Antifungal activity of novel synthetic peptides by accumulation of reactive oxygen species (ROS) and disruption of cell wall against Candida albicans

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Peptides 32 (2011) 1732–1740

Contents lists available at ScienceDirect

Peptides

j our na l ho me p age : www.elsev ier .com/ locate /pept ides

ntifungal activity of novel synthetic peptides by accumulation of reactivexygen species (ROS) and disruption of cell wall against Candida albicans

ndresh Kumar Mauryaa, Sarika Pathakb, Monika Sharmaa, Hina Sanwala, Preeti Chaudharyc,antosh Tupec, Mukund Deshpandec, Virander Singh Chauhanb,∗, Rajendra Prasada,∗∗

Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, IndiaInternational Centre for Genetic Engineering and Biotechnology, New Delhi 110067, IndiaBiochemical Sciences Division, National Chemical Laboratory, Pune 411008, India

r t i c l e i n f o

rticle history:eceived 25 February 2011eceived in revised form 6 June 2011ccepted 6 June 2011vailable online 13 June 2011

eywords:andida albicansntifungal peptides

a b s t r a c t

In the present work, we investigated the antifungal activity of two de novo designed, antimicrobial pep-tides VS2 and VS3, incorporating unnatural amino acid �,�-dehydrophenylalanine (�Phe). We observedthat the low-hemolytic peptides could irreversibly inhibit the growth of various Candida species and mul-tidrug resistance strains at MIC80 values ranging from 15.62 �M to 250 �M. Synergy experiments showedthat MIC80 of the peptides was drastically reduced in combination with an antifungal drug fluconazole.The dye PI uptake assay was used to demonstrate peptide induced cell membrane permeabilization. Intra-cellular localization of the FITC-labeled peptides in Candida albicans was studied by confocal microscopyand FACS. Killing kinetics, PI uptake assay, and the intracellular presence of FITC-peptides suggested that

ell wallOS

growth inhibition is not solely a consequence of increased membrane permeabilization. We showed thatentry of the peptide in Candida cells resulted in accumulation of reactive oxygen species (ROS) leading tocell necrosis. Morphological alteration in Candida cells caused by the peptides was visualized by electronmicroscopy. We propose that de novo designed VS2 and VS3 peptides have multiple detrimental effectson target fungi, which ultimately result in cell wall disruption and killing. Therefore, these peptidesrepresent a good template for further design and development as antifungal agents.

. Introduction

Systemic fungal infections have drastically increased over theast three decades due to the rising immunocompromised popu-

ation as a result of transplantation, cancer chemotherapy, steroidherapy and, in particular, HIV infection (AIDS) [40,41]. There is arowing need to discover new antifungal therapies because of theimited number of clinically available antifungals and the fact that

any antifungals either lack potency or are toxic to the host cells.

lthough, newer azole derivatives such as voriconazole are moreffective and have cidal activity against filamentous fungi suchspergillus fumigatus [5,43], these derivatives are fungistatic and

Abbreviations: ABC, ATP-binding cassette; MFS, major facilitator superfamily;DR, multidrug resistance; SEM, scanning electron microscopy; TEM, transmission

lectron microscopy; FICI, fraction inhibitory concentration index; ROS, reactivexygen species.∗ Corresponding author. Present address: Membrane Biology Laboratory, Schoolf Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.el.: +91 11 26704509; fax: +91 11 26741081.∗∗ Corresponding author. Tel.: +91 11 26704509; fax: +91 11 26741081.

E-mail addresses: [email protected] (V.S. Chauhan), [email protected],[email protected] (R. Prasad).

196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.peptides.2011.06.003

© 2011 Elsevier Inc. All rights reserved.

not fungicidal against pathogenic yeasts; the inability to kill yeastsleads to resistance to azole in prolonged infections and increasesthe likelihood that these agents will lack efficacy in severe Candidainfections in immunosuppressed patients [4,17]. Amphotericin Bhas also been commonly used to treat serious fungal infections.Nevertheless, resistance to amphotericin B is slowly developing inselected Candida species [5] and there are significant side effectsassociated with its use, including nephrotoxicity [39]. Although,recent antifungal agents, including the peptide-based agents, mica-fungin and caspofungin, have been developed but their clinicalutility is limited by their potential sensitivity to proteases, bioavail-ability, toxicity to host cell and high cost [3,22,23,25,26,30]. Thedevelopment of resistance to current antifungal agents, the lim-ited efficacy, and the side effects associated with several of theseagents increase the importance of continued development of newantifungal agents [29].

