The Aspergillus giganteus antifungal protein AFPNN5353 activates the cell wall integrity pathway ...

13 Pages·2011·1.61 MB·English

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RESEARCH ARTICLE Open Access
The Aspergillus giganteus antifungal protein
AFP activates the cell wall integrity
NN5353
pathway and perturbs calcium homeostasis
Ulrike Binder1,5, Mojca Bencina2,3, Andrea Eigentler1, Vera Meyer4 and Florentine Marx1*
Abstract
Background: The antifungal protein AFP is a defensin-like protein of Aspergillus giganteus. It belongs to a
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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.
Results: We determined an AFP hypersensitive phenotype of non-functional A. nidulans mutants in the
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protein kinase C (Pkc)/mitogen-activated protein kinase (Mpk) signalling pathway and the induction of the a-
glucan synthase A (agsA) promoter in a transgenic A. niger strain which point at the activation of the cell wall
integrity pathway (CWIP) and the remodelling of the cell wall in response to AFP . The activation of the CWIP
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by AFP , however, operates independently from RhoA which is the central regulator of CWIP signal
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transduction in fungi.
Furthermore, we provide evidence that calcium (Ca2+) signalling plays an important role in the mechanistic
function of this antifungal protein. AFP increased about 2-fold the cytosolic free Ca2+ ([Ca2+] ) of a transgenic
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A. niger strain expressing codon optimized aequorin. Supplementation of the growth medium with CaCl
2
counteracted AFP toxicity, ameliorated the perturbation of the [Ca2+] resting level and prevented protein
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uptake into Aspergillus sp. cells.
Conclusions: The present study contributes new insights into the molecular mechanisms of action of the A.
giganteus antifungal protein AFP . We identified its antifungal activity, initiated the investigation of pathways
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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.
Background antimicrobial activity is not limited to higher eukaryotes,
All organisms have evolved several defence systems in but also found in microorganisms, including fungi. The
order to protect themselves against bacteria, fungi and diversity of these proteins is reflected in their mode of
viruses. Higher organisms have developed a complex action and their species-specificity. Some of them form
network of humoral and cellular responses, called adap- pores in the membrane, others are known to inhibit cell
tive immunity. A second defence system, the innate wall synthesis or interfere with nucleic acids and their
immunity, consists of many components, including synthesis [3,4]. They can be involved in the inhibition of
small peptides with a broad antimicrobial spectrum protein synthesis or interfere with cell cycle control
[1,2]. The production of such proteins with [3,4]. A relatively new group of antimicrobial proteins
secreted by filamentous ascomycetes includes small,
cationic and cysteine-rich proteins. So far, only few anti-
*Correspondence:[email protected]
1Biocenter,DivisionofMolecularBiology,InnsbruckMedicalUniversity,Fritz- fungal proteins have been characterized, namely AFP
PreglStrasse3,Innsbruck,A-6020,Austria from Aspergillus giganteus, ANAFP from Aspergillus
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Binderetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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niger, PAF from Penicillium chrysogenum and NAF from of 51 aa and differs only in 5 aa from AFP (Figure 1).
Penicillium nalgiovense [5-8]. Three aa exchanges belong to structurally related aa,
The mode of action of these proteins is not fully one aa exhibits weak similarity and one aa is different
understood. Nevertheless, there is evidence, that their (position 4). These aa exchanges do not influence the
toxicity is mediated by interaction with distinct mole- theoretical isoelectric point (pI) of AFP , which is
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cules or receptors at the outer layers of the cell, e.g. cell the same as for AFP (pI 9.3, http://expasy.org/tools/
wall or plasma membrane. Deleterious effects can then protparam.html). Most importantly, AFP still con-
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be induced either by transmitting signals from the outer tains the putative chitin-binding domain CKYKAQ pre-
layers into the cell, or by internalization of the protein sent in AFP but not in PAF or ANAFP and also harbors
and interaction with internal molecules [9-15]. Similar all conserved cysteine residues important for protein
to substances that perturb the cell wall, such as caspo- stabilization [10,23].
fungin, congo red or calcofluor white (CFW) [10,16],
the A. giganteus antifungal protein AFP was found to Antifungal activity of the protein AFP
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modulate the cell wall composition by enhancing the To investigate the antifungal specificity of AFP ,
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expression of the a-1,3-glucan synthase A gene (agsA), fifteen filamentous fungi were tested for their suscept-
possibly by the activation of the cell wall integrity path- ibility to the protein. Since antifungal proteins might be
way (CWIP), and inhibiting chitin synthesis in sensitive useful for biotechnological applications, filamentous
fungi [10]. This, however, stands in contrast to the human and plant pathogenic fungi were selected as test
mode of action of the P. chrysogenum antifungal protein organisms (e.g. Fusarium oxysporum, Botrytis cinerea,
PAF which fails to activate the CWIP [9]. However, the Mucor sp. and A. fumigatus) in addition to the model
central players that trigger cell wall remodelling in AFP- organisms A. nidulans and A. niger. As shown in Table
sensitive fungi have not been investigated so far. 1, thirteen out of fifteen tested moulds were found to be
Another mechanistic function of antifungal proteins is sensitive against AFP . A. nidulans wild type, N.
