The Penicillium chrysogenum-derived antifungal peptide shows no toxic effects on mammalian cells in the intended therapeutic concentration

The Penicillium chrysogenum-derived antifungal peptide shows no toxic effects on mammalian cells in the intended therapeutic concentration
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   Naunyn-Schmiedeberg ’ s Arch Pharmacol (2005) 371: 122  –  132DOI 10.1007/s00210-004-1013-7 ORIGINAL ARTICLE Henrietta Szappanos . Gyula Péter Szigeti . Balázs Pál .Zoltán Rusznák  . Géza Sz ű cs . Éva Rajnavölgyi .József Balla . György Balla . Em ő ke Nagy. Éva Leiter.István Pócsi . Florentine Marx . László Csernoch The  Penicillium chrysogenum  -derived antifungal peptide showsno toxic effects on mammalian cells in the intended therapeutic concentration Received: 30 August 2004 / Accepted: 1 December 2004 / Published online: 9 February 2005 # Springer-Verlag 2005 Abstract  Certain filamentous fungi, such as the penicillin- producing strain  Penicillium chrysogenum , secrete small,highly basic and cysteine-rich proteins with antifungaleffects. Affected fungi include a number of important zoo- pathogens, including those infecting humans. Recent stud-ies, however, have pointed to a membrane-perturbing effect of these antifungal compounds, apparent as a potassiumefflux from affected fungal cells. If present on mammaliancells, this would severely hinder the potential therapeuticuse of these molecules. Here we studied the effects of the  P. chrysogenum -derived antifungal peptide (PAF) on anumber of mammalian cells to establish whether the pro-tein has any cytotoxic effects, alters transmembrane cur-rents on excitable cells or activates the immune system.PAF, in a concentration range of 2  –  100  μ  g/ml, did not cause any cytotoxicity on human endothelial cells from theumbilical vein. Applied at 10  μ  g/ml, it also failed to mod-ify voltage-gated potassium channels of neurones, skeletalmuscle fibers, and astrocytes. PAF also left the hyperpo-larization-activated non-specific cationic current (  I  h ) andthe L-type calcium current unaffected. Finally, up to 2  μ  g/ ml, PAF did not induce the production of pro-inflammatorycytokines such as IL-6, IL-8, and TNF- α  . These resultssuggest that PAF should have only minor, if any, effects onmammalian cells in the intended therapeutic concentrationrange. Keywords  Potassium current  . Calcium current  . Patchclamp . Cytotoxicity . Inflammatory action . Antifungal proteins Introduction Filamentous fungi secrete a large number of proteinswhich might be part of their defense mechanism. Recent-ly, a number of reports have focused on antifungal pro-teins secreted by  Penicillium chrysogenum  (Marx et al.1995),  Penicillium nalgiovense  (Geisen 2000),  Aspergil-lus niger   (Lee et al. 1999), and  Aspergillus giganteus (Wnendt et al. 1994). These peptides, while unrelated tocysteine-rich antimicrobial proteins from other species(Lehrer and Ganz 1996; Dimarcq et al. 1998; Fritig et al. 1998; Garcia-Olmedo et al. 1998), have a high homology including their amino acid sequence and relatively basic H. Szappanos . G. P. Szigeti . B. Pál . Z. Rusznák . G. Sz ű csDepartment of Physiology, RCMM, MHSC,University of Debrecen,P.O. Box 22 Debrecen, Hungary, 4012É. RajnavölgyiDepartment of Immunology, RCMM, MHSC,University of Debrecen,P.O. Box 22 Debrecen, Hungary, 4012J. BallaDepartment of Medicine, MHSC, University of Debrecen,P.O. Box 22 Debrecen, Hungary, 4012G. Balla . E. NagyDepartment of Neonatology, MHSC, University of Debrecen,P.O. Box 22 Debrecen, Hungary, 4012É. Leiter  . I. PócsiDepartment of Microbiology and Biotechnology,Faculty of Sciences, University of Debrecen,P.O. Box 22 Debrecen, Hungary, 4012F. MarxDepartment of Molecular Biology,Medical University of Innsbruck,Innsbruck, AustriaL. CsernochCell Physiology Research Group of the Hungarian Academy of Sciences, University of Debrecen,P.O. Box 22 Debrecen, Hungary, 4012L. Csernoch ( * )Department