Synthetic Multivalent Antifungal Peptides Effective against Fungi Rajamani Lakshminarayanan1,2*., Shouping Liu1,2., Jianguo Li1,3, Muruganantham Nandhakumar1, Thet Tun Aung1, Eunice Goh1, Jamie Ya Ting Chang1, Padhmanaban Saraswathi1, Charles Tang5¤, Siti Radiah Binte Safie1, Lim Yih Lin1, Howard Riezman4, Zhou Lei1,2, Chandra S. Verma3,6,7, Roger W. Beuerman1,2,6* 1 Singapore Eye Research Institute, Singapore, Singapore, 2 Department of Neuroscience and Behavioural Disorders, Duke-NUS Graduate Medical School, Singapore, Singapore, 3 Bioinformatics Institute (A*STAR), Singapore, Singapore, 4 Department of Biochemistry and NCCR Chemical Biology, University of Geneva, Geneva, Switzerland, 5 Department of Pathology, Singapore General Hospital, Singapore, Singapore, 6 School of Biological Sciences, Nanyang Technological University, Singapore, Singapore, 7 Department of Biological Sciences, National University of Singapore, Singapore, Singapore
Abstract Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010) which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is nonhaemolytic and non-toxic to mice by intraperitoneal (200 mg/kg) or intravenous (100 mg/kg) routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2–4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides. Citation: Lakshminarayanan R, Liu S, Li J, Nandhakumar M, Aung TT, et al. (2014) Synthetic Multivalent Antifungal Peptides Effective against Fungi. PLoS ONE 9(2): e87730. doi:10.1371/journal.pone.0087730 Editor: Rizwan H. Khan, Aligarh Muslim University, India Received October 16, 2013; Accepted December 28, 2013; Published February 3, 2014 Copyright: ß 2014 Lakshminarayanan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research is supported by the Singapore Ministry of Health’s National Medical Research Council under its Translational Research Innovations in Ocular Surgery (TRIOS) Programme: Defensins project NMRC/TCR/002-SERI/2008 (RWB) and NMRC/NIG/1020/2010 (RL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (RL); [email protected] (RWB) ¤ Current address: ALS Food and Pharmaceuticals, Singapore, Singapore. . These authors contributed equally to this work.
Since current antifungals have limited number of targets and increasing reports of resistance to the available drugs, peptidebased antifungals, which disable the membrane physiology, offer an important opportunity for rational drug design [8–10]. However, their antimicrobial properties are antagonized by physiological concentration of salts and polyanionic polymers (e.g., DNA, glycosaminoglycans and mucins) as well as being rapidly degraded by proteolytic enzymes in complex biological milieu, thus limiting their therapeutic potential [11,12]. Several different strategies have been attempted to circumvent the drawbacks of antimicrobial peptides [13–17]. The development of multivalent peptides by assembling multiple copies of monomeric peptides around a core molecule has attracted significant interest as a new molecular phenotype for potential therapeutic use [18–20]. However, the antimicrobial properties of multivalent peptides have been limited to Gram-negative and/or Grampositive bacteria and their utility as potent antifungals has not been explored in great detail [21–24].
Introduction Resistance to current antifungal drugs has become common in recent years, mainly due to increased number of immunocompromised patients, and has underscored the need for new classes of antifungals [1,2]. Pathogenic fungal infections are the 7th most common cause of infection-related deaths in the USA [3]. In fact, recent surveys suggest that fungal diseases are posing a growing threat to the global biota [4]. The number of available antifungals is limited and developing new antifungal drugs is challenging since common drug targets in fungi are homologues of similar molecular types in human which might also be inhibited. Among the five different classes of antifungals polyenes, azoles and allylamines target ergosterol or ergosterol biosynthetic pathways whereas 5-fluorocytosine (5-FC) targets DNA synthesis and caspofungins inhibit b-glucan synthase [5]. The extent of drug resistance also varies, as resistance to 5-FC and azoles are more common when compared to polyenes, although there are now many reports of resistance to polyenes as well [6,7].