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the interference with ion, especially Ca2+ ion homeosta- crassa wild type and A. niger wild type were the most
sis and signalling [15,17,18]. We could recently show sensitive strains to AFP . The minimal inhibitory
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that the P. chrysogenum antifungal protein PAF severely concentration (MIC) of AFP (the concentration that
perturbed the Ca2+ homeostasis of Neurospora crassa by completely inhibited conidial germination in liquid
rapidly elevating the cytoplasmic Ca2+ [Ca2+] resting growth assays) was 0.2 μg/ml for A. nidulans, 0.5 μg/ml
c
level [17]. Numerous reports indicate that the activity of for N. crassa and 1 μg/ml for A. niger. Two strains were
antifungal proteins can be antagonized by the external unaffected at the protein concentrations tested: M. circe-
addition of Ca2+ ions to the test medium [15,17-21] nelloides and M. genevensis were insensitive against
pointing towards the induction of adaptive responses AFP when concentrations up to 500 μg/ml were
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which may be triggered by Ca2+ signalling [15,17]. used.
The aim of this study was to characterize in more
detail the mode of action of the A. giganteus AFP var- AFP interferes with the cell wall integrity of A.
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iant protein AFP and to investigate the pathways nidulans
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that might be affected/modulated by this antifungal pro- It is known that antifungal compounds such as congo
tein. Therefore, we focussed our interest on the involve- red, caffeine, CFW or caspofungin interfere with cell
ment of the CWIP and the Ca2+ signalling in the wall biosynthesis and weaken the cell wall in fungi
toxicity of AFP . To address these questions, we (reviewed by [24]). The remodeling of the cell wall by
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used the highly AFP sensitive model organisms A. these antifungal compounds is mediated by the activa-
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nidulans and A. niger for which appropriate mutant tion of the CWIP. In fungi, extracellular signals are
strains were available. transmitted via the membrane bound small GTPase
RhoA to the central regulators Pkc and Mpk, which are
Results regulated by phosphorylation/dephosphorylation. The
In silico analysis of AFP signal transduction cascade eventually enforces tran-
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CLUSTALW amino acid (aa) sequence analysis of scription of cell wall synthesis genes, partly via the tran-
AFP with other known antifungal proteins scription factor RlmA [16,25]. Respective loss-of-
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revealed that AFP from A. giganteus strain A3274 function or conditional mutants show hypersensitive
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is a protein homologous to AFP from A. giganteus strain phenotypes in the presence of cell wall perturbing
MDH 18894 [8,22]. AFP exhibits > 90% identity agents [9,24-26]. Similar to substances that weaken the
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with AFP, but only 42% identity with the P. chryso- cell wall, the A. giganteus antifungal protein AFP modu-
genum PAF and 27% identity with the A. niger ANAFP. lates the cell wall composition by inhibiting chitin
In fact, the secreted mature form of AFP consists synthesis in sensitive fungi (e.g. A. niger, A. oryzae) and
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Figure1Clustalwsequencealignmenthttp://www.ebi.ac.uk/Tools/msa/clustalw2/oftheantifungalproteinsAFP andAFPfrom
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A.giganteus,ANAFPfromA.nigerandPAFfromP.chrysogenum.Identicalaminoacids(aa)aremarkedwith(*),aawithstrongsimilarity
areindicatedwith(:)andaawithweaksimilarityaremarkedwith(.).
inducing the expression of agsA most likely by the acti- a nuclear-targeted GFP protein fused to the A. niger
vation of the CWIP [10]. agsA promoter. RD6.47 germlings were treated with
To study the involvement of the CWIP in AFP AFP (conc. 10 to 100 μg/ml) for 2 h and analyzed
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toxicity, we first tested whether the osmotic stabilizer microscopically. As shown in Additional file 1, a nuclear
sorbitol counteracts the toxicity of AFP . In the signal was clearly detectable in germlings of RD6.47
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absence of AFP A. nidulans proliferated less well treated with ≥ 50 μg/ml AFP , similar to that when
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in the presence of 1 M sorbitol and reached only 30% exposed to 10 μg/ml caspofungin. In untreated germl-
growth compared to the growth in standard medium ings, however, no signal could be observed. These obser-
(100%). Nevertheless, the addition of 1 M sorbitol to the vations perfectly match with the data obtained for AFP
growth medium strongly reduced the activity of [10]. It has to be noted here that antifungal protein con-
AFP on A. nidulans wild type. The osmotic stabi- centrations higher than the MIC determined for conidia
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lizer ameliorated growth in the presence of 0.05 μg/ml (> 10-50 fold) are needed to inhibit the growth of
AFP by 80% compared to a 10% growth rate in germlings or hyphae of sensitive fungi [10,27] (data not
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the absence of sorbitol (Table 2). This was even more shown).
