This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013 New J. Chem., 2013, 37, 1569--1577 1569 Cite this: New J. Chem., 2013, 37, 1569 A molecular analysis of the toxicity of alkyltributylphosphonium chlorides in Aspergillus nidulans† Diego O. Hartmann and Cristina Silva Pereira* Investigating ionic liquids in vivo effects at a molecular level is crucial for the deeper understanding of their toxicity and the development of new biological applications. In the present study, we propose the use of qRT-PCR to analyse the expression of Aspergillus nidulans genes after exposure to alkyltributylphosphonium chlorides ([P 4 4 4 n ]Cl, where n = 1, 4, 8 or 12). The selected genes are involved in plasma membrane and cell wall biosynthesis and repair mechanisms. The data strongly indicate cell wall damage as the common mechanism of toxicity amongst these ionic liquids, while plasma membrane permeabilisation is dependent on the alkyl substituent length. Considering their effects on the fungal cell walls, the knowledge herein produced opens doors for several possible applications of quaternary phosphonium ionic liquids, in particular their potential use in antifungal formulations. Introduction Over the last two decades, the potential of ionic liquids has been extensively explored. Their inherent potential comes from their structural diversity and tunable physical and chemical prop- erties. From the estimated millions of possible formulations, 1 several hundred ionic liquids are already well known and characterised. 2 A great amount of data is available on their chemical 1 and physical properties, 2 and numerous applications have been proposed. 3 Ionic liquids are generally defined as salts that are liquid below 100 1C, 1 and some are regarded as green solvents due to their excellent solvation capacity, negli- gible vapour pressure, and bulk non-flammability. 4 However, ionic liquids comprise a very heterogeneous group of fluids that are not intrinsically green. Recent reviews on their environ- mental impact and biodegradability highlight the toxic nature and recalcitrance of some of these compounds. 5,6 Although great amount of data have been produced focusing on imidazolium-based ionic liquids, in recent years, other cationic groups have been receiving more attention. Amongst them are the quaternary phosphonium ionic liquids. They are generally considered thermally and chemically more stable (the latter due to the absence of an acidic proton) than the quaternary ammonium or imidazolium salts. 7 Within the quaternary phosphonium ionic liquids, the tetraalkylphos- phonium ones are, to present, the most studied. They have already been investigated for diverse applications, 8–15 but there are only few studies regarding their environmental impact. Their apparent high toxicity was observed in various aquatic organisms. 16–18 Recently, our group performed a systematic study on the toxicity and biodegradability of alkyltributyl- phosphonium chlorides, namely [P 444 n ]Cl, where n = 1, 3–8, 10, 12 or 14 (Fig. 1). 19 Their inhibitory and lethal effects on Aspergillus nidulans were, as expected, determined by the length of the alkyl substituent in the cation, and correlated well with the cation lipophilicity. 20 Fluorescence microscopy suggested that membrane permeabilisation and cell wall damage are the basis of the toxic mechanism of action of [P 4 4 4 n ]Cl, when n Z 4, while for n o 4 it remained to be solved. 19 Fig. 1 The structure of the alkyltributylphosphonium cation. Instituto de Tecnologia Quı ´mica e Biolo´gica, Universidade Nova de Lisboa, Av. da Repu ´blica, Oeiras, 2780-157, Portugal. E-mail: [email protected]† Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3nj00167a Received (in Porto Alegre, Brazil) 11th February 2013, Accepted 6th March 2013 DOI: 10.1039/c3nj00167a www.rsc.org/njc NJC PAPER Published on 07 March 2013. Downloaded by INSTITUTO DE TECNOLOGIA QUIMICA E BIOLOGICA on 28/10/2014 20:31:07. View Article Online View Journal | View Issue
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This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013 New J. Chem., 2013, 37, 1569--1577 1569
Cite this: NewJ.Chem.,2013,37, 1569
A molecular analysis of the toxicity ofalkyltributylphosphonium chlorides inAspergillus nidulans†
Diego O. Hartmann and Cristina Silva Pereira*
Investigating ionic liquids in vivo effects at a molecular level is crucial for the deeper understanding of
their toxicity and the development of new biological applications. In the present study, we propose the
use of qRT-PCR to analyse the expression of Aspergillus nidulans genes after exposure to
alkyltributylphosphonium chlorides ([P4 4 4 n]Cl, where n = 1, 4, 8 or 12). The selected genes are
involved in plasma membrane and cell wall biosynthesis and repair mechanisms. The data strongly
indicate cell wall damage as the common mechanism of toxicity amongst these ionic liquids, while
plasma membrane permeabilisation is dependent on the alkyl substituent length. Considering their
effects on the fungal cell walls, the knowledge herein produced opens doors for several possible
applications of quaternary phosphonium ionic liquids, in particular their potential use in antifungal
formulations.