In this study, we describe the antifungal activity of two denovo designed cationic peptides, VS2 and VS3 against Candida albi-cans, non-albicans, filamentous and non-filamentous pathogenic

fungi. The designed peptides have already been tested for theirantibiotic activity against E. coli and Staphylococcus aureus [36].The synthesized incorporated a non-natural amino acid �,�-didehydrophenylalanine (�Phe) on hydrophobic face and lysine on

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Table 1Amino acids sequences of VS2 and VS3 peptides.

Peptides Sequences Length Net charge Mw (calculated) Mw (observed) Retention time (min)

4

4

ta�osowFada

2

2

w((2pioM(

2

FMr

2

TieRpteo

2

bt(us(Vitr

VS2 Ac-K-W-�F-W-K-�F-V-K-�F-V-K-NH2 11 +VS3 Ac-K-W-�F-W-K-�F-W-K-�F-V-K-NH2 11 +

he hydrophilic face. In addition to Lys and �Phe, these peptideslso incorporated amino acid valine and tryptohan. The presence ofPhe in peptides has been shown to increase the relative stability

f the peptides towards proteolytic degradation and induce helicaltructures [2,10,11,45,46]. Peptide VS2 and VS3 carried a net chargef +4 and were identical except for one residue, where valine in VS2as replaced with tryptophan in VS3 (Table 1 and Supplementary

ig. S1). We show that the two peptides act synergistically with well-known antifungal drug fluconazole on C. albicans. We alsoescribe the killing kinetics and the detailed analysis of the mech-nism of antifungal action on C. albicans.

. Materials and methods

.1. Materials

Amino acids derivatives and resin for peptides synthesisere obtained from Nova Biochem. N,N′-diisopropylcarbodiimide

DIPCDI), piperidine, dimethylformamide (DMF), dichloromethaneDCM), N-hydroxybenzotriazole (HOBT), trifluoroacetic acid (TFA),′,7′-dichlorofluorescein diacetate (DCFH-DA), ascorbic acid (AA),olyethylene glycol (PEG), triisopropylsilane (TIS), fluorescein 5-

socyanate (FITC), dimethylsulfoxide (DMSO), sodium chloride andther chemicals were obtained from Sigma Chemical Co. (St. Louis,O). Propidium iodide (PI) was purchased from Molecular Probes

Eugene).

.2. Peptide synthesis

Peptides were synthesized by the solid-phase methods usingmoc (9-fluorenyl-methxycarbonyl) chemistry on rink amide-BHA resin in the manual mode using DIPCDI and HOBT as coupling

eagents as described previously [33,36,47].

.3. Fungal strains and growth media

Various fungal strains used are listed in Supplementaryable S1. Cryptococcus neoformans, Aspergillus niger, Fusar-

um oxysporum, Neurospora crassa were cultured in yeastxtract–peptone–dextrose (YEPD) broth (BIO101, Vista, CA) andPMI 1640 media (Gibco BRL, Gaitharsburg, MD) [7–9,19]. For agarlates, 2.5% (w/v) Bactoagar (Difco, BD Biosciences, NJ) was added tohe medium. All strains were stored as frozen stocks with 15% glyc-rol at −80 ◦C. Before each experiment, cells were freshly revivedn YEPD plates from the stock.

.4. Antifungal activity

The in vitro antifungal assays were performed in RPMI mediumy broth microdilution methods according to the recommenda-ions of the National Committee for Clinical Laboratory standardsNCCLS) [12,32]. C. albicans (ATCC-90028) (∼106 cells) were inoc-lated in RPMI 1640 medium. Appropriate amount of peptideolutions were added to the wells of a 96-well microtiter plate100 �l/well) and serially diluted twofold. Final concentrations of

S2 and VS3 peptides ranged between 0.35 �M and 250 �M. After

noculation, the microtiter plate was incubated at 30 ◦C for 48 h, andhe absorbance was measured at 600 nm by using microtiter plateeader to assess cell growth. The MIC was defined as the lowest

1577.6 1575.1 421665.7 1665 43

concentration exhibiting 100% inhibition visible growth comparedto growth control cell.