accentuated when 0.1 and 0.2 μg/ml AFP were Next, we tested several A. nidulans mutant strains
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applied, suggesting that AFP indeed weakens the affected in central players of the CWIP for their sus-
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cell wall of A. nidulans. ceptibility to AFP by determining their radial
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To investigate whether AFP induces agsA gene growth in the presence or absence of the antifungal pro-
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transcription similar to AFP via the Pkc/Mpk signalling tein. Since RhoA is an essential protein in A. nidulans,
pathway, we tested the effect of the antifungal protein two strains with ectopic copies of the constitutively
on the transgenic A. niger strain RD6.47 which expresses active rhoAG14V allele and the dominant rhoAE40I allele
[28] were tested in comparison to the wild type strain
(GR5). The rhoAG14V mutation prevents the hydrolysis
Table1Minimal inhibitory concentrations (MIC; μg/ml)of of GTP and therefore renders RhoA constantly active
AFP against differentfilamentous fungi [28]. Similarly, the GTP hydrolysis is inhibited in the
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organism MIC(μg/ml) RhoAE40I strain, but this mutation also perturbs the
AspergillusflavusATCC9643 50 binding of the GTPase activating protein (GAP) to
AspergillusfumigatusATCC46645 50 RhoA and possibly disturbs downstream effectors of
AspergillusgiganteusAG090701 50 RhoA-GAP [28]. The constitutively active RhoAG14V
AspergillusnidulansFGSC4 0.2 and the dominant RhoAE40I strain exhibited the same
AspergillusnigerCBS120.49 1 sensitivity towards AFPNN5353 as the wild type strain at
Aspergillusterreus304 5 low protein concentrations (≤ 0.2 μg/ml) (Figure 2A).
BotrytiscinereaBC080801 10 Interestingly, the dominant RhoAE40I strain was more
FusariumoxysporumFO240901 5
FusariumsambucinumFS210901 5 Table 2The effect of1Msorbitol on the growth
GliocladiumroseumGR210901 100 inhibiting activity ofAFPNN5353 onA. nidulans
MucorcircinelloidesMC080801 insensitivea AFP (μg/ml) CM CM+1Msorbitol
NN5353
MucorgenevensisMG080801 insensitivea 0 100( ±10) 100( ±11)
SD SD
PenicilliumchrysogenumATCC10002 10 0.05 10.4( ±1) 79.3( ±6)
SD SD
TrichodermakoningiiTC060901 20 0.1 5.5( ±2) 68.3( ±0.8)
SD SD
NeuropsoracrassaFGSC2489 0.5 0.2 nogrowth 17.8( ±0.8)
SD
aupto500μg/mlAFPNN5353wastested 1×104conidia/mlwereincubatedinCMwith0-0.2μg/mlAFPNN5353for24h.
1×104conidia/mlwereincubatedin200μlCMmediuminthepresenceof PercentvalueswerecalculatedfrompercentchangesinOD ofAFP
620 NN5353
variousconcentrationsofAFPNN5353at30°Cfor24h.Growthwasdetermined treatedA.nidulanscomparedtountreatedcontrols(=100%).Resultsare
bymeasuringtheOD620nm. expressedasmean±SD(n=3).
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Figure 2 AFP susceptibility of A. nidulans mutants RhoAG14V, RhoAE40I, alcA-PkcA and ΔmpkA compared to the respective
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recipientstrainsGR5andR153.(A)Atotalof2×103conidiawerepointinoculatedonagarplates(CMforGR5,RhoAG14V,RhoAE40Iand
ΔmpkA,repressiveMMcontaining1%glucoseaccordingto[26]forR135andalcA-PkcA)containingtheappropriatesupplementsand0,0.2and
1μg/mlAFP forGR5,RhoAG14V,RhoAE40I,R135andalcA-PkcA.TheΔmpkAmutantanditsreferencestrainGR5wereexposedto0,0.5and
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1μg/mlAFP .Theplateswereincubatedat37°Cfor48h.(B)1×104conidia/mloftheΔmpkAmutantandGR5weretreatedwith0.05
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μg/mlAFP orwithoutprotein(controls)inatotalvolumeof200μlofappropriatelysupplementedCMin96-wellplates.