Introduction
Over the last two decades, the potential of ionic liquids hasbeen extensively explored. Their inherent potential comes fromtheir structural diversity and tunable physical and chemical prop-erties. From the estimated millions of possible formulations,1
several hundred ionic liquids are already well known andcharacterised.2 A great amount of data is available on theirchemical1 and physical properties,2 and numerous applicationshave been proposed.3 Ionic liquids are generally defined assalts that are liquid below 100 1C,1 and some are regarded asgreen solvents due to their excellent solvation capacity, negli-gible vapour pressure, and bulk non-flammability.4 However,ionic liquids comprise a very heterogeneous group of fluids thatare not intrinsically green. Recent reviews on their environ-mental impact and biodegradability highlight the toxic natureand recalcitrance of some of these compounds.5,6
Although great amount of data have been produced focusingon imidazolium-based ionic liquids, in recent years, othercationic groups have been receiving more attention. Amongstthem are the quaternary phosphonium ionic liquids. Theyare generally considered thermally and chemically more stable
(the latter due to the absence of an acidic proton) than thequaternary ammonium or imidazolium salts.7 Within thequaternary phosphonium ionic liquids, the tetraalkylphos-phonium ones are, to present, the most studied. They havealready been investigated for diverse applications,8–15 but thereare only few studies regarding their environmental impact.Their apparent high toxicity was observed in various aquaticorganisms.16–18 Recently, our group performed a systematicstudy on the toxicity and biodegradability of alkyltributyl-phosphonium chlorides, namely [P4 4 4 n]Cl, where n = 1, 3–8,10, 12 or 14 (Fig. 1).19 Their inhibitory and lethal effectson Aspergillus nidulans were, as expected, determined bythe length of the alkyl substituent in the cation, and correlatedwell with the cation lipophilicity.20 Fluorescence microscopysuggested that membrane permeabilisation and cell walldamage are the basis of the toxic mechanism of action of[P4 4 4 n]Cl, when n Z 4, while for n o 4 it remained tobe solved.19
Fig. 1 The structure of the alkyltributylphosphonium cation.
Instituto de Tecnologia Quımica e Biologica, Universidade Nova de Lisboa,
Av. da Republica, Oeiras, 2780-157, Portugal. E-mail: [email protected]
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nj00167a
Received (in Porto Alegre, Brazil)11th February 2013,Accepted 6th March 2013
1570 New J. Chem., 2013, 37, 1569--1577 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
While many studies on ionic liquids toxicity have alreadybeen published, towards a broad range of organisms, themajority of these are based on the determination of theinhibitory and lethal concentrations of these compounds.6 Afew works tried to escape this trend, by investigating ionicliquids e.g. antibiofilm activity in clinically relevant bacterialstrains,21 induction of higher diversity in the metabolic foot-print of filamentous fungi,22 and induction of the production ofreactive oxygen species and programmed cell death in mam-malian cells.23,24 Other studies focused on the moleculartoxicity of ionic liquids, namely investigating their inhibitoryeffects on the activity of enzymes involved in important cellularprocesses, e.g. acetylcholinesterase,25 adenosine monophos-phate deaminase26 and cytochrome P450.27 However, in vitroanalyses not always reflect the effects produced in livingorganisms. Recently, the first gene expression study on ionicliquids was performed with the soil bacterium Enterobacterlignolyticus after exposure to 1-ethyl-3-methylimidazoliumchloride.28 The molecular mechanisms underlying the resis-tance of this bacterial strain to considerably high concentra-tions of the ionic liquid were discussed. The study opensdoors to new applications of these compounds, such asfermentation processes with engineered strains resistant toionic liquids.28
In the present study, we propose the use of quantitative real-time polymerase chain reaction (qRT-PCR) to analyse theexpression of specific genes involved in plasma membraneand cell wall biosynthesis and repair mechanisms in A. nidulans,after exposure to [P4 4 4 n]Cl, where n = 1, 4, 8 or 12. Aspergillusnidulans has its genome completely sequenced, which allowsthe selection of genes of interest and the design of specificoligonucleotides. Moreover, this halotolerant strain is closelyrelated to an important human pathogen, A. fumigatus. Thedata herein produced complement previous microscopic obser-vations on the mechanisms of toxicity of this class of ionicliquids,19 and reveal that damage to the fungal cell wall plays apivotal role in their toxicity.
ExperimentalChemicals
All compounds used in the preparation of minimal media,with the exception of NaCl (Panreac, 99.5%), were purchasedfrom Sigma Aldrich: D(+)-glucose, K2HPO4, ZnSO4�7H2O,CuSO4�5H2O, FeSO4�7H2O, MgSO4�7H2O, NaNO3, and KCl.Poly(vinylpolypyrrolidone), Calcofluor White M2R (CFW) andglycerol (Z99.5%) were also purchased from Sigma Aldrich.Sodium dodecyl sulphate (SDS, 99%) was purchased from AcrosOrganics.