of Physiology, Medical and Health Science Center,University of Debrecen,P.O. Box 22 Debrecen, Hungary, 4012e-mail: csl@phys.dote.huTel.: +36-52-416634Fax: +36-52-432289  nature (Marx 2004; Theis and Stahl 2004). They were shown to inhibit the growth of different fungi (Wnendt et al. 1994; Marx et al. 1995; Lee et al. 1999; Geisen 2000; Theis et al. 2003), including human pathogens. Since pep-tides derived from various ascomycetes have long beenused in human therapy, this latter finding clearly identifiesthese antifungal proteins as possible candidates for futureantifungal drug development.While their structure and the pathway of their synthesishave been studied in detail (Wnendt et al. 1994; Marxet al. 1995), little is known about how these antifungal proteins exert their detrimental effects on target organisms.Kaiserer et al. (2003) have recently reported that the anti-fungal protein derived from  P. chrysogenum  (PAF) triggersthe formation of reactive oxygen intermediates in  A. niger  together with reducing its metabolic rate. Besides the re-duced metabolism, PAF was found to induce an efflux of  potassium from cultured  A. nidulans  having intact hyphaltips, indicating a specific efflux pathway for potassium ions(Kaiserer et al. 2003). These effects are alarming for a potential therapeutic use if present on mammalian cells.Furthermore, cells of the human innate immune systemexpress pattern recognition receptors, which are specificfor various microbial compounds. The highly conservedfamily members of toll-like receptors (TLR) are expressedon neutrophils, monocytes, macrophages, and dendriticcells circulating in peripheral blood and respond to minuteconcentrations of cell wall components derived from bac-teria and/or fungi (Barton and Medzhitov 2002). Ligand-induced activation through TLR results in the rapid production of pro-inflammatory cytokines, such as IL-6,IL-1 β , TNF- α  , and IL-8, which initiate inflammation andthe acute phase response (O ’  Neill 2002). Proteins of mi-crobial srcin or their contamination with pg/ml amounts of TLR ligands, such as lipopolysaccharides (LPS) of Gram( − ) bacteria (Triantafilou and Triantafilou 2002) or fungalcomponents (Bellocchio et al. 2004; Braedel et al. 2004), may induce severe inflammatory responses.We therefore set out to explore the potential toxic ef-fects of PAF on a wide variety of mammalian cells, spe-cifically focusing on potassium currents, cell toxicity andinflammatory effects. Here we report that PAF, applied at 10  μ  g/ml, a concentration which was shown to induce potassium efflux from target fungi, failed to modify the potassium currents of all cells examined. In addition, PAFneither affected the total membrane current of hippocam- pal neurones nor changed the L-type calcium current of skeletal muscle fibers. In the concentration range of 2  –  100 μ  g/ml, where it was shown to severely inhibit the growthof a number of filamentous fungi, PAF had no toxic effectson human endothelial cells. Finally, PAF had only little, if any, effects on the production of IL-6, IL-8, and TNF- α  ,indicating that its inflammatory effects should be minor.These results establish PAF as a potential new antifungaltherapeutic agent. Materials and methods Preparation of tissue and cell samples  Preparation of brain slices  The basic steps of the prep-aration were the same as described earlier (Rusznák et al.1997). Briefly, 7- to 14-day-old Wistar rats were decap-itated, and 150- to 200- μ  m-thick, sagittal slices of thecochlear nucleus were cut by employing a Campden vi- bratome (Campden Instruments, Loughborough, UK). Theslicesweremaintainedinanincubationchambercontainingnormal artificial cerebrospinal fluid (aCSF; in mM: NaCl,125; KCl, 2.5; glucose, 10; NaH 2 PO 4 , 1.25; NaHCO 3 , 26;CaCl 2 , 2; MgCl 2 , 1; myo-inositol, 3; ascorbic acid, 0.5;sodium pyruvate, 2). The pH was 7.2 when gassed with95% O 2 /5% CO 2 ; its osmolarity was set to 320 mOsm/l.During the electrophysiological experiments, the sliceswere continuously perfused ( ∼ 1 ml/min) with gassed aCSFsolution. In some cases the extracellular solution con-tained 1 μ  Mtetrodotoxin(TTX;AlomoneLabs,Jerusalem,Israel) during the recordings, to prevent the activation of voltage-gated Na + channels.  