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We have previously reported the properties of a 10-residue peptide (RGRKVVRRKK) which showed potent activity against P. aeruginosa, but poor activity against fungi [25]. The antipseudomonas activity was attributed to the concentration-dependent formation of a non-covalent dimer in buffer as well as in liposome model of a prokaryotic membrane [26]. The present work extends the analysis to higher order covalently linked peptides. The antifungal properties of a potent tetravalent peptide (B4010) have been examined in physiological concentrations of salts and in complex biological fluids. We have also assessed the biocompatibility of B4010 including its effect on corneal reepithelialization in rabbit and systemic toxicity in mice. The antifungal action of this molecule on fungal membrane was ascertained using environmental-sensitive fluorescent probes. Finally, we demonstrate that coupling MD simulations with in vitro experiments shed light on the important residues that are essential for mediating membrane-lipid interactions and rationalization of the antifungal activity.
out as described above. C. albicans ATCC10231 cells and salts in broth were taken as a positive control. Salts alone (NaCl, KCl, and CaCl2 and MgCl2) in broth or with 22 mM peptide served as a negative control. The effect of 5% human serum on the activities of B4010 against two C. albicans clinical isolates was determined. The human male serum was centrifuged at 13,000 rpm for 10 mins in order to remove the lipids and the supernatant was collected [27]. The MIC values against clinical isolates of C. albicans 2672R and C. albicans 1976R were determined both in standard medium (SD broth) as well as in standard medium containing 5% serum supernatant as before.
Determination of MIC against Wild Type (WT) and Mutant S. cerevisiae Strains A few identical colonies from WT and mutant strains were immediately inoculated and grown overnight in SD broth containing 0.1 mg/mL ampicillin at 30uC [28,29]. Cells were harvested by centrifugation, washed with sterile water and frozen. Cells were plated again on SD agar plates and incubated at 37uC for 48 h. Two or three identical colonies of the mutant strains were collected and inoculated in SD broth. Peptides were dissolved in sterile water and mixed with WT and mutant strains and incubated at 37uC for 48 or 72 h in a test tube. The lowest concentration of the peptide that inhibited the growth of the yeast strain was determined by visual inspection.
Materials and Methods Ethics Statement All animals used in this study were treated in agreement with the tenets of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and the protocol was approved by the Singhealth Institutional Animal Care and Use Committee (IACUC; AALAC accredited, #2012/SHS/775).
Time-kill Kinetics Assay The time-kill kinetics of B4010 was determined against two strains of C. albicans (ATCC 10231 and DF2672R). The cultures were grown overnight in SD broth and the cell concentration was adjusted to 105–106 CFU/mL with phosphate buffer. The peptide or antifungals were added to the individual cultures. Appropriate final concentrations of B4010 (1.4–11 mM for ATCC and 0.37– 3.7 mM for 2672R) or amphotericin B (0.55–11 mM for 2672R strains) and natamycin (4.7–75 mM for ATCC and 30 mM for 2672R strains) were adjusted for the inocula. The test solution was incubated at 37uC with constant shaking. 100 mL of the suspension was withdrawn at predetermined time points, serially diluted (102 or 103 fold) and poured into the SDA plate. The plate was incubated for 48 h at 37uC for colony counting. The data were expressed in terms of % fungal viability relative to the positive control.
Chemicals and Peptides Sabouraud’s Dextrose Agar was purchased from Acumedia (Michigan, MI, USA). Peptides were purchased from EZBiolabs Inc., (Carmel, IN, USA). Lipids such as L-a-phophatidylcholine (PC), L-a-phostidylethanolamine (PE), L-a-phosphatidylserine (PS) and L-a-phosphatidylinositol (PI) were bought from Avanti Polar Lipids Inc. (AL, USA). Ergosterol, sodium azide (NaN3), Carbonyl cyanide m-chloro phenylhydrazone (CCCP), 4-aminopyridine (4AP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), Gadolinium III Chloride, tetra ethyl ammonium chloride (TEA) and 39,39-dipropylthiadicarbocyanine (diS-C3-5) dye were purchased from Sigma-Aldrich Corp. (MO, USA). ATP bioluminescence kit was obtained from Molecular Probes Inc (OR, USA). Amphotericin B and Natamycin were obtained in powder form from SigmaAldrich (S) Pte Ltd (Singapore).