Ionic liquids
All ionic liquids used in this study were prepared by QUILL(Queen’s University Ionic Liquids Laboratory, The Queen’sUniversity of Belfast, UK), except for [P4 4 4 4]Cl, which wassupplied by Cytec Industries, Canada. Ionic liquids were charac-terised by 1H, 13C and 31P NMR spectroscopy, mass spectrometry,
CHN elemental analysis, and halide and water contentanalyses. The comprehensive study on the synthesis andphysicochemical properties of these ionic liquids was pre-viously published by Adamova et al.29
Fungal strain
Aspergillus nidulans strain FGSC A4 was cultivated on dichloran-glycerol (DG18) agar (Oxoid), and suspensions of fungal conidia(asexual spores), prepared as previously described,30 werestored at �80 1C in cryoprotective solution containing 0.85%w/v NaCl and 10% v/v glycerol.
Experimental conditions
Aspergillus nidulans was cultivated on DG18 agar, at 27 1C, inthe dark, for 5 days prior to harvest. Conidia were harvestedwith a saline solution (0.85% w/v NaCl) and used immediately.A suspension of 106 conidia per ml of medium was incubated in25 ml of minimal medium for 24 hours at 27 1C, withoutagitation. The minimal culture medium containing glucose(10.0 g l�1) and K2HPO4 (1.0 g l�1) was dissolved in distilledwater, sterilised in an autoclave (10 min; 115 1C), and supple-mented with the mixture of essential salts, previously sterilisedby filtration: NaNO3 (3.0 g l�1), ZnSO4�7H2O, (0.01 g l�1),CuSO4�5H2O (0.005 g l�1), MgSO4�7H2O (0.5 g l�1), FeSO4�7H2O(0.01 g l�1) and KCl (0.5 g l�1). After 24 hours of growth, ionicliquids ([P4 4 4 n]Cl, where n = 1, 4, 8 or 12) were added to theculture media to obtain a final concentration corresponding to80% of the minimal inhibitory concentration (MIC) of eachcompound and incubated for one, two or four hours. As positivecontrols two compounds known to cause plasma membrane andcell wall damage (SDS and CFW, respectively) were also testedunder the same conditions. A control without ionic liquids wasalso included. MICs and the concentrations used in this studyfor all tested compounds are shown in Table 1. MICs of ionicliquids towards A. nidulans were reported previously, while forSDS and CFW were determined following the same protocol.19
Total RNA extraction and cDNA synthesis
Mycelia from control or exposed to ionic liquids, SDS or CFWwere recovered by filtration (0.45 mm membrane filters, Millipore)and immediately frozen in liquid nitrogen. Approximately 100 mgof frozen mycelia was ground with poly(vinylpolypyrrolidone)
Table 1 Minimal inhibitory concentrations (MICs) and sub-inhibitory concentra-tions used in the gene expression experiments for alkyltributylphosphoniumchlorides [P4 4 4 n]Cl (where n = 1, 4, 8 or 12), SDS and CFW, defined forAspergillus nidulans
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(0.4 mg per mg of mycelia) using a mortar and pestle. The finalpowder was used in the extraction and purification of total RNAusing the RNeasy Plant Mini Kit (QIAGEN), according to themanufacturer’s protocol. Genomic DNA digestion was donewith the RNase-Free DNase Set (QIAGEN). Quality, integrityand quantity of the total RNA were analysed using a NanoDrop1000 Spectrophotometer (Thermo Scientific) and by running2 mg of RNA into 1% agarose gels in TBE buffer 1�. Thecomplementary DNA (cDNA) was synthesised from 100 ng ofthe total RNA using an iScript cDNA Synthesis Kit (Bio-Rad) inan Applied Biosystems 2720 Thermal Cycler. The reactionprotocol consisted of 5 min at 25 1C, 30 min at 42 1C and5 min at 85 1C.
Oligonucleotides design
Based on the sequences of A. nidulans genes (AspergillusGenome Database, http://www.aspergillusgenome.org/), alloligonucleotide pairs were designed using the GeneFisher2web tool (http://bibiserv.techfak.uni-bielefeld.de/genefisher2),with the exception of those for chsB, fksA, agsB and mpkA, whichwere previously designed by Fujioka et al.31 All oligonucleotideswere produced by Thermo Fisher Scientific (see ESI† for the listof the oligonucleotides used in this study).
Quantitative real-time PCR analysis
The qRT-PCR analysis was performed in a CFX96 ThermalCycler (Bio-Rad), using the SsoFast EvaGreen Supermix(Bio-Rad), 250 nM of each oligonucleotide and the cDNAtemplate equivalent to 1 ng of total RNA, at a final volume of5 ml per well, in three technical replicates. The PCR conditionswere: enzyme activation at 95 1C for 30 s; 40 cycles of denatura-tion at 95 1C for 10 s and annealing/extension at 59 1C for 30 s;and melting curve obtained from 65 1C to 95 1C, consisting of0.5 1C increments for 5 s. Data analyses were performed usingthe CFX Manager software (Bio-Rad). The expression of eachgene was taken as the relative expression compared to the timezero (before incubation with the tested compounds). Theexpression of all target genes was normalised by the expressionof g-actin and b-tubulin (internal controls).