Isolation of hippocampal neurones  The neurone isolation procedure was similar to that described earlier (Rusznáket al. 2001). Briefly, after the decapitation of the 6- or 9-day-old rat the brain was removed, and the hippocampusseparated. The enzymatic dissociation was achieved byusing normal aCSF containing 0.03 mg/ml collagenase(type IA) plus 0.12 mg/ml pronase (type XIV), for 50 minat 31°C. The enzyme treatment was terminated by trans-ferring the tissue to normal aCSF containing 1 mg/ml tryp-sin inhibitor (type I-S). Neurones were isolated by gentletrituration with fire-polished Pasteur pipette in HEPES buffered aCSF, containing (in mM): NaCl, 135; KCl, 3;glucose, 10; HEPES, 10; sucrose, 30; CaCl 2 , 2; MgCl 2 , 1.After dissociation, the cells were allowed to settle for 30min prior to the experiments.  Enzymatic isolation and tissue culturing of astrocytes Astrocyte cultures were prepared from the hippocampus of the rat. The preparation of the hippocampus was per-formed in ice-cold dissecting medium (D1; in mM: NaCl,136; KCl, 5.2; Na 2 HPO 4 ·H 2 O, 0.64; KH 2 PO 4 , 0.22; glu-cose, 16.6; sucrose, 22; HEPES, 10; plus 0.06 U/ml pe-nicillin and 0.06  μ  g/ml streptomycin), followed by itsenzymatic dissociation in D1 solution, containing trypsin(0.025 g/ml; 30 min, 37°C). At the end of the incubation period the tissue pieces were transferred to minimum es-sential medium (MEM) supplemented with 10% fetal calf serum (FCS) for 5 min (room temperature). Individualcells were separated by gentle agitation with fire polishedPasteur pipettes. The cell suspension was then diluted to100,000 cells/ml, and 0.5 ml of this suspension wastransferred onto cover-slips situated in 12 wells of a 24-well tray (marginal wells were not used in order to reducethe risk of infection). Cells were allowed to grow at 37°Cin a 5% CO 2  atmosphere. The feeding medium (MEM,supplemented with 10% FCS) was changed on the fol- 123  lowing day, and on every other day, thereafter. Four- to 5-day-old ( ∼ 70  –  80% confluent) cultures were employed for the electrophysiological experiments.  Isolation and voltage clamp of rat skeletal muscle fibers Skeletal muscle fibers isolated enzymatically from the  ex-tensor digitorum communis  muscles of rats were mountedinto a double Vaseline gap chamber as described earlier (Szentesi et al. 1997). Briefly, rats were anaesthetized andkilled by cervical dislocation. The muscles were removedand were treated with collagenase (Sigma, St. Louis, MO,USA; Type I) for 60  –  90 min at 37°C. The isolated fiber was transferred into a recording chamber filled with re-laxing solution containing (in mM) K-glutamate, 150;MgCl 2 , 2; HEPES, 10; and EGTA, 1. Fiber segments in thetwo end pools were permeabilized by a brief exposure to0.01% saponin. After completing the permeabilization, thesolutions were exchanged to internal solution in the open-end pools (containing in mM Cs-glutamate, 120; MgCl 2 ,5.5; Na 2 -ATP, 5, Na-phosphocreatine, 10; glucose, 10;HEPES, 5; and EGTA, 5) and to external solution inthe middle pool (containing in mM TEA-CH 3 SO 3 , 140;MgCl 2 , 2; HEPES, 5; tetrodotoxin, 0.0003 ’  and 3,4-dia-minopyridine, 1). For calcium current measurements 10mM CaCl 2  was added to the external solution and TEA-CH 3 SO 3  was reduced appropriately, while the EGTA con-centration in the internal solution was increased to 20 mMwith reducing Cs-glutamate. For potassium current mea-surements the internal solution contained 120 mM K-glu-tamate instead of Cs-glutamate and the external solutionwas modified to have 140 mM  N  -methyl- D -glucamine in-stead of tetraethyl ammonium (TEA), and 4 mM MgCl 2 instead of 2 mM. All solutions were adjusted to pH 7.2 and300 mOsm/l.  