Antifungal Activity of B4010 in Presence of Trypsin and 50% Tear Fluid (TF)
Determination of Minimum Inhibitory Concentration (MIC)
For all the experiments, unless otherwise stated, C. albicans ATCC 10231 strain was used. B4010 (1 mg/mL) was incubated with trypsin (enzyme:peptide ratio 1:100) at 37uC. 20 mL aliquot of this mixture was withdrawn at various time intervals (0.5, 1, 2, 4 and 6 h) and mixed with 1 mL of trypsin inhibitor. The antifungal activity of the peptide was determined by adding this mixture to 180 mL of inoculum and monitoring the growth at OD600 for 24 h. Similar experiments performed in the presence of trypsin/ trypsin inhibitor (without B4010) were used as a positive control. For assessing the antifungal activity in TF, the peptide was dissolved in freshly collected rabbit TF and incubated at 37uC for 6 h. After incubation, an equal volume of an overnight culture of C. albicans (,106 CFU/mL) was mixed with peptides in tear fluid and incubated at 37uC for 24 h. The final concentrations of the peptide were at 4.4, 8.8 and 22 mM. A 100 mL aliquot of serial dilutions (102 or 103 times) of this mixture were inoculated on a SD agar plate and then incubated for 48 h at 37uC. Culture alone and culture with 50% tear fluid served as the positive control. The
The yeast strains were cultivated and suspended in Sabouraud’s Dextrose (SD) broth diluted to one sixth at a starting OD600 = ,0.08 in a flat-bottomed microtitre plate. A serial dilution of peptide in the same broth was mixed with the inoculum to give a final peptide concentration of 0.4–22 mM. The antifungal activity was assessed by monitoring the OD600 in cycles of 30 minutes and an orbital shaking at 100 rpm using an Infinite M200 microplate reader (Tecan Group Ltd., Switzerland) for 24/48 h at 37uC. Cultures without peptides were used as positive controls and broth alone or with 22 mM peptide served as negative controls. The minimum concentration required for complete inhibition was assessed by both visible observations as well as by measuring the OD600 and taken as the MIC. Each experiment was repeated in triplicates. For studying the effects of metal ions, SD broth was adjusted with appropriate concentrations of salts to yield a final concentration of 100 and 135 mM for NaCl and KCl and 0.5– 2 mM for CaCl2 and MgCl2. MIC determinations were carried PLOS ONE | www.plosone.org
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data was expressed in % killing with respect to culture without tear fluid. About 6–10% killing was observed in the presence of 50% tear fluid.
Candidacidal Activity of B4010 in Presence of Metal Ions, Energy Poisons and Ion Channel Inhibitors To study the effects of metal ions, the broth solution containing C. albicans (OD600 = 0.4–0.6) was adjusted to appropriate final concentrations of metal ions, incubated with 5.5 mM of B4010 for 6 h and the cell viability was determined as before after 48 h. For the effects of energy poisons and ion channel inhibitors, yeast cells (105–106 CFU/mL) were incubated for 2 h with or without additives at 37uC. A final concentration of 5.5 mM B4010 peptide was added to the cell suspension and incubated further for another 1.5 h at 37uC. Fungal viability was obtained by counting the number of colonies formed in each plate after 48 h incubation at 37uC. SDA plates with additives (and no peptide) served as the positive control. The final concentrations of the additives were: CCCP (5 mM), NaN3 (5 mM), 4-AP (1 mM), NPPB (0.5 mM), Gadolinium II chloride (1 mM) and tetra ethyl ammonium chloride (15 mM). The reported values were averages of two independent duplicate experiments. Control experiments with all the additives were also performed to assess the toxicity of the additives to C. albicans.
Haemolytic Assay Haemolytic activity of peptides and antifungal drugs were determined against rabbit red blood cells [30]. Briefly, serial dilution of peptides/antifungals in PBS was mixed with rRBC (final concentration 4% v/v), incubated at 37uC for 1 h and centrifuged at 3000 rpm for 10 minutes. The release of hemoglobin in the supernatant was monitored by measuring the hemoglobin absorbance at 576 nm. The readings from cell suspension in PBS (without any additives) or 1% Triton-X100 were used as 0% or 100% haemolysis.
Cytotoxicity on Human Conjunctival Epithelial Cells Immortalized normal human conjunctival epithelial cells (IOBANHC) were a gift from Yolanda Diebold at the University of Valladolid, Spain [31]. IOBA-NHC cells were cultured under standard conditions (humidified atmosphere of 5% CO2 at 37uC) in DMEM/F12 supplemented with 1 mg/ml bovine pancreas insulin, 2 ng/ml mouse epidermal growth factor, 0.1 mg/ml cholera toxin, 5 mg/ml hydrocortisone, 10% fetal bovine serum (FBS), 50 UI/ml penicillin, and 50 UI/ml streptomycin. Cells from passages 50–80 were used in all experiments. Every day, normal culture development was observed by phase-contrast microscopy. Cells were removed by gentle trypsin incubation at confluence and counted. They were seeded into 96 well culture plates (Corning, Schiphol-Rijk, The Netherlands) for microtitration analysis of flow cytotoxicity assay (,10,000 cells/well). Cultures were kept at 37uC for 24 h. Subconfluent cells (culture surface covering nearly 70%) were then exposed to various peptide concentrations (0.22–225 mM). Cytotoxicity was measured periodically using MultiTox-Fluor Multiplex assay kit (Promega, WI, USA) by measuring the AFC fluorescence (lex = 485 and lem = 520 nm). We report the cell viability after 24 h incubation of the cells with the peptides since there was no detectable toxicity even after 8 h of incubation.