Statistical analysis
Four biological replicates were performed. Statistical analysis ofthe qRT-PCR data was performed using the GraphPad Prismv6.0 software. Treatments with ionic liquids, SDS or CFW werecompared with the control for every respective hour of exposureby multiple Student’s t-test. Differences with a p-value below0.05 were considered statistically significant.
Microscopic analyses
Aspergillus nidulans was cultivated and grown for 24 hours asdescribed above. From the grown cultures, aliquots of 1 mlwere exposed to ionic liquids for one, two and four hours attesting concentrations (0.01, 0.1, 1, 10 and 100 mM) that rangedbelow and above the previously obtained MIC values.19 Thetreated cultures were centrifuged (21 000 � g, 4 1C) and washedfive times with a saline solution (0.85% w/v NaCl). Mycelia were
stained with CFW, at the final concentration of 25 mM, incu-bated for 30 min at 27 1C, in the dark and under agitation(90 rpm). Residual dye was removed by centrifugation (21 000� g,4 1C) and washing (3�), and mycelia were resuspended in 100 mlof saline solution with 10% v/v glycerol. Slides were mountedwith 10 ml of the obtained suspension. Hyphae were observedusing a DM5500 B fluorescence microscope (Leica) with a49 DAPI filter set and a 63�magnification objective and imagescaptured using an Andor Luca R EMCCD camera. This assayprovided only qualitative analysis of the alterations in thehyphal cell wall.
Results and discussion
To further understand the toxic mechanisms of alkyltributyl-phosphonium chlorides we decided to analyse by qRT-PCR theexpression levels of genes involved in plasma membrane andcell wall biosynthesis. In particular, 24-hour-grown A. nidulanswas exposed to sub-inhibitory concentrations of [P4 4 4 n]Cl(where n = 1, 4, 8 or 12), SDS and CFW for one, two and fourhours (Table 1). No biodegradation of the cation is expectedunder these conditions, since, as previously reported, it was notobserved after 21 days of incubation.19 It is important to high-light that damage to the plasma membrane and the cell wall arenot completely separated effects. Since these are interconnectedstructures, the damage imposed to the cell wall can consequentlylead to alterations in the organisation of the plasmamembrane.32 In turn, perturbations of the plasma membranecan also affect the integrity of the fungal cell wall.33
Plasma membrane biosynthetic genes
The fungal plasma membrane, as for most cellular organisms,is a lipid bilayer composed mainly of phospholipids. A commonfeature of all phospholipids is the presence of fatty acids intheir structures; thus synthesis of fatty acids is required for theformation of new molecules of phospholipids. Aspergillusnidulans has two genes involved in the de novo synthesis offatty acids, fasA and fasB.34 They encode for the two subunitsnecessary to form the fatty acid synthase, the enzymaticcomplex that catalyses the formation of long-chain fatty acids.35
In a recent proteomic study, the a subunit of the fattyacid synthase, FasA, of the filamentous fungus Pleurotustuber-regium has been reported to be amongst the proteins withincreased accumulation after treatment with the nonionicsurfactant Tween 80.36 In the present study, exposure to[P4 4 4 n]Cl (where n = 1, 4, 8 or 12) led to an increase in theexpression of fasA in A. nidulans for all the tested ionic liquids,with the highest levels obtained after two hours of exposure(Fig. 2). It is likely that after two hours A. nidulans producedlevels of the fatty acid synthase complex which were sufficientfor the initial response to the imposed damage. The pattern inthe expression of fasA was apparently correlated with the lengthof the alkyl substituent of the ionic liquid. After two hours ofexposure, [P4 4 4 8]Cl and [P4 4 4 12]Cl led to a 2.4-fold increase inthe expression levels of fasA. [P4 4 4 4]Cl provoked a 2.1-foldincrease, while [P4 4 4 1]Cl treatment only increased the
1572 New J. Chem., 2013, 37, 1569--1577 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
expression of fasA 1.7-fold after two hours. Interestingly, theincrease in the fasA expression promoted by any of the testedionic liquids was higher than the one observed for the anionicsurfactant SDS. The higher effect of alkyltributylphosphoniumchlorides in the plasma membrane, when compared to the oneprovoked by a commonly used anionic surfactant, is mostcertainly related to their cationic nature. This relation has beenpreviously observed for cationic surfactants, usually more toxicthan anionic ones,37,38 and for other cationic chemicals,e.g. chitosan.39 One suggested explanation is that the cationiccompounds interact with the negatively-charged headgroups ofphospholipids and, due to their uneven distribution in theplasma membrane, permeabilisation occurs.39,40 As mentionedabove, damage to the cell wall can also affect the organisationof the plasma membrane,32 which explains the observedincrease (although not exceeding 1.5-fold) in the expressionlevels of fasA after exposure to CFW.