2.5. Checkerboard assay

The interaction of VS peptides with fluconazole was evalu-ated by the checkerboard method as recommended by the NCCLSand was expressed as the sum of the fractional inhibitory con-centration index (FICI) for each agent. The fluconazole of eachagent is calculated as the MIC of this agent in combinationdivided by the MIC of this agent alone. After making drug dilu-tions, a 100 �l suspension of Candida strains adjusted to 104

colony forming unit (CFU/ml) was added to each well and cul-tured at 30 ◦C for 48 h in RPMI 1640 medium. Then visual readingof MICs was performed and OD492 values were measured. Thebackground OD was subtracted from the OD of each well. Eachcheckerboard test generates many different combinations and byconvention the FIC value of the most effective combination isused in calculating the FIC index. FICI was calculated by addingboth FICs: FICI = FICA + FICB = CA

comb/MICAalone+ CB

comb/MICBalone,

where MICAalone and MICB

alone are the MICs of drug A and B whenacting alone and CA

comb and CBcomb are concentrations of drugs A

and B at the isoeffective combinations, respectively. Off-scale MICswere converted to the next highest or next lowest doubling concen-tration. The FICI was interpreted as synergistic when it was ≤0.5,as antagonistic when >4.0, and any value in between as indifferent[33,38,44,45].

2.6. Membrane permeabilization assay by FACS analysis

For the analysis of the permeabilization of fungal membraneafter peptide treatment, C. albicans (ATCC-90028) cells (∼1 × 106)were first harvested at the logarithmic phase and suspended inRPMI 1640 medium. Cells were incubated in water bath set at30 ◦C at 200 rpm with 1.49 �M (PI) and peptides at MIC80 values,for 5–60 min. After incubation, cells were harvested by centrifu-gation and suspended in (phosphate buffer saline) PBS [31]. Flowcytometry was performed via a FACS, Calibur flow cytometer(Becton–Dickinson, San Jose, CA, USA). Untreated cells with PIserved as a control. For the analysis of the permeabilization of fun-gal membrane after FITC-labeled VS peptide treatment, C. albicans(ATCC-90028) (∼106 cells) were first harvested at the logarithmicphase and suspended in RPMI 1640 medium. Cells were incu-bated in water bath at 200 rpm and peptides at MIC80 values, for30 min at 30 ◦C. After incubation, cells were harvested by centrifu-gation and suspended in PBS. Flow cytometry was performed viaa FACS, Calibur flow cytometer (Becton–Dickinson, San Jose, CA,USA). Untreated cells served as a control.

2.7. Time kill assay

C. albicans (ATCC-90028) cell (∼106 cells) were inoculated inRPMI 1640 medium containing VS2 (78.12 �M), VS3 (39.06 �M)and fluconazole (4.01 �M). At pre-determined time points (0, 5,

10, 15, 30, 45 and 60 min at 30 ◦C incubation; agitation 200 rpm),a 100 �l aliquot was removed, serially diluted (10 fold) in salinewater and plated on YEPD agar plates. Colony counts were deter-mined after incubation at 30 ◦C for 24 h.

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Table 2MIC values (�M) of VS2 and VS3 peptides against various Candida albicans, non-albicans, filamentous, non-filamentous fungi and azole sensitive (AS) as well as azoleresistance (AR) isolates (show increased resistance to azole due to an overexpressionof either CaCDR1 (Gu4, Gu5) or CaMDR1 (F2, F5).

Strains MIC(�M)

VS2 VS3

C. albicans 90028 78.12 39.06C. kefyr 78.12 39.06C. glabrata 312.5 312.5C. utilis 39.06 19.53C. dublinienesis 156.2 156.2C. tropicalis 19.53 19.53C. krusei 19.53 19.53C. parapsilosis 78.12 39.06Gu4 62.50 15.62Gu5 62.50 15.62F2 62.50 15.62F5 62.50 15.62Cryptococcus neoformans 92.90 75.1Aspergillus niger 46.40 >152Fusarium oxysporium 46.40 152Neurospora crassa 91.53 >152.73

Fig. 1. Time kill assay confirmed growth inhibitory effect of VS2 and VS3 peptides.Time kill kinetics of C. albicans (ATCC-90028) by VS2, VS3 and fluconazole at theirMIC concentrations; � represents killed cells at 78.12 �M VS2, � represents killingcurve at MIC of 39.06 �M VS3, � represents killing curve at MIC of 4.01 �M FLC and�t1

2

(pi(scaF4Fp

ultramicrotome (Ultramicrotome Lecia EM UC6). Sections were

TT

represents control cells (without any drug). Samples withdrawn at the indicatedimes were evaluated for colony forming units (CFU). Assay was performed in RPMI640 with 2% MOPS and incubated at 30 ◦C for 24 h.