SDS-PAGE Pull down Assay B4010 (5.5–22 mM) was incubated with chitin (from shrimp shells) or b-D-glucan for 2 h at 37uC. The mixture was centrifuged at 10,000 g and the supernatant was analyzed by SDS-PAGE (4– 20%). A control experiment without chitin/b-D-glucan was also performed to quantify peptide binding to the carbohydrate polymers.
DiS-C3-5 Membrane Depolarization and SYTOX Green (SG) uptake Assays Changes in membrane potential of C. albicans upon addition of B4010 were monitored by the release of potential sensitive probe diS-C3-5 dye. Briefly, 1 ml of overnight grown mid-log phase Candida cells in 5 mM HEPES buffer (pH 7.0) was mixed with 10 mM diS-C3-5 and incubated at 37uC for 1–2 h in a thermo shaker. 800 mL of the dye-loaded cell suspension was transferred to a stirred quartz cuvette and the change in fluorescence intensity was monitored at an emission wavelength (lem) of 670 nm (excitation wavelength, 622 nm) using a Quanta Master spectrofluorimeter (Photon Technology International, NJ, USA). The excitation and emission bandwidths were set at 1 and 2 nm, respectively. Once a constant fluorescence level was achieved, 10 mL of concentrated peptide solution in HEPES buffer was added so that the final concentration of peptide was 0.22 - 22 mM. The change in fluorescence intensity was monitored continuously for 1 or 2 h. To study the effects of energy poisons and ionchannel inhibitors, the additives were added prior to the addition of 5.5 mM B4010. The change in fluorescence intensity was monitored as before. For SG uptake assay, overnight cultures of Candida were harvested by centrifugation, washed three times with HEPES buffer and resuspended in the same buffer. The cells were incubated with 1 mM SG in the dark prior to the addition of peptide. The increase in emission intensity at 520 nm was monitored after addition of various concentrations of peptide (lex = 485 nm). 1% Triton-X100 was added at the end of the experiment to determine the %uptake.
Rabbit Model of Corneal wound Healing Four New Zealand white rabbits were divided into two groups, two each for control (saline) and two each for the study group treated with B4010. Rabbits were tranquilized by intra muscular injection of 1 mL of ketamine (100 mg/mL) and 0.5 mL of xylazil (20 mg/mL). Corneas were anesthetized by topical administration of 1% xylocaine. A 5-mm trephine was used to outline the wound margin and mechanical removal of epithelial cells was carried out by sterile mini blade (BD-Beaver) leaving the basal lamina intact as previously described [32]. The animals were treated by topical administration of 22 mM of B4010 at 3 times/day. Cornea wound was visualized by staining with fluorescein sodium, which is used in the ophthalmology clinic as a non-toxic dye to disclose wounds of the cornea, and using a cobalt-blue filter with slit lamp biomicroscopy. Measurements of the residual wound area were performed during the reepithelialization process by Image-J 1.440.
In vivo Toxicity Acute toxicity of B4010 was assessed with C57BL6 (6–8 weeks old) wild type mice. Two healthy wild type mice were chosen for each routes of administration. B4010 was delivered through intraperitoneal (200 mg/kg) or (100 mg/kg) intra-venous routes. Two animals were used for each administration and monitored through 24/48 h to determine mortality, or signs of toxicity. PLOS ONE | www.plosone.org
Measurement of Extracellular Cations Overnight cultured late logarithmic phase C. albicans was harvested by centrifugation, washed 5 times with 10 mM HEPES (pH 7.0), resuspended in the same buffer and adjusted to an 3
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Percentage calcein released at each time point was calculated using the equation,
OD600 = 0.4. To 5 mL of this suspension, B4010 (final concentration 5.5 mM) was added and incubated at 37uC for 2 h. The mixture was centrifuged at 3,000 g and the presence of K+, Ca2+ and Mg2+ in the supernatant was estimated by Perkin Elmer Dualview Optima 5300 DV inductively coupled plasma – optical emission spectrometry (Massachusetts, USA) available at CMMAC facilities (Department of Chemistry, National University of Singapore).