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resistant to AFP than the wild type strain or the promoter, which is repressed by glucose but derepressed
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RhoAG14V strain at higher protein concentrations (1 μg/ by glycerol [26]. Both the conditional alcA-PKC mutant
ml) (Figure 2A). Therefore, we suggest that the toxicity (cultivated under repressive conditions) and a ΔmpkA
of AFP is transmitted by RhoA-GAP targets and mutant were hypersensitive to AFP compared to
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not by RhoA itself. These mutants performed similarly their recipient strains R153 and GR5, respectively, indi-
when exposed to the orthologous P. chrysogenum anti- cating that the activity of PkcA and MpkA confers a
fungal protein PAF [9]. certain resistance to AFP (Figure 2A). The hyper-
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In addition, mutants defective in PkcA and MpkA sensitive phenotype of the ΔmpkA mutant was also con-
activity were tested for their AFP susceptibility. firmed by liquid growth inhibitory assays. In
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As pkcA is an essential gene in A. nidulans, a condi- unchallenged liquid condition, the GR5 and the ΔmpkA
tional alcA-PKC mutant strain was used, where the mutant showed a comparable proliferation rate (Figure
pkcA gene was put under the control of the alcA 2B). In the presence of 0.05 μg/ml AFP , however,
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the mpkA deletion strain did not germinate whereas the addition, the resting level of the intracellular Ca2+ was
GR5 strain still exhibited 11% growth. Note that growth 0.08 μM. We could show, however, that the [Ca2+] rest-
c
inhibition in liquid conditions requires less antifungal ing level was significantly increased in twelve h old A.
protein to monitor its toxicity than on solid media prob- niger cultures that were treated with 20 μg/ml
ably due to less diffusion in the latter case (data not AFP . The [Ca2+] resting level rose to a maximum
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shown). of 0.19 μM within the first 8 min and stayed elevated
From these data we conclude that AFP inter- throughout the time of measurement (60 min), whereas
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feres with the cell wall homeostasis of A. nidulans and the Ca2+ level of the untreated control remained at 0.08
that this interaction is mediated by PkcA/MpkA signal- μM (Figure 3). This indicated that AFP indeed
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ling, although independently from RhoA. disrupts Ca2+ homeostasis in A. niger.
To exclude the possibility that the AFP induced
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AFP disrupts calcium homeostasis in A. niger rise in the [Ca2+] resting level is due to membrane per-
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Supplements other than osmotic stabilizers can also meabilization and/or pore formation, we studied the
antagonize the activity of antifungal proteins from plants effects of AFP on germlings in the presence of
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and ascomycetes. For example, the addition of cations CMFDA, a membrane permeant dye that is metabolized
such as Ca2+ ions to the growth medium reversed the by viable cells, and the membrane impermeant dye pro-
antifungal activity of the P. chrysogenum PAF [17], the pidium iodide (PI). Additional file 2 shows that samples
A. giganteus AFP [15,21] and of plant defensins [29,30] treated with 20 μg/ml AFP for 10 min metabo-
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which are usually positively charged due to their high lized CMFDA but did not take up PI, resulting in green
pI. A cation-sensitive antifungal mode of action can for but no red fluorescence, similar to untreated controls.
example be associated with the perturbation of the This indicated that the plasma membrane was still intact
intracellular Ca2+ homeostasis by antifungal peptides after 10 min of protein treatment. Samples exposed to
[17,18] but might also result from the interference of ethanol did not metabolize CMFDA but appeared bright
cations with antifungal-target interaction(s). red due to PI internalization, indicating that here the
Therefore,wetestedtowhichextendtheseeffectsalso membrane was permeabilized. We therefore conclude
accountforthe antifungalactivityof AFP .Tothis that the rapid increase in [Ca2+] within the first 10 min
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end,weselectedA.nigerasmodelorganismbecausethis of protein treatment is not the result of uncontrolled
mould was highly sensitive to AFP and a trans- Ca2+ influx due to plasma membrane permeabilization.
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genicstrainwasavailablethatexpressedtherecombinant
codon optimized Ca2+-sensitive photoprotein aequorin The calcium chelator BAPTA abrogates the AFP -
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for measuring the [Ca2+] resting level in response to induced calcium signature
c
AFP [31].First,wetestedtheactivityofAFP Theincreased[Ca2+] inresponsetoAFP treatment
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in Vogels* medium supplemented with 5-20 mM CaCl couldoriginatefromextracellularand/orfromintracellu-
2
orwithoutCaCl asacontrol(datanotshown).Addition lar Ca2+ stores, such as mitochondria, vacuoles,
2
ofCaCl didnotinfluencethegrowthofA.nigeruptoa
2
concentrationof20mM.ThegrowthofA.nigerexposed
to AFP , however, ameliorated in the presence of
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increasing concentrations of CaCl . 20 mM CaCl neu-
2 2
tralized the toxicity of 0.5-1.0 μg/ml AFP and the
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treatedsamplesresumedgrowthto100%(Table3).
Next, we determined the influence of AFP on
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the intracellular Ca2+ signature. Before AFP
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Table 3The effect of20mM externalCaCl (in Vogels*
2
medium) onthe growth inhibitory activity ofAFP
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on A.niger strainA533.