.8. Permeability of FITC-labeled VS peptides

To determine, if the peptides enter Candida cells, C. albicansATCC-90028) (∼106 cells) were first harvested at the logarithmichase and suspended in a RPMI 1640 medium. Cells were then

ncubated with FITC-labeled VS2 and VS3 peptides at their MIC8062.5 �M and 31.25 �M, respectively) for indicated time with con-tant shaking (200 rpm). After incubation, cells were harvested byentrifugation and suspended in PBS. A smear was made, sealednd visualized under a confocal microscopy (Olympus FluoviewTM

V1000). The excitation and emission wavelength for FITC were

88 nm and 515 nm. Cells without VS peptides served as control.or the analysis of the permeabilization of fungal membrane aftereptide treatment, the impermeant dye PI was used [31]. For this, C.

able 3he peptides VS2 and VS3 demonstrate synergy with fluconazole against C. albicans (ATC

Peptides Fluconazole concentration (�M) Peptid

VS2 1.14 7.81

VS3 0.82 1.92

2 (2011) 1732–1740

albicans (ATCC-90028) (∼106 cells) were first harvested at the log-arithmic phase and suspended in a RPMI 1640 medium. Cells wereincubated with VS2 (62.5 �M), VS3 (31.25 �M), TM1 peptides andPI (3 �M) with constant shaking (200 rpm). After incubation at indi-cated time, cells were harvested by centrifugation and suspendedin PBS. The cells were then directly viewed by a confocal micro-scope (Olympus FluoviewTM FV1000) and a wavelength >560 nmfor PI. Cells without peptide served as control.

2.9. Measurement of reactive oxygen species (ROS) production

Amount of ROS was measured by fluorometric assay with DCFH-DA as described earlier [24]. Briefly, the cells were adjusted to anOD600 of 1 in 10 ml of PBS and centrifuged at 5000 × g for 10 min.The cell pellet was then resuspended in PBS and treated with VS2and VS3 peptides alone and in combination with ascorbic acid for30 min or was left untreated at room temperature. After incubationwith the peptides at 50 ◦C for different time intervals (0–60 min),10 �M 2′,7′-dichlorofluorescein diacetate (DCFH-DA) in PBS wasadded. The fluorescence intensities (excitation 485 nm and emis-sion 540 nm respectively) of the resuspended cells were measuredwith a Spectrofluorometer (Varian, Cary Eclipse) and the images of2,7-dichlorofluorescein (DCF) fluorescence were taken by using afluorescence microscope (Carl Zeiss, Axiovert 40 CFL, USA).

2.10. Analysis of apoptotic markers

Protoplast of C. albicans was stained with PI and FITC labeledAnnexin V by using the Annexin V-FITC apoptosis detection kit(BD Biosciences, USA) to assess cellular integrity and the external-ization of phosphatidylserine (PS) [1,36]. The cells were analyzedby using FACS at 488 nm excitation and a 515 nm band pass fil-ter for FITC detection and wavelength >560 nm for PI detection. Atotal of 10,000 events were counted at the flow rate. Data anal-ysis was performed using cell Quest software (Becton–DickinsonImmunocytometry System) [20].

2.11. Electron microscopy of Candida cells

C. albicans (ATCC-90028) cell suspension from overnight cul-tures was prepared in RPMI 1640 (pH 7). Peptides at equivalent totheir MIC80 concentrations were added to the (∼106 cell) and incu-bated at 30 ◦C. All Candida cells were fixed with 2% glutaraldehydein 0.1% phosphate buffer for 1 h at room temperature (20 ◦C) [5,28].Washed with 0.1 M phosphate buffer (pH 7.2) and post-fixed 1%OSO4 in 0.1 M phosphate buffer for 1 h at 4 ◦C. For SEM, dehydratedin acetone and dropped on round glass cover slip with hexam-ethyldisilizane (HMDS) and dried at room temperature then sputtercoating with gold and observed under the SEM (Zeiss EV040). Forultrastructure study, samples were dehydrated with graded ace-tone, clearing with toluene and infiltrated with toluene and aralditemixture at room temperature then finally in pure araldite at 50 ◦Cand imbedded in Eppendoff tube (1.5 ml) with pure araldite mix-ture at 60 ◦C. Semithin and ultrathin section cutting was done with

taken on the 3.05 mm diameter and 200 mess copper grid, stainedwith uranyl acetate and lead acetate before observing under theTEM (JEOL, JEOL2100F) [21].

