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Peptides 32 (2011) 1741–1747

Contents lists available at ScienceDirect

Peptides

j ourna l ho me pa ge: www.elsev ier .com/ locate /pept ides

dentification of an antifungal peptide from Trapa natans fruits with inhibitoryffects on Candida tropicalis biofilm formation

anti M. Mandala,∗, Ludovico Migliolob, Octavio L. Francob, Ananta K. Ghosha

Central Research Facility, Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, WB, IndiaCentro de Análises Proteomicas e Bioquimicas, Pós-Graduac ão em Ciências Genômicas e Biotecnologia UCB, Brasília-DF, Brazil

r t i c l e i n f o

rticle history:eceived 6 May 2011eceived in revised form 21 June 2011ccepted 21 June 2011vailable online 28 June 2011

a b s t r a c t

Due to recent emergence of fungal pathogens resistant to current antifungal therapies, several studieshave been focused on screening of plant peptides to find novel compounds having antifungal activities.Here, a novel antifungal plant peptide, with molecular mass of 1230 Da was purified from fruits of Trapanatans by reverse phase high performance liquid chromatography using 300SB-C18 column and named asTn-AFP1. Determination of complete amino acid sequences of this peptide by tandem mass spectrometry

eywords:ntifungal peptideandida tropicalisruitsrapa natansn-AFP1

showed to contain following eleven amino acid residues: LMCTHPLDCSN. Purified Tn-AFP1 showed theinhibition of Candida tropicalis growth in vitro and disrupted the biofilm formation in a concentrationdependent manner. It also showed downregulation of MDR1 and ERG11 gene expression in real time-PCRanalysis. In silico molecular modeling predicted the structure of Tn-AFP1 as a single coil attached by aunique disulfide bond. Characterization of Tn-AFP1 could contribute in designing novel derivative(s) ofthis peptide for the development of more effective antimycotic compounds.

© 2011 Elsevier Inc. All rights reserved.

. Introduction

Antifungal protein and peptides are produced by a wide varietyf organisms in order to protect themselves from fungal infection.umerous antifungal peptides have been isolated from insects,mphibians, mammals, bacteria [9] and plants [6,11,14,25]. Amonghem, plants are important sources of antimicrobial and antifun-al compounds due to their inherent ability to defend themselvesgainst natural pathogens [8]. Compounds like plant defensinshich are small cysteine-rich peptides act as host innate immune

ystem to control their natural microbial flora and to combatathogens [32]. They are non-cytotoxic and suitable candidateolecules to be used as antifungal therapeutics [32]. A number of

lant defensins have been purified and their activities on Candidapp. have been reported [31,33]. Antifungal peptides are classifiedn the basis of their structures or mode of action. Some excellent

Abbreviations: AFP, antifungal peptides; Tn-AFP1, Trapa natans-antifungaleptide 1; HPLC, high performance liquid chromatography; LC–MS/MS, liquid chro-atography tandem mass spectrometry; MALDI ToF MS, matrix assisted laser

esorption ionization time of flight mass spectrometry; MIC, minimum inhibitoryoncentration; RPMI-1640, Roswell Park Memorial Institute-1640 medium; TFA,rifluoroacetic acid; CHCA, �-cyano-4-hydroxycinnamic acid; MDR1, multidrugesistant gene1; ERG11, lanosterol 14-alpha-demethylase.∗ Corresponding author. Tel.: +91 3222 282486; fax: +91 3222 282481.

E-mail address: sm crf@yahoo.com (S.M. Mandal).

196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.peptides.2011.06.020

reviews of antifungal peptides with their mode of action have beenpreviously published [2,8].

Fungal infections remain a significant cause of morbidity andmortality despite advances in medicine and the emergence ofnew antifungal agents [21]. Among fungi affecting human health,Candida tropicalis found in normal human mucocutaneous flora,is mainly responsible for septicemia and disseminated candidia-sis, especially in patients with lymphoma, leukemia and diabetes[23,27]. C. tropicalis also shows a high incidence of antifungal drugresistance [17] and demand efforts to find new antifungal com-pounds for the treatment of this fungal infection.

The plant species used in this study, Trapa natans, commonlyknown as water chestnut, is a floating annual aquatic plant, andits fruits are widely used as food [16]. Here we report purificationand biochemical characterization of a novel small peptide from thefruits of T. natans, and show that the isolated peptide possessesantifungal activity against C. tropicalis.