AFP (μg/ml) Vogels* Vogels*+20mMCa2+
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0 100( ±10) 100( ±8)
SD SD
0.5 12( ±3) 101( ±9)
SD SD
1.0 nogrowth 105(SD±6) Figure 3 Increase in resting [Ca2+]c of twelve h old A. niger
OD620wasmeasuredafter24hofincubation.Thegrowthofuntreated germlingstreatedwithAFPNN5353ornoprotein(controls).
controlswasnormalizedto100%toevaluatethepercentgrowthofsamples Measurementsweretakenevery1.4minutes.Valuesrepresent
inthepresenceofAFPNN5353.Vogels*mediumwithoutCaCl2supplementation averageofsixsamples.
contains0.7mMCa2+.Resultsareexpressedasmean±SD(n=3).
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endoplasmicreticulumortheGolgiapparatus.Todiscri- AFP treatment in the presence or absence of high
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minatebetweentheextracellular andintracellularsource Ca2+ concentration (20 mM versus 0.7 mM) are sum-
ofthe[Ca2+] increase,wetestedtheinfluenceoftheCa2 marized in Table 4. The average of the [Ca2+] of the
c c
+-selectivemembraneimpermeablechelatorBAPTA.On controls which were not exposed to AFP was
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itsown,BAPTAdidnotinfluencetherestinglevelof[Ca2 0.039 μM in the presence of 0.7 μM CaCl (standard
2
+] intwelveholdA.nigercultures(Figure4).However,a condition) and 0.062 μM in the presence of 20 mM
c
pretreatment ofthe sampleswith10 mMBAPTA before CaCl . When AFP was added, there was no signif-
2 NN5353
the addition of AFP inhibited the protein-specific icant elevation of the [Ca2+] in high-Ca2+ medium (20
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increase in [Ca2+] resting level (Figure 4). Interestingly, mM) (0.057 μM) whereas the [Ca2+] rised to 0.146 μM
c c
the elevated [Ca2+] in response to a 40 min AFP - at standard CaCl concentration (0.7 mM). These results
c NN5353 2
treatment dropped to the resting level immediately after suggest that Ca2+ externally added prior to the addition
theadditionof10mMBAPTA(Figure4),indicatingthat of AFP counteracts the AFP induced per-
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the AFP -induced elevation of the [Ca2+] resting turbation of the [Ca2+] and growth inhibitory effect, at
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level requires the continuousinfluxof extracellular Ca2+ least partly, by controlling the [Ca2+] resting level.
c
andeventuallyresultsinlossof[Ca2+] homeostasis.
c
AFP decreases the amplitude of the [Ca2+]
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Extracellular calcium ameliorates the AFP -induced response to mechanical perturbation in A. niger
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rise in [Ca2+] It is known that a range of external stimuli transiently
c
To decipher the observation that high external CaCl increase [Ca2+] levels in Aspergilli and other fungi
2 c
concentrations counteracted AFP toxicity (Table [31,32]. One of these physiological stimuli is mechanical
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3), we monitored the effect of externally added Ca2+ on perturbation, which is achieved by the rapid injection of
the AFP -induced Ca2+ signature. To this end, A. isotonic medium into the test system. This stimulus
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niger germlings were preincubated with 20 mM CaCl results in a unique Ca2+ signature, likely involving differ-
2
for 10 min before 20 μg/ml AFP was added and ent components of the Ca2+-signalling and Ca2+ homeo-
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the changes in the [Ca2+] resting level were monitored static machinery. Changes in this specific Ca2+ signature
c
over a time course of 60 min. This treatment resulted in in the presence of compounds, such as AFP , can
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a less pronounced rise of the [Ca2+] resting level com- give insights into the mode of action of these com-
c
pared to samples without preincubation with CaCl . In pounds. In our study, twelve h old cultures of A. niger
2
contrast, the presence of 20 mM CaCl alone had no were pre-incubated with AFP for 60 min and
2 NN5353
major effect on the intracellular [Ca2+] resting level thereafter subjected to mechanical perturbation (rapid
c
which resembled that of the control without AFP injection of 100 μl Vogels medium). The resulting Ca2+
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(data not shown). The values of the [Ca2+] resting levels signature, including [Ca2+] resting level, kinetics and
c c
of the last 10 min (50 to 60 min) measurement of amplitude, were determined and compared with controls
that were not exposed to the protein but also subjected
to mechanical perturbation. As shown in Figure 5,
AFP provoked a less pronounced [Ca2+] ampli-
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tude; however, the [Ca2+] level remained elevated even
c
after the stimulus specific response had stopped.
AFP binding and uptake are essential for protein
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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
Table 4The effect ofhighexternal CaCl concentration
2
on theAFP induced Ca2+ signaturein response to
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AFP .