%Calcein Release~
Where A0 is the observed fluorescence intensity upon addition of peptides, Amin is the average intensity at the baseline (before peptide addition), Amax is the average intensity at the saturation phase after adding 1% Triton X-100. Similar protocol was followed for the preparation of PC/Cholesterol SUVs.
ATP Bioluminescence Assay The extracellular ATP levels upon challenging C. albicans with B4010 were determined as reported before [33]. Cells (OD600
Abstract Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010) which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is nonhaemolytic and non-toxic to mice by intraperitoneal (200 mg/kg) or intravenous (100 mg/kg) routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2–4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides. Citation: Lakshminarayanan R, Liu S, Li J, Nandhakumar M, Aung TT, et al. (2014) Synthetic Multivalent Antifungal Peptides Effective against Fungi. PLoS ONE 9(2): e87730. doi:10.1371/journal.pone.0087730 Editor: Rizwan H. Khan, Aligarh Muslim University, India Received October 16, 2013; Accepted December 28, 2013; Published February 3, 2014 Copyright: ß 2014 Lakshminarayanan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research is supported by the Singapore Ministry of Health’s National Medical Research Council under its Translational Research Innovations in Ocular Surgery (TRIOS) Programme: Defensins project NMRC/TCR/002-SERI/2008 (RWB) and NMRC/NIG/1020/2010 (RL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (RL); [email protected] (RWB) ¤ Current address: ALS Food and Pharmaceuticals, Singapore, Singapore. . These authors contributed equally to this work.
Since current antifungals have limited number of targets and increasing reports of resistance to the available drugs, peptidebased antifungals, which disable the membrane physiology, offer an important opportunity for rational drug design [8–10]. However, their antimicrobial properties are antagonized by physiological concentration of salts and polyanionic polymers (e.g., DNA, glycosaminoglycans and mucins) as well as being rapidly degraded by proteolytic enzymes in complex biological milieu, thus limiting their therapeutic potential [11,12]. Several different strategies have been attempted to circumvent the drawbacks of antimicrobial peptides [13–17]. The development of multivalent peptides by assembling multiple copies of monomeric peptides around a core molecule has attracted significant interest as a new molecular phenotype for potential therapeutic use [18–20]. However, the antimicrobial properties of multivalent peptides have been limited to Gram-negative and/or Grampositive bacteria and their utility as potent antifungals has not been explored in great detail [21–24].
Introduction Resistance to current antifungal drugs has become common in recent years, mainly due to increased number of immunocompromised patients, and has underscored the need for new classes of antifungals [1,2]. Pathogenic fungal infections are the 7th most common cause of infection-related deaths in the USA [3]. In fact, recent surveys suggest that fungal diseases are posing a growing threat to the global biota [4]. The number of available antifungals is limited and developing new antifungal drugs is challenging since common drug targets in fungi are homologues of similar molecular types in human which might also be inhibited. Among the five different classes of antifungals polyenes, azoles and allylamines target ergosterol or ergosterol biosynthetic pathways whereas 5-fluorocytosine (5-FC) targets DNA synthesis and caspofungins inhibit b-glucan synthase [5]. The extent of drug resistance also varies, as resistance to 5-FC and azoles are more common when compared to polyenes, although there are now many reports of resistance to polyenes as well [6,7].
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We have previously reported the properties of a 10-residue peptide (RGRKVVRRKK) which showed potent activity against P. aeruginosa, but poor activity against fungi [25]. The antipseudomonas activity was attributed to the concentration-dependent formation of a non-covalent dimer in buffer as well as in liposome model of a prokaryotic membrane [26]. The present work extends the analysis to higher order covalently linked peptides. The antifungal properties of a potent tetravalent peptide (B4010) have been examined in physiological concentrations of salts and in complex biological fluids. We have also assessed the biocompatibility of B4010 including its effect on corneal reepithelialization in rabbit and systemic toxicity in mice. The antifungal action of this molecule on fungal membrane was ascertained using environmental-sensitive fluorescent probes. Finally, we demonstrate that coupling MD simulations with in vitro experiments shed light on the important residues that are essential for mediating membrane-lipid interactions and rationalization of the antifungal activity.
out as described above. C. albicans ATCC10231 cells and salts in broth were taken as a positive control. Salts alone (NaCl, KCl, and CaCl2 and MgCl2) in broth or with 22 mM peptide served as a negative control. The effect of 5% human serum on the activities of B4010 against two C. albicans clinical isolates was determined. The human male serum was centrifuged at 13,000 rpm for 10 mins in order to remove the lipids and the supernatant was collected [27]. The MIC values against clinical isolates of C. albicans 2672R and C. albicans 1976R were determined both in standard medium (SD broth) as well as in standard medium containing 5% serum supernatant as before.