C-90028).

es concentration (�M) FIC index Effect

0.471 Synergic0.32 Synergic

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I.K. Maurya et al. / Peptides 32 (2011) 1732–1740 1735

Fig. 2. (A) Confocal microscopy analysis of membrane permeablization assay by PI uptake. (Panel I) C. albicans (ATCC-90028) (∼106 cells) was incubated (5–60 min) withV 028)

1 and Vp

3

ofi

3

ssCfiwsMdptVb(

S2 peptide (62.5 �M) at 30 ◦C in RPMI 1640 medium. (Panel II) C. albicans (ATCC-90640 medium. (B) Flow cytometry analysis of membrane permeablization by VS2eptide (62.5 �M) at 30 ◦C in RPMI 1640 medium.

. Results

We evaluated the fungicidal activity of VS2 and VS3 peptidesn human pathogenic yeast C. albicans and filamentous and non-lamentous fungi.

.1. Anti-fungal activity of the peptides

We determined anti-fungal activity of the peptides against 16trains of fungi, including clinical isolates of C. albicans, non-albicanspecies of Candida (C. krusei, C. glabrata, C. utilis, C. dublinienesis,. tropicalis, C. parapsilosis), the filamentous and non-filamentousungi (A. niger, C. neoformans, F. oxysporum, N. crassa). The minimumnhibitory concentration (MIC) is defined as the concentration,

hich inhibits the growth of cells by 100%. As shown in Table 2, VS2howed MIC ranging from 39.06 �M to 312.5 �M and VS3 showedIC from 19.53 �M to 312.5 �M against the tested strains. We also

etermined the candidacidal activity of these peptides against twoairs of genetically matched azole sensitive (Gu4 and F2) and resis-

ant (Gu5 and F5) clinical isolates of C. albicans. Peptides VS2 andS3 showed an MIC of 62.5 �M and 15.62 �M respectively, againstoth, the azole sensitive strain (Gu4 and F2) and the resistant strainGu5 and F5) (Table 2).

(∼106 cells) was incubated (5–60 min) with TM1 peptide (250 �M) at 30 ◦C in RPMIS3 in PI uptake assay. C. albicans (∼106 cells) was incubated (5–60 min) with VS2

3.2. Peptides show synergy with fluconazole against C. albicans

Since the peptides were found to be active against fluconazole-resistant C. albicans, non-candidacidal concentrations of thepeptides were used to determine whether they act synergisti-cally with fluconazole to kill fluconazole-resistant C. albicans. Theresults revealed that combinations of non-candidacidal concen-trations of VS2 or VS3 and fluconazole were highly active againstfluconazole-resistant C. albicans. Combination of non-candidacidalconcentrations of 7.81 �M VS2 and 1.14 �M fluconazole and1.92 �M VS3 with 0.82 �M fluconazole showed complete killing ofCandida cells. We observed that in combination with the flucona-zole the MIC80 of the peptides were drastically reduced. As shown inTable 3, fraction inhibitory concentration index (FICI) values of VS2and VS3 with FLC were calculated to be 0.47 and 0.32, respectively.

3.3. Killing kinetics assay

In the time kill assay, the CFUs of the C. albicans (ATCC-90028)

cells were rapidly reduced after treatment with the peptides at con-centrations equal to their MIC. However, VS2 and VS3 were able tokill C. albicans cells in 4 h killing kinetics of these peptides werecompared with fluconazole (MIC 4.01 �M), a well-known potent

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Fig. 3. (A) Intracellular localization of FITC-labeled VS2 and VS3 peptides. Confocal microscopic of the uptake of FITC-labeled VS2 and VS3 peptides (at MIC80 concentrations6 experO FITCF ium. C

acd

3

3(

uccpdmiCPwT

3

tc

2.5 �M and 31.25 �M) by C. albicans (ATCC-90028) at time point (5–60 min). TheD (∼106 cells). (B) Flow cytometry analysis of membrane permeablization assay byITC labeled VS2 (62.5 �M) and VS3 peptide (31.25 �M) at 30 ◦C in RPMI 1640 med

ntifungal agent. It was observed that fluconazole was unable toompletely kill C. albicans cells. In comparison to fluconazole theesigned peptides showed faster killing kinetics (Fig. 1).