Endothelial cell isolation and culture  Human umbilicalveinendothelialcells(HUVECs)wereremovedfromhumanumbilical vein by exposure to dispase II (Boehringer Mannheim, Vienna, Austria) as described earlier (Ballaet al. 1993). Cells were cultured in medium 199 containing15% fetal bovine serum (both from Life Technologies,Vienna, Austria), supplemented with  L -glutamine, sodium pyruvate, endothelial growth factor, a mixture of penicillinstreptomycin (100 U/ml each), and 5 U/ml heparin (Bio-chemie, Vienna, Austria). Endothelial cells were identifiedas described earlier (Balla et al. 1993).  Activation of white blood cells  Fresh blood samples of healthy donors were obtained from the Regional BloodTransfusion Center. Blood clotting was inhibited by citrate- buffered dextrose. Three milliliters of blood were incu- bated with 300 μ  l of the test samples at 0.2  –  20 μ  g/ml finalconcentrations of PAF. Negative controls were incubatedwith 300 μ  l saline. Positive control samples were incubatedwith 300 μ  l LPS (  E. coli  0128, Difco Laboratories, Sparks,MI, USA) solutions to give a final concentration of 0.1 μ  g/ml. Samples were incubated for 1 h at room temperatureon a shaker and the in vitro culture was continued for 24 hat 37°C in a CO 2  incubator (5% CO 2 ). Plasma was col-lected by centrifugation at room temperature (1,500 rpm,23 min) and stored at   − 70°C until cytokine determination.Functional measurements  Recording of ionic currents in neurones and astrocytes Whole-cell patch-clamp pipettes were fabricated from thin-walled borosilicate glass (Clark Electromedical Instru-ments, Reading, UK), and filled with a solution containing(in mM): KCl, 120; HEPES, 40; MgCl 2 , 1; EGTA, 10; Na 3 -GTP, 0.5; MgATP, 2. The resistance of these patch pipettesvaried between 2 and 4 M Ω  when filled with the pipettesolution. The series resistance was usually between 3 and7 M Ω  and it was compensated by 40  –  80%. The seriesresistance was kept as constant as possible during themeasurements by occasional light suction or reposition-ing of the electrodes. For the whole-cell voltage-clamp ex- periments an Axopatch 200A patch-clamp amplifier wasused, together with a DigiData 1200 interface (Axon In-struments, Foster City, CA, USA). Data acquisition andanalysis were performed by employing the pClamp 6.0software. Unless otherwise stated, cells were incubated inPAF for at least 5 min prior to the measurements.  Recording of ionic currents in isolated rat skeletal muscle fibers  The experimental setup and data acquisition have been described in detail in earlier reports (e.g., Csernoch et al. 1999). Fibers were voltage-clamped and the holding potential was set to  − 100 mV. All experiments were per-formed at 16  –  18°C. Calcium current (  I  Ca  ) was measuredusing 800-ms depolarizing voltage pulses exploring the − 50 to +60 mV voltage range. Potassium current (  I  K  )was measured using 100- or 200-ms depolarizing voltagesteps exploring the  − 80 to +40 mV voltage range. Thelinear capacitive component was subtracted by applying 20mV hyperpolarizing pulses (Szentesi et al. 2001). Cellswere incubated in PAF for at least 5 min prior to themeasurements.  Endothelial cell cytotoxicity assay  Endothelial cell mono-layers were grown in 24-well tissue-culture plates. Prior toexposure to PAF, cells were washed twice with Hanks balanced salt solution (HBSS, Life Technologies). Cellswere treated with PAF at concentrations from 2 to 100 μ  g/ ml in HBSS and in medium 199. After incubation for 7and 16 h, respectively, the test solutions were replacedwith 550  μ  l 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tet-razolium-bromide (MTT) in HBSS and cells were in-cubated for additional 6 h (Mosmann 1983). The amount of reduced MTT was measured spectrophotometrically at 570 nm after the formazan was dissolved in 100  μ  l 10%sodium dodecyl sulfate and 500  μ  l hot isopropanolcontaining 