Determination of MIC against Wild Type (WT) and Mutant S. cerevisiae Strains A few identical colonies from WT and mutant strains were immediately inoculated and grown overnight in SD broth containing 0.1 mg/mL ampicillin at 30uC [28,29]. Cells were harvested by centrifugation, washed with sterile water and frozen. Cells were plated again on SD agar plates and incubated at 37uC for 48 h. Two or three identical colonies of the mutant strains were collected and inoculated in SD broth. Peptides were dissolved in sterile water and mixed with WT and mutant strains and incubated at 37uC for 48 or 72 h in a test tube. The lowest concentration of the peptide that inhibited the growth of the yeast strain was determined by visual inspection.
Materials and Methods Ethics Statement All animals used in this study were treated in agreement with the tenets of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and the protocol was approved by the Singhealth Institutional Animal Care and Use Committee (IACUC; AALAC accredited, #2012/SHS/775).
Time-kill Kinetics Assay The time-kill kinetics of B4010 was determined against two strains of C. albicans (ATCC 10231 and DF2672R). The cultures were grown overnight in SD broth and the cell concentration was adjusted to 105–106 CFU/mL with phosphate buffer. The peptide or antifungals were added to the individual cultures. Appropriate final concentrations of B4010 (1.4–11 mM for ATCC and 0.37– 3.7 mM for 2672R) or amphotericin B (0.55–11 mM for 2672R strains) and natamycin (4.7–75 mM for ATCC and 30 mM for 2672R strains) were adjusted for the inocula. The test solution was incubated at 37uC with constant shaking. 100 mL of the suspension was withdrawn at predetermined time points, serially diluted (102 or 103 fold) and poured into the SDA plate. The plate was incubated for 48 h at 37uC for colony counting. The data were expressed in terms of % fungal viability relative to the positive control.
Chemicals and Peptides Sabouraud’s Dextrose Agar was purchased from Acumedia (Michigan, MI, USA). Peptides were purchased from EZBiolabs Inc., (Carmel, IN, USA). Lipids such as L-a-phophatidylcholine (PC), L-a-phostidylethanolamine (PE), L-a-phosphatidylserine (PS) and L-a-phosphatidylinositol (PI) were bought from Avanti Polar Lipids Inc. (AL, USA). Ergosterol, sodium azide (NaN3), Carbonyl cyanide m-chloro phenylhydrazone (CCCP), 4-aminopyridine (4AP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), Gadolinium III Chloride, tetra ethyl ammonium chloride (TEA) and 39,39-dipropylthiadicarbocyanine (diS-C3-5) dye were purchased from Sigma-Aldrich Corp. (MO, USA). ATP bioluminescence kit was obtained from Molecular Probes Inc (OR, USA). Amphotericin B and Natamycin were obtained in powder form from SigmaAldrich (S) Pte Ltd (Singapore).
Antifungal Activity of B4010 in Presence of Trypsin and 50% Tear Fluid (TF)
Determination of Minimum Inhibitory Concentration (MIC)
For all the experiments, unless otherwise stated, C. albicans ATCC 10231 strain was used. B4010 (1 mg/mL) was incubated with trypsin (enzyme:peptide ratio 1:100) at 37uC. 20 mL aliquot of this mixture was withdrawn at various time intervals (0.5, 1, 2, 4 and 6 h) and mixed with 1 mL of trypsin inhibitor. The antifungal activity of the peptide was determined by adding this mixture to 180 mL of inoculum and monitoring the growth at OD600 for 24 h. Similar experiments performed in the presence of trypsin/ trypsin inhibitor (without B4010) were used as a positive control. For assessing the antifungal activity in TF, the peptide was dissolved in freshly collected rabbit TF and incubated at 37uC for 6 h. After incubation, an equal volume of an overnight culture of C. albicans (,106 CFU/mL) was mixed with peptides in tear fluid and incubated at 37uC for 24 h. The final concentrations of the peptide were at 4.4, 8.8 and 22 mM. A 100 mL aliquot of serial dilutions (102 or 103 times) of this mixture were inoculated on a SD agar plate and then incubated for 48 h at 37uC. Culture alone and culture with 50% tear fluid served as the positive control. The
The yeast strains were cultivated and suspended in Sabouraud’s Dextrose (SD) broth diluted to one sixth at a starting OD600 = ,0.08 in a flat-bottomed microtitre plate. A serial dilution of peptide in the same broth was mixed with the inoculum to give a final peptide concentration of 0.4–22 mM. The antifungal activity was assessed by monitoring the OD600 in cycles of 30 minutes and an orbital shaking at 100 rpm using an Infinite M200 microplate reader (Tecan Group Ltd., Switzerland) for 24/48 h at 37uC. Cultures without peptides were used as positive controls and broth alone or with 22 mM peptide served as negative controls. The minimum concentration required for complete inhibition was assessed by both visible observations as well as by measuring the OD600 and taken as the MIC. Each experiment was repeated in triplicates. For studying the effects of metal ions, SD broth was adjusted with appropriate concentrations of salts to yield a final concentration of 100 and 135 mM for NaCl and KCl and 0.5– 2 mM for CaCl2 and MgCl2. MIC determinations were carried PLOS ONE | www.plosone.org
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data was expressed in % killing with respect to culture without tear fluid. About 6–10% killing was observed in the presence of 50% tear fluid.