.4. Mechanism of peptide action

.4.1. Peptide induced fungal membrane permeabilizationpropidium iodide (PI) uptake assay)

PI is a membrane impermeable, nucleic acid staining dye, whichpon binding to double stranded nucleic acid gives a red fluores-ence when excited by a 480 nm laser. PI can only enter into thoseells, which have permeable membranes. Fungal cell membraneermeabilization was studied for VS3. Incubation of VS3 with Can-ida cells resulted in PI uptake by the cells as monitored by confocalicroscopy and FACS (Fig. 2A and B). We observed that increase

n the PI associated fluorescence was time dependent. VS3 treatedandida cells showed red fluorescence after 5 min of incubation. NoI fluorescence was observed when Candida cells were incubatedith PI alone or when treated with a non-fungicidal control peptide

M1.

.4.2. Intracellular localization of FITC-labeled peptidesWe monitored intracellular localization of the FITC-labeled pep-

ides in a time dependent assay by confocal microscopy. C. albicansells were incubated with VS2 and VS3 peptides at their respective

iment was conducted in RPMI 1640 media on log phase Candida cell at A600 of 0.1-labeled VS2 and VS3 peptides. C. albicans (∼106 cells) was incubated (30 min) withells without peptides treatment served as control.

MIC80 concentrations. Fig. 3A shows that VS2 was able to perme-abilize the Candida cells in 15 min. However, VS3 was relativelyfaster and was able to permeabilize the cells in 5 min of incuba-tion. A time dependent increase in intracellular fluorescence of theFITC-conjugated peptides was observed. In FACS study (Fig. 3B), itwas observed that after 30 min of incubation VS2 and VS3 werecompletely internalized in Candida cells.

3.4.3. Generation of reactive oxygen species by the VS2 and VS3The formation of reactive oxygen species (ROS) has been sug-

gested to be one of the fungicidal mechanisms of a numberof antimicrobial peptides [23,36]. In order to determine if themechanism of killing by VS2 and VS3 was ROS mediated, weused fluorescence based assay in combination with fluorescentmicroscopy, to monitor the generation of ROS in the Candida cellsafter incubation with the peptides (Fig. 4). The cell-permeant dyeDCFH-DA is oxidized by ascorbic acid, peroxinitrite (ONOO−), andhydroxyl radicals (OH•) to yield the fluorescent molecule 2′,7′-dichlorofluorescein. Generation of ROS by peptides was monitoredby incubation of VS2 and VS3 at their respective MIC80 (62.5 �M,31.25 �M) with the Candida cells for 30 min. A green fluorescence,

resulting from oxidation of dye DCFH-DA was observed indicatingthe presence of ROS. No fluorescence was observed when the cellswere incubated with peptide VS2 and VS3 along with a well-knownantioxidant ascorbic acid.

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I.K. Maurya et al. / Peptides 32 (2011) 1732–1740 1737

Fig. 4. ROS levels in the presence of VS2 and VS3 peptides in C. albicans cells. (A) Amounts of ROS produced in VS2 (62.5 �M) and VS2 (62.5 �M) + ascorbic acid (5 mM) treatedcells. The fluorescence emitted by the cells was measured using a Spectrofluorometer (Varian, Cary Eclipse). An excitation and emission of 485 nm and 540 nm respectively,were used. Inset shows the level of ROS at different time points (5–60 min). The values are means of three independent experimental triplicates showing the error bars (Pvalue < 0.05). The black, white and gray colors in histogram show cells only, cells + VS2 peptide, cells + VS2 peptide + ascorbic acid (antioxidant), respectively. (B) Amounts ofROS produced in VS3 (31.25 �M) and VS3 (31.25 �M) + ascorbic acid (5 mM), treated cells. The fluorescence emitted by the cells was measured using a Spectrofluorometer(Varian, Cary Eclipse). An excitation and emission of 485 nm and 540 nm were used respectively. Inset shows the level of ROS at different time points (5–60 min). The valuesare means of three independent experimental triplicates showing the error bars (P value < 0.05). The black, white and gray colors in histogram show cells only, cells + VS3peptide, cells + VS3 peptide + ascorbic acid (antioxidant), respectively. (C) Fluorescent images after VS2 (62.5 �M) and VS2 (62.5 �M) + ascorbic acid (5 mM) treatment weret cent imw

