity of the intracellular immunofluorescent signals: no intracellular fluorescent signals were detected in samples where either afpnn or the primary antibody or the secondary antibody was omitted. to analyse the afpnn localization in more detail, a. nidulans was incubated with afpnn in the presence of latrunculin b, a potent inhibitor of actin polymerization and endocytosis. at low latrunculin b concentrations, protein uptake was severely reduced compared to the positive control without latrunculin b, whereas μg latrunculin b/ml completely inhibited the uptake of μg/ml afpnn the solvent of latrunculin b, dmso, had no adverse effect on protein uptake. this indicates that afpnn enters the a. nidulans cells by an endocytotic mechanism. based on our observation that ca2+ ions antagonize the growth inhibitory activity of afpnn we questioned whether ca2+ prevents actin-mediated internalisation of the antifungal protein. indeed, the presence of mm cacl inhibited protein uptake. most interestingly, no specific fluorescent signals were detectable in m. circinelloides when treated with up to μg/ml of antifungal protein, indicating that afpnn does not bind to insensitive strains. discussion in this study we provide important insights into the mechanistic basis of afpnn a afp homologous protein. species specificity tests revealed that afpnn is active against a broad range of filamentous fungi, including human and plant pathogens. although the proteins afpnn and afp are almost identical and show a similar toxicity, mics for afpnn 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 afpnn 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 afpnn this is in agreement to the reported function of cell wall stressing agents, such as cfw or caffeine in s. cerevisiae and a. nidulans and to the penicillium antifungal protein paf. importantly, mpk function is essential for cwip activation in both, unicellular and filamentous fungi 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. 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, or rhoa-independent way, the latter proved for paf and caffeine and for afpnn. as proposed by the dominant rhoae40i 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 afpnn similarly to the mode of action of the p. chrysogenum paf. our assumption of the activation of the cwip by afpnn was further strengthened by the fact, that afpnn 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 afpnn 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. afpnn and paf - despite their homology. this phenomenon was also reported for other closely related antifungal proteins, such as the plant defensins msdef and mtdef 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. the intracellular ca2+ response to afpnn in a. niger reflected that of the penicillium antifungal protein paf in n. crassa. the rapid and sustained increase of the c resting level depended on a sustained influx of ca2+ ions from the external medium. moreover, the afpnn induced changes in the ca2+ signature of mechanically perturbed a. niger cells further underlines the disruption of the ca2+ response and homeostasis by afpnn the addition of cacl to the growth medium reduced the susceptibility of a. niger towards the antifungal protein and decreased the afpnn specific rise in the 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 c resting level when challenged with the antifungal protein, e.g. by the increase of the activity of existing ca2+ pumps/transporters to counteract the afpnn5353-specific intracellular ca2+ perturbation, or the modulation of the expression of ca2+ channels/pumps/exchangers. the former hypothesis might be supported by the observation that the addition of cacl only min before a. niger was challenged with afpnn restored the low c resting level. however, the perturbation of the ca2+ homeostasis by a sustained elevation of the c resting level indicates that a. niger is not able to restore the low c resting level after exposure to afpnn and this might trigger programmed cell death 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 min of exposure to afpnn when the c resting level reaches its maximum. this result is further corroborated by the fact that higher external concentrations of ca2+ reduced the afpnn specific rise in 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 afpnn is characteristic for sensitive but not resistant moulds. a lack of binding of afpnn to insensitive fungi might point towards the absence or inaccessibility of a putative interacting molecule at the cell surface. afpnn 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. importantly, we observed that afpnn was internalized by hyphae even under sub-inhibitory concentrations 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 afpnn 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. one possible explanation might be that extracellular ca2+ ions compete with afpnn for the same molecular target on the fungal surface which might represent a first binding receptor or even a "gate" for protein uptake or, alternatively, that the interacting target is repressed under these conditions. an additional explanation might be that the primary cell-surface localized afpnn 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 afpnn 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.  