In addition to phospholipids, two other classes of lipids playfundamental roles in the plasma membrane structure: sterolsand sphingolipids. Ergosterol is the major sterol present in thefungal plasma membrane. It is very similar, in structure andfunction, to cholesterol.41 However, ergosterol is only found infungi, which renders this lipid and its biosynthetic pathwayvery important targets in the development of antifungaldrugs.42 Exposing A. nidulans to [P4 4 4 n]Cl (where n = 1, 4, 8or 12) for two or four hours led to a significant increase(approximately from 1.5- to 2.1-fold) in the expression levelsof HMGR1 (Fig. 2). This gene encodes a putative 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which isresponsible for the rate-limiting step in ergosterol biosynthesis.43
Interestingly, differently from what was observed for fasA,
the alterations in the expression levels of HMGR1 after treat-ment with [P4 4 4 n]Cl (where n = 1, 4, 8 or 12) did not seem to berelated to the increase in the chain length of the alkyl sub-stituent. Despite some differences, the four tested ionic liquidsseemed to induce a similar response, by increasing the expres-sion of HMGR1 when the exposure time was prolonged. Theincrease provoked by SDS was not significant, and only afterfour hours of exposure, the cell-wall-damaging agent CFWincreased 1.8-fold the expression of HMGR1.
Fluidity of the plasma membrane is crucial for deter-mining its sensitivity or resistance to agents that can cause itspermeabilisation.39 Both fatty acids composition and ergosterolamounts are important factors influencing plasma membranefluidity. Those enriched in saturated fatty acids and with higheramounts of ergosterol are less fluid, and seem to be moreresistant to agents that cause membrane damage.39 Theincrease in the expression of both the fasA (which is involvedin the synthesis of saturated fatty acids) and HMGR1 afterexposure supports that plasma membrane permeabilisation isone of the main mechanisms of toxicity of alkyltributylphos-phonium chlorides.19 One can reasonably hypothesise thatfungi respond to the imposed damage by altering the plasmamembrane fluidity.
In filamentous fungi, ceramides are the most common typeof sphingolipids; they are composed of two moieties: a sphin-goid base, phytosphingosine; and a very-long-chain fatty acid.44
In A. nidulans, two enzymes, encoded by barA and lagA, areresponsible for their condensation. After exposure to [P4 4 4 n]Cl(where n = 1, 4, 8 or 12), there was no increase in the expressionlevels of either barA or lagA, with the exception of [P4 4 4 1]Cl(1.4-fold increase of lagA) (Fig. 2). However, SDS and CFW led to
Fig. 2 Analyses of relative expression of four genes involved in plasma membrane biosynthesis (HMGR1, fasA, barA and lagA) by qRT-PCR. Aspergillus nidulans wasexposed to [P4 4 4 n]Cl (n = 1, 4, 8 or 12), SDS or CFW for one (white), two (grey) or four hours (black). The y axes represent the fold-change in gene expression relative tothe culture at time zero (before exposure). g-Actin and b-tubulin genes were used as internal controls. The asterisk marks significant difference (p-value o 0.05) inexpression of each treatment when compared to the control for the same period of incubation.
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an increase of approximately 1.5- and 2.2-fold, respectively, inthe expression levels of lagA. In fact, exposure to either of thetested ionic liquids led, in general, to a slight decrease inthe expression levels of barA and lagA at the first hour ofincubation. Sphingolipids, together with ergosterol, participatein the formation of lipid rafts in the plasma membrane.45
These are special domains responsible for anchoring severalproteins such as structural elements, receptors and sensors.In filamentous fungi, sphingolipids and lipid rafts are pre-sent, particularly, at the tip of the hyphae, playing a vital role inthe establishment of polarity and hyphal growth.44,46 Theexpression levels of barA and lagA observed here further high-light the toxic nature of these ionic liquids. As an initialresponse to the presence of these toxicants, the fungus mostlikely undergoes an arrest of the vegetative growth (polarisedgrowth), directing its biosynthetic machinery towards the pro-duction of molecules that can help it to overcome the toxiceffects of ionic liquids.
Cell wall biosynthetic genes
The fungal cell wall accounts for a great percentage of the celldry weight, and is responsible for maintaining its shape,counteracting the turgor pressure and protecting the plasmamembrane against mechanical damage.47 The cell wall ofA. nidulans is composed of an inner layer of polysaccharides,mainly chitin, 1,3-b-glucans and 1,3-a-glucans, and an outerlayer composed of highly glycosylated proteins.48 Upon damageto the cell wall, fungi respond by activating several genesinvolved in its biosynthesis, creating conditions that allowthem to re-establish its integrity, through the so-called cell wallintegrity (CWI) pathway (see below).