NN5353
[CaCl ]inVogels* 0μg/mlAFP 20μg/mlAFP
2 NN5353 NN5353
Figure 4 Effect of the extracellular chelator BAPTA on the
0.7mM 0.039( ±0.001) 0.146( ±0.009)
AFP induced[Ca2+] restinglevel.10mMBAPTA(final SD SD
NN5353 c 20mM 0.062( ±0.003) 0.057( ±0.004)
conc.)wereapplied40minbeforeor40minaftertreatmentwith SD SD
20μg/mlAFP .Sampleswithoutsupplementswereusedas Twelveholdgermlingswerepreincubatedwith20mMCaCl for10min
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controls.SD(n=6)waslessthan10%ofthevaluespresented. beforeexposuretoAFPNN5353.ValuesrepresenttheaverageμMconcentration
of[Ca2+]cwithinthelast10min(50-60min)ofmeasurement.
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Figure5EffectsofAFP onthe[Ca2+] responsetomechanicalperturbation.TwelveholdA.nigerculturesweretreatedwith20μg/
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mlAFP for60minbeforestimulationbymechanicalperturbation(additionof100μlVogelsmedium).The[Ca2+] signaturewas
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monitoredfor5min.Valuesrepresenttheaverageofsixsamples.
localization of the homologous A. giganteus AFP pro- polymerization and endocytosis [35-37]. At low latrun-
tein. AFP has been detected to bind to outer layers, e.g. culin B concentrations (5 μg/ml), protein uptake was
the cell wall or the plasma membrane of sensitive fungi severely reduced compared to the positive control with-
[20,21] and a time- and concentration-dependent intra- out latrunculin B (data not shown), whereas 20 μg
cellular localization was reported [20]. In another study, latrunculin B/ml completely inhibited the uptake of 0.2
Alexa-labelled AFP was shown to be internalized by the μg/ml AFP . The solvent of latrunculin B, DMSO,
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fungal cell and to localize to the nucleus [33]. had no adverse effect on protein uptake (data not
To dissect the uptake and localization of AFP , shown). This indicates that AFP enters the A.
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we performed indirect immunofluorescence staining nidulans cells by an endocytotic mechanism (Figure 6E,
with A. nidulans wild type exposed to a sublethal con- F).
centration of AFP (0.2 μg/ml). We applied a pro- Based on our observation that Ca2+ ions antagonize
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tein amount below the toxic concentration for hyphae the growth inhibitory activity of AFP , we ques-
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to maintain the cellular structure and to avoid apoptotic tioned whether Ca2+ prevents actin-mediated internali-
cell disruption [34]. Our study revealed that the protein sation of the antifungal protein. Indeed, the presence of
was internalized after 90 min of incubation, mostly in 10 mM CaCl inhibited protein uptake (Figure 6G, H).
2
hyphal tips, but also within hyphal segments (Figure 6A, Most interestingly, no specific fluorescent signals were
B). The protein seemed not to localize to cell compart- detectable in M. circinelloides when treated with up to
ments, but was distributed in the cytoplasm. Similar 500 μg/ml of antifungal protein (data not shown), indi-
results were obtained with A. niger wild type (data not cating that AFP does not bind to insensitive
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shown). Control experiments proved the specificity of strains.
the intracellular immunofluorescent signals: no intracel-
lular fluorescent signals were detected in samples where Discussion
either AFP (Figure 6C, D) or the primary antibody In this study we provide important insights into the
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or the secondary antibody was omitted (data not mechanistic basis of AFP , a AFP homologous
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shown). protein.
To analyse the AFP localization in more detail, Species specificity tests revealed that AFP is
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A. nidulans was incubated with AFP in the pre- active against a broad range of filamentous fungi,
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sence of latrunculin B, a potent inhibitor of actin including human and plant pathogens. Although the
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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 anti-
fungal protein PAF [9]. 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 regu-
late 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 [25]. Importantly, the
activation of the CWIP can occur in a RhoA-dependent,
e.g. with CFW [9,43], or RhoA-independent way, the lat-
ter proved for PAF and caffeine [9,16] and for
AFP (this study). As proposed by [28] the domi-
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nant 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
AFP similarly to the mode of action of the P.
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chrysogenum PAF [9]. Our assumption of the activation
of the CWIP by AFP was further strengthened by
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the fact, that AFP treatment induced agsA expres-
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sion in the A. niger reporter strain. This result was con-
sistent with the activity of AFP and caspofungin [10],
but differed to the function of PAF, where no CWIP
activation and no induction of cell wall biosynthesis
genes occurred [9].
Therefore, we conclude that AFP triggers cell
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wall remodeling via Pkc/Mpk signalling. We further
deduce from our data that similarities and differences
Figure6IndirectimmunofluorescencestainingofA.nidulans
exist in the molecular targets and the mode of action of
withrabbitanti-AFP antibody.Fungiwereincubatedwith
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0.2μg/mlAFP (A,E,G)orwithoutantifungalprotein(C).20 antifungal proteins from filamentous fungi, e.g.