2. Materials and methods

2.1. Sample preparation

T. natans L. fruit was collected from a local market in Kharag-pur, India. Fresh fruits were washed thoroughly under running tapwater, and fruit coat was removed. Thereafter, the fleshy, whiteedible part was gently rinsed with distilled water and air dried.

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ried fruits (500 g) were homogenized with 100 mL of 5% acetic acidolution and kept overnight with shaking at 80 rpm in a shaker. Theesulting extract was filtered and centrifuged at 5000 × g for 15 min.he supernatant was collected and precipitated with 200 mL of ace-one by incubating at 4 ◦C for overnight. Pellet was collected byentrifugation at 5000 × g for 15 min, supernatant decanted andxcess acetone was evaporated to dryness to obtain the dry crudextract.

.2. Peptide purification

Dry crude extract was dissolved in 3 mL of 0.1% aqueous TFAolution and fractionated by reverse phase-HPLC (Agilent 1100eries) with a ZORBAX-300SB-C18 column (4.6 mm × 150 mm, par-icle size 5 mm), at a flow rate of 1 mL min−1. The solvent was 0.1%queous TFA (A) and acetonitrile with 0.1% TFA (B). A step gradi-nt of solvent B used to run the column was as follows: 0–60%or 0–45 min, 60–80% for 45–55 min and 80–100% for 55–60 min.he elution from the column was monitored at 215 nm in a dioderray detector and all the peaks of the HPLC chromatogram wereollected using a fraction collector (GILSON, France) coupled withhe system. Selected peaks were concentrated by lyophilizationnd their antifungal activity was screened against C. tropicalisCIM 3110. The fraction showing antifungal activity was rechro-atographed to check the purity.

.3. Antifungal assay and inhibition of biofilm formation

Antifungal activity of the purified peptide fractions wascreened using broth microdilution assay. The minimum inhibitoryoncentration (MIC) of the selected peptide for planktonic cells of. tropicalis was determined according to the guidelines of the Clin-

cal and Laboratory Standards Institute [CLSI (M27-A2 document,CCLS, [22]. In brief, 80 �L glucose-deficient medium (RPMI 1640ithout glucose) and 20 �L fungal inocula (3.5 × 106 CFU/mL) were

dded to each flat-bottom well of a microtiter plate. Then, 100 �Lf glucose-rich medium (RPMI 1640 with glucose 2 mg mL−1,ithout bicarbonate) containing the test concentration of pep-

ide was added to each well. Two wells of the plate served asrowth (without peptide) and sterility (without inocula) con-rols. The plates were incubated at 35 ◦C for 18 h. The amountf glucose remaining after incubation was detected followinghe enzymatic assay described by Riesselman et al. [26]. Exper-ments were carried out in triplicate on three different sets.iofilm formation by C. tropicalis was evaluated in the presencend absence of peptide. Production of biofilm formation by C.ropicalis was checked following the method established by Shint al. [29] and Bizerra et al. [4]. Briefly, a 20 �L cell suspension3.5 ×106 CFU/mL) was placed in each well containing 80 �L ofPMI 1640 (Invitrogen) medium. Various concentrations of peptideolutions in the same medium (100 �L) were added to each well.he plate was incubated for 24 h at 35 ◦C. After 24 h incubation, theedium was aspirated off and non-adherent cells were removed

y washing thoroughly with sterile 0.15 M phosphate-bufferedaline (PBS) pH 7.2. Again, 200 �L of peptide (same concentra-ion as previous incubation) in the same medium was added toach well and incubated for 48 h. After incubation, each well wasashed with 1× PBS, visualized directly in an inverted micro-

cope and photographed (Lica Microsystem, CMS GmbH Wetzler,odel-304566).Biofilm formation was quantified using the 2,3-bis(2-methoxy-

-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilideXTT)-reduction assay following Ramage et al. [24]. A 100-�Lliquot of XTT-menadione [0.1 mg mL−1 XTT, 1 �M menadione]as added to each well, and the plates were incubated in the dark

32 (2011) 1741–1747

for 2 h at 37 ◦C. XTT formazan was measured at 490 nm with amicrotiter plate reader (Biorad Microplate reader 5804R).