A typical response to cell wall damage is the increasedexpression of genes related to chitin synthesis and its deposi-tion in the cell wall, to confer greater resistance.32 The first andrate-limiting step in the synthesis of chitin is catalysed byglutamine-fructose-6-phosphate transaminase.33 The expres-sion levels of its encoding gene, gfaA, have been previouslyshown to increase upon exposure to cell wall damaging agents,such as CFW, caspofungin and SDS, in A. niger,33 and micafunginin A. nidulans.31 In the former study, the authors also demon-strated that chitin biosynthesis and deposition were alsostimulated.33 Exposing A. nidulans to [P4 4 4 n]Cl (where n = 1,4, 8 or 12) led to an increased expression of gfaA (Fig. 3).Unsurprisingly, both SDS and CFW also led to an up-regulationof this gene, reaching 2.6- and 2.5-fold increase after four hoursof incubation, respectively. The ionic liquids effect seemed tobe related to the length of the alkyl substituent. Only after onehour of exposure to [P4 4 4 12]Cl, gfaA levels increased 2.0-fold,and reached a maximum of 2.3-fold after two hours. However,two hours of exposure to [P4 4 4 8]Cl or [P4 4 4 4]Cl were necessaryto significantly increase the expression levels of this gene(2.0- and 2.1-fold increase, respectively). Finally, althoughthe expression of gfaA increased after four hours of exposureto [P4 4 4 1]Cl, the data were not statistically significant.
While gfaA encodes the enzyme responsible for the first stepin chitin biosynthesis, three more steps are required for thesynthesis of UDP-N-acetylglucosamine. This monomer is thesubstrate of a group of enzymes called chitin synthases,responsible for the final step in chitin synthesis and itsdeposition in the cell wall.49 Aspergillus nidulans has a total ofeight genes coding for this type of enzymes,48 thought to bedifferentially regulated during development.49 For example,
Fig. 3 Analyses of relative expression of four genes involved in cell wall biosynthesis (gfaA, chsB, fksA and agsB) by qRT-PCR. Aspergillus nidulans was exposed to[P4 4 4 n]Cl (n = 1, 4, 8 or 12), SDS or CFW for one (white), two (grey) or four hours (black). The y axes represent the fold-change in gene expression relative to the cultureat time zero (before exposure). g-Actin and b-tubulin genes were used as internal controls. The asterisk marks significant difference (p-value o 0.05) in expression ofeach treatment when compared to the control for the same period of incubation.
1574 New J. Chem., 2013, 37, 1569--1577 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
ChsA is expressed specifically during the asexual development;ChsB is present throughout the vegetative growth; and expres-sion of ChsC and ChsD is seen during the sexual developmentof the fungus.49–51 In this study, we chose chsB as a representa-tive of chitin synthases, for its major role in A. nidulans hyphalgrowth and for its proven up-regulation upon cell walldamage.31 Exposure of A. nidulans to sub-inhibitory concentra-tions of [P4 4 4 n]Cl (where n = 1, 4, 8 or 12) led, in general, to anincrease in the levels of chsB, even if few of these observationswere statistically significant (Fig. 3). In addition, contrary towhat was observed for gfaA, neither SDS nor CFW significantlyaltered the expression levels of chsB. These observations may bepartially explained by the fact that A. nidulans possess severalchitin synthases; thus, the pool of UDP-N-acetylglucosamineproduced in the first steps of chitin synthesis might have beenutilised by different chitin synthases besides ChsB. As demon-strated by the work of Fujioka et al., several chitin synthasegenes of A. nidulans were up-regulated upon cell wall damageinduced by micafungin.31
Glucans are the more abundant polysaccharides in the cellwall.47 The main one, 1,3-b-glucan, forms long, poorly branchedchains, covalently linked to chitin and other cell wall compo-nents. While the yeast Saccharomyces cerevisiae has two genesrelated to 1,3-b-glucan biosynthesis, A. nidulans has only one,fksA.48,52 The expression of this gene has been previously reportedto increase upon treatment with cell wall damaging agents, e.g.micafungin.31 Exposing A. nidulans to [P4 4 4 n]Cl (where n = 1, 4, 8or 12) for only one hour was sufficient to increase the expressionlevels of fksA (Fig. 3). Exposure for two hours, in general, led toeven higher fold-change values that were kept constant after fourhours of incubation with the ionic liquids. The [P4 4 4 1]Cl effectwas lower than for the other tested ionic liquids after one or twohours of exposure, but after four hours it reached levels similar tothe ones observed for [P4 4 4 4]Cl and [P4 4 4 8]Cl. This suggeststhat the ionic liquids cause a significant damage to the cell wallregardless of the alkyl substituent length.