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μg/mllatrunculinB(E)and10mMCa2+(G)significantlyreduced AFP and PAF - despite their homology. This phe-
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proteinuptake.(B,D,F,H)aretherespectivelightmicroscopic nomenon was also reported for other closely related
imagesof(A,C,E,G).Scalebar10μm.
antifungal proteins, such as the plant defensins MsDef1
and MtDef4 from Medicago spp. [44].
Apart from the activation of the CWIP, the perturba-
proteins AFP and AFP are almost identical and tion of the Ca2+ homeostasis represents a major
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show a similar toxicity, MICs for AFP differed mechanistic function of antifungal proteins in sensitive
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slightly from those reported for AFP [21]. We attribute fungi [17,18]. The intracellular Ca2+ response to
this discrepancy to differences in the experimental set- AFP in A. niger reflected that of the Penicillium
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ups, e.g. fungal strains, medium composition, conidial antifungal protein PAF in N. crassa [17]. The rapid and
inoculum, incubation times, cultivation temperature etc., sustained increase of the [Ca2+] resting level depended
c
rather than to the differences in the primary sequence on a sustained influx of Ca2+ ions from the external
of both proteins. medium. Moreover, the AFP induced changes in
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It has been reported that the closely related AFP pro- the Ca2+ signature of mechanically perturbed A. niger
tein interfered with cell wall synthesis [10] and our find- cells further underlines the disruption of the Ca2+
ing that the osmotic stabilizer sorbitol neutralized response and homeostasis by AFP . The addition
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AFP toxicity further corroborated this assumption. of CaCl to the growth medium reduced the susceptibil-
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Two A. nidulans mutants, the conditional alcA-PkcA ity of A. niger towards the antifungal protein and
and the mpkA deletion mutant showed a hypersensitive decreased the AFP specific rise in the [Ca2+] rest-
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phenotype when exposed to AFP . This is in ing level. Both observations point towards an adaptive
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response which is mediated most probably via Ca2+ sig- withthePenicilliumPAF(unpublisheddata).Onepossible
nalling.First,highextracellularCa2+concentrationstrig- explanationmightbethatextracellularCa2+ionscompete
ger chitin synthesis in A. niger and thereby confer withAFP forthesamemoleculartargetonthefun-
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increasedprotectionagainstantifungalproteinsasshown galsurfacewhichmightrepresentafirstbindingreceptor
for AFP [15]. Second, it primes the Ca2+ homeostatic orevena“gate”forproteinuptake[20,21]or,alternatively,
machinery to better maintain a low [Ca2+] resting level thattheinteractingtargetisrepressedunderthesecondi-
c
when challenged with the antifungal protein, e.g. by (i) tions[17].Anadditionalexplanationmightbethatthepri-
theincreaseoftheactivityofexistingCa2+pumps/trans- mary cell-surface localized AFP target might be
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porterstocounteracttheAFP -specificintracellular masked due to a Ca2+-dependent stimulation of chitin
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Ca2+ perturbation, or (ii) the modulation of the expres- synthesisandcellwallremodelingasrecentlyobservedfor
sion of Ca2+ channels/pumps/exchangers [17]. The for- AFPinA.niger[15].Thisfurthersuggeststhattheactiva-
merhypothesis(i)mightbesupportedbytheobservation tionof the CWIP and the agsA inductiondoes not med-
that the addition of CaCl only 10 min before A. niger iate sufficient resistance to survive the toxic effects of
2
was challengedwithAFP restoredthe low [Ca2+] AFP . Instead, according to the “damage-response
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resting level. However, the perturbation of the Ca2+ framework of AFP-fungal interactions” [15], the chitin
homeostasisby a sustained elevationofthe [Ca2+] rest- response might represent the better strategy for fungi to
c
ing level indicatesthat A.nigeris not able torestore the survivetheantifungalattack.
low[Ca2+] restinglevelafterexposuretoAFP and
c NN5353
this might trigger programmed cell death (PCD) on the Conclusions
long term as it was shown to occur in A. nidulans in Based on the growth inhibitory activity, antifungal pro-
responsetotheP.chrysogenumPAF[34]. teins like AFP can be well considered as promis-
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Since AFP was shown to cause membrane permeabili- ing candidates for future antimycotic drug
zation [21], the influx of Ca2+ might be due to changes developments. However, for biotechnological exploita-
in membrane permeability for this ion, if not the forma- tion, the detailed knowledge on the mode of action is
tion of pores. However, our staining experiments with demanded. Our study shows that the detrimental effects
CMFDA and PI exclude this possibility at least in the caused by the A. giganteus antifungal protein AFP
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first 10 min of exposure to AFP when the [Ca2+] in sensitive target aspergilli are based on the interaction
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resting level reaches its maximum. This result is further of this protein with more than one signalling pathway.