20 mM HCl. Cytokine determination  Measurement of soluble IL-6, IL-8 and TNF- α   cytokine concentrations in the plasmasamples was performed by ultrasensitive double sandwichenzyme immunoassays (ELISA) according to the manu- 124  facturer  ’ s instructions (Biosource International, Camarillo,CA, USA). Cytokine concentrations were calculated fromthe linear range of the standard curves determined in alltitrations. The results were expressed as pg cytokine/mldetected in the undiluted individual plasma samples. Theoptimal dilution of the negative and positive controls wasdetermined in preliminary experiments. The inflammatoryeffect of the test samples was characterized by the relativeincrease in cytokine concentration compared with the basallevel detected in the untreated control plasma. Cytokineconcentrations measured in the LPS-treated blood sampleswere used as positive controls and reflected the magnitudeof the pro-inflammatory response in the different donors.Protocols and chemicals  —   purification of PAF  P. chrysogenum  NCAIM 00237 was grown in a sucrose (20g/l)  –   NaNO 3  (3 g/l) minimal medium for 72 h at 25°C withshaking (4.2 Hz; Marx et al. 1995). Mycelia were removedwith centrifugation and the low molecular weight proteinfraction of the supernatant was separated in Amicon StirredCells (volume=50 ml, Biomix PBTK ultrafiltration discs,size exclusion limit   M  r  =30,000; Millipore, Billerica, MA,USA). PAF was purified with ion exchange chromatogra- phy on a CM Sephadex Fast Flow column (2×18 cm,equilibrated with 50 mM sodium phosphate buffer, pH 6.6,flow rate 1 ml/min,  t  =4°C, NaCl gradient 0.05  –  1.0 M;Amersham-Pharmacia, Uppsala, Sweden). The quality of the preparation was always checked with SDS-PAGE on pre-cast Novex 16% Tris/glycine gels (Invitrogen LifeTechnologies, Carlsbad, CA, USA), where protein bandswere visualized with Coomassie Brilliant Blue R staining.All protocols followed the guidelines put forth by theDeclaration of Helsinki and have been approved by theInstitutional Ethics or Institutional Animal Care commit-tees. Chemicals, unless otherwise stated, were purchasedfrom Sigma and were of analytical grade.Averages were expressed as means±standard error (SE)of the mean. Significance between groups of data wasassessed using Student  ’ s  t   test. Results Ionic currents on neurones in brain slicesIn the first step of the experiments the effects of PAF wereinvestigated on the hyperpolarization-activated nonspecificcationic current (  I  h ) of the bushy cells situated in brainslices prepared from the ventral cochlear nucleus. Thedetailed characterization of this current component was performed in a previous study (Cuttle et al. 2001). In the present experiments 2-s hyperpolarizing pulses were de-livered from a holding potential of  − 60 mVin 10-mV steps,to a maximum hyperpolarization of   − 140 mV. As Fig. 1Aindicates, the time course of the current was not affected inthe presence of 10  μ  g/ml PAF. Although a slight decreasein the  I  h  amplitude can be noticed in Fig. 1A, a statisticallysignificant difference could not be substantiated when thedata from all cells were pooled, as demonstrated by thecurrent   –  voltage relations of the instantaneous (measured just after the capacitive transient) and steady-state (de-termined at the end of the hyperpolarizing voltage step)current components ( n =7). Note that in order to avoid theerror introduced by the different size of the cells in-vestigated, current density was calculated and plotted.In the next step of the experiments the depolarization-activated current components were investigated in slices.In these cases a holding potential of   − 60 mV was appliedand 200-ms voltage steps were delivered starting from − 80 mV up to a maximum depolarization of +40 mV. Nochannel blockers were employed in these experiments.Although this experimental approach does not allow thedetailed investigation of the Na + currents, the recordsdemonstrated