based on the growth inhibitory activity, antifungal proteins like afpnn can be well considered as promising candidates for future antimycotic drug developments. however, for biotechnological exploitation, the detailed knowledge on the mode of action is demanded. our study shows that the detrimental effects caused by the a. giganteus antifungal protein afpnn in sensitive target aspergilli are based on the interaction of this protein with more than one signalling pathway. in figure we present a tentative working model. the toxicity of afpnn is mediated via pkca/mpka signalling which occurs independently from rhoa. instead, so far unidentified rhoa-gap effector molecules might contribute to afpnn toxicity. the activation of the cwip by afpnn induces the agsa gene expression which is, however, insufficient to counteract toxicity of the protein. furthermore, afpnn leads to an immediate and significant increase of the c resting level in the cell. this sustained perturbation of the ca2+ homeostasis could lead to pcd. the presence of extracellular ca2+ neutralizes the toxic effects of afpnn and improves the resistance of the target organism possibly by decreasing the elevated c resting level and stimulating the fortification of the cell wall by the induction of chsd expression as shown for afp. further investigations are in progress to clarify how these pathways are interconnected and interfere with each other on the molecular level. 
the antifungal protein afpnn is a defensin-like protein of aspergillus giganteus. the protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims aspergillus nidulans and aspergillus niger. furthermore, we provide evidence that calcium signalling plays an important role in the mechanistic function of this antifungal protein. the present study contributes new insights into the molecular mechanisms of action of the a. giganteus antifungal protein afpnn we identified its antifungal activity, initiated the investigation of pathways that determine protein toxicity, namely the cwip and the ca2+ signalling cascade, and studied in detail the cellular uptake mechanism in sensitive target fungi. it belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. we determined an afpnn hypersensitive phenotype of non-functional a. nidulans mutants in the protein kinase c /mitogen-activated protein kinase signalling pathway and the induction of the α-glucan synthase a promoter in a transgenic a. niger strain which point at the activation of the cell wall integrity pathway and the remodelling of the cell wall in response to afpnn the activation of the cwip by afpnn however, operates independently from rhoa which is the central regulator of cwip signal transduction in fungi. this knowledge contributes to define new potential targets for the development of novel antifungal strategies to prevent and combat infections of filamentous fungi which have severe negative impact in medicine and agriculture. supplementation of the growth medium with cacl counteracted afpnn toxicity, ameliorated the perturbation of the c resting level and prevented protein uptake into aspergillus sp.
 the antifungal protein afpnn is a defensin-like protein of aspergillus giganteus. it belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. the protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims aspergillus nidulans and aspergillus niger.  we determined an afpnn hypersensitive phenotype of non-functional a. nidulans mutants in the protein kinase c /mitogen-activated protein kinase signalling pathway and the induction of the α-glucan synthase a promoter in a transgenic a. niger strain which point at the activation of the cell wall integrity pathway and the remodelling of the cell wall in response to afpnn the activation of the cwip by afpnn however, operates independently from rhoa which is the central regulator of cwip signal transduction in fungi. furthermore, we provide evidence that calcium signalling plays an important role in the mechanistic function of this antifungal protein. afpnn increased about 2-fold the cytosolic free ca2+ of a transgenic a. niger strain expressing codon optimized aequorin. supplementation of the growth medium with cacl counteracted afpnn toxicity, ameliorated the perturbation of the c resting level and prevented protein uptake into aspergillus sp. cells.  the present study contributes new insights into the molecular mechanisms of action of the a. giganteus antifungal protein afpnn we identified its antifungal activity, initiated the investigation of pathways that determine protein toxicity, namely the cwip and the ca2+ signalling cascade, and studied in detail the cellular uptake mechanism in sensitive target fungi. this knowledge contributes to define new potential targets for the development of novel antifungal strategies to prevent and combat infections of filamentous fungi which have severe negative impact in medicine and agriculture. 