Candidacidal Activity of B4010 in Presence of Metal Ions, Energy Poisons and Ion Channel Inhibitors To study the effects of metal ions, the broth solution containing C. albicans (OD600 = 0.4–0.6) was adjusted to appropriate final concentrations of metal ions, incubated with 5.5 mM of B4010 for 6 h and the cell viability was determined as before after 48 h. For the effects of energy poisons and ion channel inhibitors, yeast cells (105–106 CFU/mL) were incubated for 2 h with or without additives at 37uC. A final concentration of 5.5 mM B4010 peptide was added to the cell suspension and incubated further for another 1.5 h at 37uC. Fungal viability was obtained by counting the number of colonies formed in each plate after 48 h incubation at 37uC. SDA plates with additives (and no peptide) served as the positive control. The final concentrations of the additives were: CCCP (5 mM), NaN3 (5 mM), 4-AP (1 mM), NPPB (0.5 mM), Gadolinium II chloride (1 mM) and tetra ethyl ammonium chloride (15 mM). The reported values were averages of two independent duplicate experiments. Control experiments with all the additives were also performed to assess the toxicity of the additives to C. albicans.
Haemolytic Assay Haemolytic activity of peptides and antifungal drugs were determined against rabbit red blood cells [30]. Briefly, serial dilution of peptides/antifungals in PBS was mixed with rRBC (final concentration 4% v/v), incubated at 37uC for 1 h and centrifuged at 3000 rpm for 10 minutes. The release of hemoglobin in the supernatant was monitored by measuring the hemoglobin absorbance at 576 nm. The readings from cell suspension in PBS (without any additives) or 1% Triton-X100 were used as 0% or 100% haemolysis.
Cytotoxicity on Human Conjunctival Epithelial Cells Immortalized normal human conjunctival epithelial cells (IOBANHC) were a gift from Yolanda Diebold at the University of Valladolid, Spain [31]. IOBA-NHC cells were cultured under standard conditions (humidified atmosphere of 5% CO2 at 37uC) in DMEM/F12 supplemented with 1 mg/ml bovine pancreas insulin, 2 ng/ml mouse epidermal growth factor, 0.1 mg/ml cholera toxin, 5 mg/ml hydrocortisone, 10% fetal bovine serum (FBS), 50 UI/ml penicillin, and 50 UI/ml streptomycin. Cells from passages 50–80 were used in all experiments. Every day, normal culture development was observed by phase-contrast microscopy. Cells were removed by gentle trypsin incubation at confluence and counted. They were seeded into 96 well culture plates (Corning, Schiphol-Rijk, The Netherlands) for microtitration analysis of flow cytotoxicity assay (,10,000 cells/well). Cultures were kept at 37uC for 24 h. Subconfluent cells (culture surface covering nearly 70%) were then exposed to various peptide concentrations (0.22–225 mM). Cytotoxicity was measured periodically using MultiTox-Fluor Multiplex assay kit (Promega, WI, USA) by measuring the AFC fluorescence (lex = 485 and lem = 520 nm). We report the cell viability after 24 h incubation of the cells with the peptides since there was no detectable toxicity even after 8 h of incubation.