Two other genes contribute greatly to the total glucancomposition of the A. nidulans cell wall, namely the 1,3-a-glucan synthase encoding genes, agsA and agsB.48 These twogenes respond to cell wall stress in a very distinct manner:while agsB, as other cell wall related genes, is up-regulatedupon cell wall damage, agsA is down-regulated. Regardless ofthis different regulation, an overall response to this stress is theincrease in the synthesis of 1,3-a-glucans and their depositionin the cell wall.31 Exposing A. nidulans to any of the tested ionicliquids led to an up-regulation of agsB that seemed to be relatedto the length of the alkyl substituent of the ionic liquid (Fig. 3).While one-hour exposure to [P4 4 4 1]Cl did not significantlyaffect the expression of agsB, exposure to [P4 4 4 4]Cl, [P4 4 4 8]Clor [P4 4 4 12]Cl for the same period of time led to a 1.7-, 2.1- and2.3-fold increase, respectively. These levels were, in general,kept constant for longer exposure times, except for [P4 4 4 1]Cl,that increased after two (1.7-fold) and four hours (2.5-fold). Thisobservation is highly suggestive that as rapidly as after fourhours, despite the alkyl substituent length, all ionic liquids leadto a similar increase in the expression of agsB.
In our previous study on alkyltributylphosphonium chlorides,based on the fluorescent dye CFW and its ability to bind to cellwalls, we could only observe cell wall damage for [P4 4 4 n]Clwhen n Z 4, in a relation where the effect of the ionic liquidwas more significant as the length of the alkyl substituentincreased.19 In the present study, we were able to detect, byqRT-PCR, alterations in gene expression characteristic of cellwall damage for all tested ionic liquids, including [P4 4 4 1]Cl.These differences could be due to the fact that, in our previousstudy, the microscopic assessment of cell wall damage wasperformed only with conidia (asexual spores),19 while themolecular analyses herein presented are based on mycelia. Torule out this possibility, we performed the same assay with24-hour-grown A. nidulans mycelia exposed to [P4 4 4 n]Cl (wheren = 1, 4, 8 or 12) for one, two or four hours. As exemplified inFig. 4, exposure of mycelia to alkyltributylphosphonium chlorideswith longer alkyl substituents, such as [P4 4 4 12]Cl, leads toan heterogeneous distribution of CFW, indicative of cell walldamage. On the other hand, exposure of A. nidulans to 100 mM of[P4 4 4 1]Cl, even for four hours, results in a homogeneousdistribution of fluorescence around the hyphae, similar to whatis observed in the control. The gene expression analysesreported here allowed the detection of changes, at a molecularlevel, characteristic of cell wall damage, even before it reacheslevels detectable by microscopy.
Cell wall integrity pathway
The CWI pathway is a salvage mechanism that is triggered upondamage to the cell wall.31,32 It is very well studied in the yeastS. cerevisiae,32 and, despite many knowledge gaps, it is alsoknown in a variety of filamentous fungi, such as A. nidulans.31
The activation of the CWI pathway is only possible due to thepresence of sensors in the cell surface that detect and transmitalterations in the plasma membrane tension, provoked bydamage to the cell wall.31,32 Up to now, only two sensorsinvolved in cell wall damage (WscA and WscB) were describedfor A. nidulans,53 even though additional ones have beensuggested.54 These proteins are imbedded in the plasmamembrane and bound to the cell wall glucans. In the presentstudy we specifically considered the effect of the ionic liquidson the expression of wscA, the gene coding for the majority ofthese sensors. Interestingly, exposure of A. nidulans to [P4 4 4 n]Cl(where n = 1, 4, 8 or 12) led to an increase in the expression ofwscA, which reached a maximum at two or four hours (Fig. 5).Exposure to [P4 4 4 1]Cl for four hours led to a 2.9-fold increase,
Fig. 4 Cell wall damage assay. Aspergillus nidulans grown for 24 hours andexposed to 100 mM of alkyltributylphosphonium chlorides, [P4 4 4 n]Cl (n = 1 or 12),for four hours and stained with Calcofluor White M2R (CFW). A, negative control;B, [P4 4 4 1]Cl; C, [P4 4 4 12]Cl. Scale bar: 10 mm.
This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013 New J. Chem., 2013, 37, 1569--1577 1575
while [P4 4 4 4]Cl, [P4 4 4 8]Cl and [P4 4 4 12]Cl provoked a 2.2- to2.4-fold increase in the expression of this gene. However, SDShas only led to a 1.7-fold increase in the expression of wscA, andfour hours of exposure to CFW did not significantly increase itsexpression. The functionality, i.e. the sensing spectrum, ofthis family of sensors seems to be different amongst species.53
In A. nidulans, these proteins have been discarded in thestress response to antifungal agents, such as CFW, contraryto that reported for S. cerevisiae. However, based on the pre-sent data, it is possible that they play an important role insignalling cell wall stress provoked by alkyltributylphos-phonium chlorides.