corroborated by the fact that higher external concentra- In Figure 7, we present a tentative working model. The
tions of Ca2+ reduced the AFP specific rise in toxicity of AFP is mediated via PkcA/MpkA sig-
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[Ca2+] resting level which - in our opinion - would not nalling which occurs independently from RhoA. Instead,
c
occur with leaky membranes. However, we do not so far unidentified RhoA-GAP effector molecules might
exclude changes in membrane permeability at longer contribute to AFP toxicity. The activation of the
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exposure times to this antifungal protein and more stu- CWIP by AFP induces the agsA gene expression
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dies are needed to answer this question. which is, however, insufficient to counteract toxicity of
Finally, we observed that the internalization of the protein. Furthermore, AFP leads to an
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AFP ischaracteristicforsensitivebutnotresistant immediate and significant increase of the [Ca2+] resting
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moulds. A lack of binding of AFP to insensitive level in the cell. This sustained perturbation of the Ca2+
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fungi might point towards the absence or inaccessibility homeostasis could lead to PCD [17,34]. The presence of
of a putative interacting molecule at the cell surface. extracellular Ca2+ neutralizes the toxic effects of
AFP localizedtothecytoplasmoftargetfungionly AFP and improves the resistance of the target
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when actin filaments were formed. This is inagreement organism possibly by decreasing the elevated [Ca2+]
c
withtheendocytoticuptakeandintracellularlocalization resting level and stimulating the fortification of the cell
oftheP.chrysogenumantifungalproteinPAFinsensitive wall by the induction of chsD expression as shown for
filamentousfungi[14,45].Importantly,weobservedthat AFP [15]. Further investigations are in progress to clar-
AFP was internalized by hyphae even under sub- ify how these pathways are interconnected and interfere
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inhibitory concentrations (0.2 μg/ml for A. nidulans) with each other on the molecular level.
whichsuggeststhatathresholdconcentrationisrequired
tocauseseveregrowthdefectsintargetfungi. Methods
ThepresenceofhighconcentrationsofextracellularCa2 Strains, Media and Chemicals
+ counteracted AFP uptake. This finding parallels Fungal strains used in this study are listed in Table 5.
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well with the report of [20] that the presence of cations, All strains were obtained from the culture collections
such as Ca2+, interfered with the binding of AFP to the FGSC, ATCC, CBS, from the Institute of Microbiology,
surface of F. oxysporum and with our observations made Division of Systematics, Taxonomy and Evolutionary
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Figure7TentativemodelofthemechanisticfunctionoftheA.giganteusantifungalproteinAFP onAspergillussp.Theresponse
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againstAFP attackismediatedviaPkcA/MpkAsignallingandresultsinincreasedagsAtranscription.However,theactivityoftheCWIP
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occursindependentlyfromRhoAandsofarunidentifiedRhoA-GAPeffectormoleculesmightcontributetotheAFP toxicity.Furthermore,
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AFP leadstoanimmediateandsignificantincreaseofthe[Ca2+] restinglevelinthecell.ThesustainedperturbationoftheCa2+
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homeostasiscouldleadtoPCD[17,34].ThepresenceofelevatedconcentrationsofextracellularCa2+counteractsthetoxiceffectsofAFP
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andimprovestheresistanceofthetargetorganismbydecreasingtheelevated[Ca2+] restinglevel.WhereascellwallremodellingviaCWIP
c
seemstobeinsufficienttocounteractAFP activity,thefortificationofthecellwallbytheinductionofchsDexpressionmightrepresentan
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adequateresponsetoincreaseresistance[15].
Biology at the Leopold Franzens University of Mogens T. Hansen, Novozymes, Denmark. The antifun-
Innsbruck, or the strain collection of the Department of gal protein was isolated from A. giganteus strain A3274
Biotechnology, National Institute of Chemistry, Ljubl- (CBS 526.65), purified and analyzed by HPLC as
jana, Slovenia. Unless otherwise stated, all fungi were described in the patent application WO94/01459 [47].
grown in complete medium (CM) [19] with the respec-
tive supplements [28,38]. R153 and alcA-PkcA were Growth inhibition assays
grown in defined minimal medium (MM) according to Antifungalactivityassayswereperformedin96-wellplates
[26]. Ca2+ response experiments were performed in inCMorVogelsmediuminoculatedwith1×104conidia/
Vogels medium [46]. For experiments with CaCl sup- ml and supplemented with various concentrations of
2
plementation, the KH PO concentration of the culture AFP or with equivalent amounts of buffer
2 4 NN5353
media was reduced from 37 mM to 10 mM to avoid (untreatedcontrols).Fungalgrowthwasmonitoredmicro-
precipitation of supplemental Ca2+ and these media scopically with an Olympus CK40 microscope equipped
were called CM* and Vogels*. Chemicals were pur- with a Zeiss MRc digital camera and the growth rates
chased from Sigma. AFP and polyconal rabbit weredeterminedspectrophotometricallyasdescribedpre-
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anti-AFP antibody were generous gifts from viously[19].Alternatively,2×103conidiawerespottedin
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