SDS-PAGE Pull down Assay B4010 (5.5–22 mM) was incubated with chitin (from shrimp shells) or b-D-glucan for 2 h at 37uC. The mixture was centrifuged at 10,000 g and the supernatant was analyzed by SDS-PAGE (4– 20%). A control experiment without chitin/b-D-glucan was also performed to quantify peptide binding to the carbohydrate polymers.
DiS-C3-5 Membrane Depolarization and SYTOX Green (SG) uptake Assays Changes in membrane potential of C. albicans upon addition of B4010 were monitored by the release of potential sensitive probe diS-C3-5 dye. Briefly, 1 ml of overnight grown mid-log phase Candida cells in 5 mM HEPES buffer (pH 7.0) was mixed with 10 mM diS-C3-5 and incubated at 37uC for 1–2 h in a thermo shaker. 800 mL of the dye-loaded cell suspension was transferred to a stirred quartz cuvette and the change in fluorescence intensity was monitored at an emission wavelength (lem) of 670 nm (excitation wavelength, 622 nm) using a Quanta Master spectrofluorimeter (Photon Technology International, NJ, USA). The excitation and emission bandwidths were set at 1 and 2 nm, respectively. Once a constant fluorescence level was achieved, 10 mL of concentrated peptide solution in HEPES buffer was added so that the final concentration of peptide was 0.22 - 22 mM. The change in fluorescence intensity was monitored continuously for 1 or 2 h. To study the effects of energy poisons and ionchannel inhibitors, the additives were added prior to the addition of 5.5 mM B4010. The change in fluorescence intensity was monitored as before. For SG uptake assay, overnight cultures of Candida were harvested by centrifugation, washed three times with HEPES buffer and resuspended in the same buffer. The cells were incubated with 1 mM SG in the dark prior to the addition of peptide. The increase in emission intensity at 520 nm was monitored after addition of various concentrations of peptide (lex = 485 nm). 1% Triton-X100 was added at the end of the experiment to determine the %uptake.
Rabbit Model of Corneal wound Healing Four New Zealand white rabbits were divided into two groups, two each for control (saline) and two each for the study group treated with B4010. Rabbits were tranquilized by intra muscular injection of 1 mL of ketamine (100 mg/mL) and 0.5 mL of xylazil (20 mg/mL). Corneas were anesthetized by topical administration of 1% xylocaine. A 5-mm trephine was used to outline the wound margin and mechanical removal of epithelial cells was carried out by sterile mini blade (BD-Beaver) leaving the basal lamina intact as previously described [32]. The animals were treated by topical administration of 22 mM of B4010 at 3 times/day. Cornea wound was visualized by staining with fluorescein sodium, which is used in the ophthalmology clinic as a non-toxic dye to disclose wounds of the cornea, and using a cobalt-blue filter with slit lamp biomicroscopy. Measurements of the residual wound area were performed during the reepithelialization process by Image-J 1.440.
In vivo Toxicity Acute toxicity of B4010 was assessed with C57BL6 (6–8 weeks old) wild type mice. Two healthy wild type mice were chosen for each routes of administration. B4010 was delivered through intraperitoneal (200 mg/kg) or (100 mg/kg) intra-venous routes. Two animals were used for each administration and monitored through 24/48 h to determine mortality, or signs of toxicity. PLOS ONE | www.plosone.org
Measurement of Extracellular Cations Overnight cultured late logarithmic phase C. albicans was harvested by centrifugation, washed 5 times with 10 mM HEPES (pH 7.0), resuspended in the same buffer and adjusted to an 3
February 2014 | Volume 9 | Issue 2 | e87730
Multivalent Antifungal Peptides
Percentage calcein released at each time point was calculated using the equation,
OD600 = 0.4. To 5 mL of this suspension, B4010 (final concentration 5.5 mM) was added and incubated at 37uC for 2 h. The mixture was centrifuged at 3,000 g and the presence of K+, Ca2+ and Mg2+ in the supernatant was estimated by Perkin Elmer Dualview Optima 5300 DV inductively coupled plasma – optical emission spectrometry (Massachusetts, USA) available at CMMAC facilities (Department of Chemistry, National University of Singapore).
%Calcein Release~
Where A0 is the observed fluorescence intensity upon addition of peptides, Amin is the average intensity at the baseline (before peptide addition), Amax is the average intensity at the saturation phase after adding 1% Triton X-100. Similar protocol was followed for the preparation of PC/Cholesterol SUVs.
ATP Bioluminescence Assay The extracellular ATP levels upon challenging C. albicans with B4010 were determined as reported before [33]. Cells (OD600