3

iHbsat(bFt2c

3

c(vwimb

aken with a fluorescence microscope (Carl Zeiss, Axiovert 40 CFL, USA). (D) Fluoresere taken with a fluorescence microscope (Carl Zeiss, Axiovert 40 CFL, USA).

.4.4. Induction of necrosis in Candida cellsGeneration of ROS in cells have been shown to be one of the lead-

ng steps towards apoptosis which is a natural cell death process.owever, In order to know if the generation of ROS was inducedy peptides, and more so, if the cell killing occurred due to necro-is and not due to apoptosis, we used an Annexin V-FITC basedpoptosis detection assay [37]. In a FACS based apoptosis detec-ion assay, we monitored the externalization of phosphatidylserinePS) to the outer monolayer of the lipid bilayer of the plasma mem-rane (PM), which is an early marker of apoptosis. As shown inig. 5, after 1 h of incubation of cells with VS2 (62.5 �M) pep-ide, while there was no visible apoptotic population, we observed2.27% necrotic population as compared to control (untreated)ells, 2.96%.

.4.5. Candida cell lysis observed by electron microscopyTo understand the mode of action, we looked for morphological

hanges in Candida cells by scanning electron microscopy (SEM)Fig. 6A). Cells treated with VS2 and VS3, at their respective MIC80alues for 30 min, showed wrinkling of the cell surface, compared

ith the smooth surface of untreated cells (Fig. 6A). The peptide-

nduced breakage in the cell wall was also clearly visible. The TEMicrograph of ultrathin section of VS2 treated cells also showed

reakage in the cell wall and cell membrane (Fig. 6B).

ages due to VS3 (31.25 �M) and VS3 (31.25 �M) + ascorbic acid (5 mM) treatment

4. Discussion

We have studied the antifungal activity of two peptides, VS2and VS3, incorporating a non-natural amino acid, �Phe. Our resultsshowed that the peptide VS2 and VS3 have antifungal activityagainst a variety of pathogenic fungal species including, flucona-zole resistant Candida sp. albeit at relatively high concentrations(Table 2). VS2 and VS3 have already been tested for hemoly-sis and cytotoxicity [36]; at the MIC80 concentrations they showlow hemolysis and cytotoxicity. Fluconazole is one of the azole-based antifungal agents, which inhibit the formation of ergosterol(the major component of fungal cell membrane) via inhibition ofenzyme(s) involved in ergosterol biosynthesis [13,14]. Our resultsdemonstrated that both, VS2 and VS3 act synergistically withfluconazole at concentrations much lower than their respectiveMIC80 against fluconazole resistant C. albicans clinical isolates.We also found that in combination with fluconazole, the effec-tive killing concentration (MIC80) for VS2 was reduced by 8 folds,from 31.25 �M to 7.81 �M while for VS3, it was reduced by 16folds, from 31.25 �M to 1.92 �M. It has been shown that manynatural and synthetic antimicrobial peptides act synergistically

with peptide or non-peptide antibiotic. For example, combinationof polymyxin B and fluconazole at low concentrations have beenshown to be fungicidal to a variety of pathogenic fungal speciesincluding fluconazole-resistant strain [6]. Combination therapy or

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1738 I.K. Maurya et al. / Peptides 32 (2011) 1732–1740

F nstain( ell wi6

sisk(c

F(a

ig. 5. Externalization of phosphatidylserine in VS2 peptides treated cells. (A) (i) UB) (i) Cell + Annexin V-FITC + VS2 (62.5 �M), (ii) cell + PI + VS2 (62.5 �M) and (iii) c0 min and analyzed by flow cytometry.