in Fig. 1B do not imply any change of theamplitude of the Na + current. The amplitude of the de- polarization-activated outward current was not affectedsignificantly either, as indicated by both the individualcurrent traces and the normalized current   –  voltage relation-ships ( n =12). The latter observation can be clearly assessedon the bases of the data presented in Fig. 1C. In this (andsimilar) experiment a single, 200-ms depolarizing voltagestep was applied from a holding potential of   − 60 to 0 mVdepolarization in every 10 s. TTX was regularly employedin these experiments. After recording the control current traces (and making sure, therefore, that the seal and thecurrent were stable enough) the application of 10  μ  g/mlPAF was commenced. The cell was exposed to the drug for 5 min then a wash-out period followed. As the current traces (recorded prior to and during the application of PAF) as well as the time course of the current amplitudeindicate, no major change was induced by the presenceof the drug.Ionic currents on isolated neurones andastrocytes in cultureThe effect of PAF on the voltage-gated currents of neu-roneswasinvestigatedinamoreconventionalexperimentaldesign as well. In these cases enzymatically separated hip- pocampal neurones were employed. The pulse protocolwas similar to that applied in conjunction with Fig. 1B, but the holding potential was set to  − 90 mV. To excludethe interfering effects of the Na + currents, TTX was addedto the bath solution. As Fig. 2A demonstrates, PAF didnot influence the depolarization activated K  + currents of the hippocampal neurones. Similar experiments were car-ried out on four more cells, and the normalized, pooleddata are presented in Fig. 2B, indicating that neither theactivation thresholds of these components nor their ampli-tudes was affected by PAF application.To have an overview of the potential effects of PAF onthe function of the central nervous system, its effects onastrocytes maintained in tissue culture were also tested.Figure 3A shows that the drug did not seem to induce anychange in the amplitude of the currents present on the 125  Fig. 1  Effect of   Penicilliumchrysogenum -derived antifungal peptide (PAF) on bushy cellssituated in brain slices.  A  Ef-fects on the hyperpolarization-activated non-specific cationiccurrent (  I  h ) of the bushy cells.The holding potential ( V  H ) wasset to  − 60 mV and 2-s hyper- polarizing voltage steps weredelivered to a maximum hyper- polarization of   − 140 mV in10-mV steps. Actual current recordings from the same cellare presented prior to ( control  )and during (  PAF  ) the applicationof 10  μ  g/ml PAF. The  bottom panel   presents pooled data(mean ± SE;  n =7) from similar experiments. Here and in allsubsequent figures current am- plitudes in pooled data werenormalized to the cell capaci-tance.  Squares  indicate the in-stantaneous, while  circles  thesteady-state current   –  voltage re-lations (here and in all subse-quent figures  filled symbols control,  open symbols  PAF ap- plication).  B  Effect of PAF onthe depolarization-activated cur-rents of bushy cells.  V  H = − 60mV was used and 200-ms de- polarizing voltage steps wereemployed to a maximum depo-larization of +30 mV in 10-mVsteps. Current recordings fromthe same cell are presented prior to ( control  ) and during (  PAF  )the application of 10 μ  g/ml PAF.For clarity only every secondtrace is shown.  Bottom panel  displays pooled data from simi-lar experiments ( n =12).  Trian- gles  indicate the peak, while  squares  the steady-state current   –  voltage relations.  C  The cell wasdepolarized to 0 mV for 200 ms( V  H = − 60 mV) once every 10 s.The current amplitude wasmeasured and plotted as thefunction of time.  Open symbols demonstrate current valuesobtained during the applicationof the drug. Current tracesare representative recordingsfrom before and during PAFapplication.126
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