From what is well known in S. cerevisiae, the information ofcell wall stress received by the surface sensors is transmitted toa series of proteins that act as transducers in the signallingcascade. One of the key proteins in this pathway is the mitogen-activated protein kinase, MAPK.32 Its gene is up-regulated uponcell wall stress, in a positive feedback manner. MAPK isresponsible for activating transcription factors that regulatethe expression of several cell wall related genes, such as thoseinvolved in chitin, glucan and cell wall protein synthesis.31,32
In A. nidulans, only the expression of agsA and agsB (genesinvolved in 1,3-a-glucan biosynthesis) and, partially, of gfaA(involved in chitin biosynthesis) is regulated by a MAPK,MpkA.31 The other cell wall related genes are regulated by anunknown signalling cascade. In fact, when exposing A. nidulansto any of the tested ionic liquids, no alterations in the
expression levels of mpkA were observed (Fig. 5). Only treatmentwith SDS for four hours or CFW for one, two or four hoursinduced the up-regulation of this gene. This last observationcorrelates well with the increase also observed for agsB and gfaAupon exposure to SDS and CFW (Fig. 3). However, even thoughboth genes increased upon treatment with [P4 4 4 n]Cl (wheren = 1, 4, 8 or 12), the expression levels of mpkA remained, ingeneral, unaltered. This observation indicates that the trans-cription factor involved in the positive-feedback expression ofmpkA is not activated upon ionic liquid stress, or could evensuggest the existence of an alternative signalling cascade for theregulation of agsB and gfaA upon cell wall stress in A. nidulans.
Conclusions
Exposure of A. nidulans to alkyltributylphosphonium chlorides([P4 4 4 n]Cl, where n = 1, 4, 8 or 12) led to an up-regulation offasA and HMGR1, genes involved in the synthesis of saturatedfatty acids and ergosterol, respectively. This suggests thatA. nidulans alters the plasma membrane fluidity in responseto the membrane permeabilisation provoked by these ionicliquids. Particularly for fasA, there is a correlation between thelength of the alkyl substituent and the effect provoked by theionic liquid, supporting previous observations.6,19 No increasewas observed in the expression of barA and lagA, two genesrelated to the synthesis of sphingolipids, which might be due toan arrest in the vegetative growth of the fungus as a firstresponse to the toxic nature of these ionic liquids. In goodagreement with what is observed under cell wall stress condi-tions, the expression levels of the genes involved in chitin,1,3-b-glucan and 1,3-a-glucan synthesis increased upon expo-sure to alkyltributylphosphonium chlorides. The ionic liquidsalso led to the up-regulation of the gene of one membranesensor (wscA), but do not seem to alter the expression of theprotein kinase gene mpkA. If ionic liquids are indeed activatingalternative signalling pathways, these compounds could beexploited to unravel unknown biological processes; however,further investigation is necessary.
The data herein presented highlight that, after a preliminaryscreening, gene expression analysis is a powerful tool to inves-tigate, in depth, the mechanisms of toxicity of ionic liquids.Moreover, taken together, the data strongly indicate that cellwall damage is the common mechanism of toxicity of alkyl-tributylphosphonium chlorides, and plasma membrane per-meabilisation appears only as a mechanism dependent on theincrease in the length of the alkyl substituent. Having inconsideration their effects on the fungal cell walls (a structurewhich is absent in mammalian cells), the knowledge hereinproduced opens new doors for the possible applications of thisfamily of ionic liquids, e.g. as antifungal surface agents. Studieson the molecular effects of ionic liquids can further elucidatetheir mechanisms of toxicity and those underlying microbialresistance to these compounds. Moreover, it paves the way to abroad range of new applications in biological sciences,55 inparticular, ionic liquids use in altering the expression of genesof interest.
Fig. 5 Analyses of relative expression of two genes related to the cell wallintegrity pathway (wscA and mpkA) by qRT-PCR. Aspergillus nidulans wasexposed to [P4 4 4 n]Cl (n = 1, 4, 8 or 12), SDS or CFW for one (white), two (grey)or four hours (black). The y axes represent the fold-change in gene expression(with standard deviations) relative to the culture at time zero (before exposure).g-Actin and b-tubulin genes were used as internal controls. The asterisk markssignificant difference (p-value o 0.05) in expression of each treatment whencompared to the control for the same period of incubation.
1576 New J. Chem., 2013, 37, 1569--1577 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
Acknowledgements
D. O. H. is grateful to Fundaçao para a Ciencia e a Tecnologia(FCT) for the fellowship SFRH/BD/66396/2009. The work waspartially supported by a grant from Iceland, Liechtenstein andNorway through the EEA financial mechanism (Project PT015)and by FCT through the grant PEst-OE/EQB/LA0004/2011. Theauthors thank Prof. Luıs P.N. Rebelo and Dr Ricardo O. Louro(ITQB) for their continuous support and critical reading of themanuscript. D. O. H. is indebted to Dr Marija Petkovic (ITQB)for the productive discussions during the preparation of themanuscript. The authors acknowledge Prof. Kenneth R. Seddonand Dr Gabriela Adamova (QUILL), and Cytec Industries,Canada, for the supply of the ionic liquids.
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