ynergy has been shown to be an effective strategy for combat-ng the increasing antimicrobial resistance [14,18]. In addition toynergy with fluconazole, the peptides also demonstrated very fast

illing kinetics. Results showed that at their respective MICs, VS278.12 �M) and VS3 (39.06 �M) showed very fast killing of C. albi-ans cells as compared to fluconazole (4.01 �M). Peptide VS2 and

ig. 6. (A) Scanning electron micrographs showing C. albicans (ATCC-90028) cell wall daVS2 (62.5 �M) and VS3 (31.5 �M) peptides). (B) Transmission electron micrographs shownd VS2 (62.5 �M) treated C. albicans cells.

ed cell, (ii) cell + Annexin V-FITC, (iii) cell + PI and (iv) cell with Annexin V-FITC + PI.th Annexin V-FITC + PI + VS2 (62.5 �M). Cells were incubated with VS2 peptide for

VS3 killed Candida cells in 2 h and 1 h respectively while, flucona-zole took more than 1 h to completely kill Candida cells.

Faster kinetics of the peptide action was also supported by PI

uptake assay, which showed that VS3 was able to permeabilizethe fungal membrane within 5 min. Intracellular localization of thepeptide in a time dependent manner was demonstrated by the

mage by VS2 (62.5 �M) and VS3 (31.25 �M) peptides. Untreated and treated cellsing C. albicans (ATCC-90028) cell wall damage by VS2 (62.5 �M) peptide. Untreated

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radual increase in the peptide associated fluorescence for both,ITC–VS2 and FITC–VS3. These results suggested that increasedembrane permeability might not be the sole cause of cell death

ut it might have lead to the “self promoted” uptake of the pep-ides [15,21,48]. It also suggests that the peptides might have somentracellular targets. There are several antimicrobial peptides, likealivary histatin 5 and buforin II that permeabilize the microbialembrane but their targets are located inside the cell [16,42].e further demonstrated that the activity of the peptides in Can-

ida cells was associated with accumulation of ROS. ROS inductionapacity of various antifungals has been well studied [27]. Forxample, azole such as miconazole (MCZ), polyene AMB and polyolacrolide such as niphimycin induce ROS levels in susceptible

ungi [23]. In addition, the benzo-naphthacenequinone, antibioticradimicin A, natural perylenequinoid pigments and several nat-ral antifungal peptides have been shown to induce ROS in yeastpecies [24]. In this case, ROS mediated killing was shown by oxi-ation of the fluorescent dye DCFH-DA in Candida cell followingeptide treatment. Interestingly, in the presence of an antioxidantscorbic acid, the dye was not oxidized resulting in no fluorescence.OS can damage a wide range of molecules, including nucleic acids,roteins and lipids, and with this wide range of targets it is difficulto determine which events lead to loss of viability of cells followingamage [20]. We further demonstrated by a FACS based apoptosisssay that induction of ROS by peptide lead to cell necrosis. Electronicroscopic study of the Candida cell clearly demonstrated dam-

ge to the fungal cell wall and membrane. All these results leads to conclude that, like several previously reported antimicrobialeptides, VS2 and VS3 have multimodal action.

Killing of fungal cells by both naturally and synthetic cationiceptides could either be by the disruption the structure of fun-al cell membrane (e.g., melittin), ROS formation (histatin 5)16], depolimerization of actin cytoskeleton, ultrastructure dam-ge (magainin-2) [29,35], whereas both VS2 and VS3 show rapidilling of C. albicans and non-Candida species using multiple tar-ets of their action (cell wall disruption, ROS accumulation andpoptosis). It would mean that the development of resistance byrolonged use of such peptides against Candida cells may not leado a rapid development of resistance to these peptides.

. Conclusion

Synthetic antimicrobial peptides utilize various mechanismsifferent from conventional antibiotics and provide an enormousdvantage, including broad-spectrum activity, rapid killing and lowesistance. Small peptides, like VS2 and VS3, which showed synergyith conventional antifungals against drug resistant clinical strains

f Candida and have multimodal action that are suitable candidatesor further development. Further modifications based on these find-ngs could produce a broad range of anti-infective agents suitableor clinical use.

cknowledgments

We acknowledge Advanced Instrumentation Research FacilityAIRF), JNU for providing instrumental support. I.K.M acknowledgesouncil of Scientific and Industrial Research (CSIR), India, for theward of research fellowships. The work presented in this paperas been supported by grants to R.P. and V.S. from Department ofiotechnology (BT/PR11158/BRB/10/640/2008).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.peptides.2011.06.003.

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