2.4. Hemolytic assay

Hemocompatibility study was performed using standard pro-tocol with some modification. In brief, blood was collected from6-week-old male BALB/c mice in a heparinized tube and red bloodcells (RBC) were obtained by centrifugation at1500 × g for 5 min in4 ◦C. The collected RBC pellet was diluted in 20 mM HEPES bufferedsaline (pH 7.4) to make a 5% (v/v) solution. The RBC suspensionwas added to HEPES-buffered saline (−ve control), 1.0% Triton X-100 (+ve control) and incubated with Tn-AFP1 peptide for 30 minand 60 min at 37 ◦C. After centrifugation at 12,000 rpm at 4 ◦C,the supernatants were transferred to a 96-well plate. Hemolyticactivity was determined by measuring the absorption at 570 nm(Biorad Microplate reader 5804R). Control samples of 0% lysis (inHEPES buffer) and 100% lysis (in 1% Triton X-100) were employed inthe experiment. All assays were performed in triplicate. Hemolyticeffect of each treatment was expressed as percent cell lysis relativeto the +ve control cells (% control) using the following formula:[(Abs570 of samples)/(Abs570 of (+)ve control cells)] × 100, whereabsorbance is abbreviated to Abs.

2.5. MALDI ToF mass spectrometry analysis

The lyophilized dried peptide was re-suspended in 5% (v/v) ace-tonitrile solution containing 0.01% (v/v) trifluoroacetic acid. Fourmicroliters of peptide solution was mixed with 24 �L of matrix(CHCA, 10 mg mL−1) and 1.0 �L of this mixture solution was spottedonto the MALDI 100 well stainless steel sample plate and allowed toair dry prior to the MALDI analysis [19]. To obtain MALDI mass spec-tra a Voyager time-of-flight mass spectrometer (Applied Biosystem,USA), equipped with 337 nm N2 laser and operated in acceleratingvoltage 20 kV was used. The spectra were recorded in the positiveion linear mode. Reproducibility of the spectrum was checked 5times from separately spotted samples.

2.6. LC–MS/MS analysis and amino acid sequencing

The antifungal peptide (Fr-3) was dissolved in 5% (v/v) acetoni-trile and analyzed using a LC–MS/MS system (Waters, USA) coupledto a Micromass Quattro Micro triple-quadruple mass spectrometer(Micromass, Manchester, UK) with MassLynx software. The samplewas infused into the ES ionization source using a syringe pump ata flow rate of 10 �L min−1. For the peptide analysis, the instrumentwas operated in the positive ion mode with a capillary voltage of3 kV and cone voltage of 30 V. An electrospray source block temper-ature 130 ◦C, and desolvation temperature 300 ◦C, were used. ForMS/MS analysis, argon gas was used to fragment peptide with col-lision energy of 30 eV. The data were acquired in the MS scanningmode with the scan range of 40–1400 (m/z), the scan time was 0.5 sand inter-scan delay time was 0.1 s. Sequence of the peptide wasdetermined manually following Gauri et al. [13].

2.7. Quantitative real-time PCR

Quantitative real-time PCR was performed to determine therelative mRNA expression level of ERG11 and MDR1 of C. tropicalisbiofilm. C. tropicalis was grown in the presence (16 �g mL−1, ashalf of MIC value) and absence of peptide Tn-AFP1. After 18 hincubation, the plate was washed twice with 1× PBS and then the

sessile cells from the polystyrene plate were harvested by gentlescraping with a sterile toothpick. The total RNAs were extractedusing RNA Mini kit (Invitrogen) as per manufacturer’s protocol.Purity of isolated RNA was checked by 1% agarose-formaldehyde

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tides 32 (2011) 1741–1747 1743

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S.M. Mandal et al. / Pep

enaturing gel electrophoresis. A template of 200 ng of totalNA was taken and treated with RNase free DNase, and incu-ated at 37 ◦C for 5 min followed by heat inactivation at 65 ◦Cor 15 min. Superscript III reverse transcriptase (Invitrogen) wassed for cDNA synthesis as per manufacturer’s protocol. Fifteenanograms of cDNA and the recommended concentration ofYBR Green Master Mix (Applied Biosystems, USA) were addedo a 25 �L reaction mixture. The specific primers were added at

concentration of 200 nM in all cases and PCR was performednder the following step: Initiated denaturation at 95 ◦C with0 min, followed by 45 cycles of 95 ◦C for 15 s, 55 ◦C for 30 s and2 ◦C for 30 s. Quantitative PCR analyses (verb)/analyses (noun)ere performed in real time using ABI PRISM 7000 sequenceetection system (Applied Biosystems). All primers and probesf two target genes (ERG11 and MDR1) and an internal controlene (ACT1) were designed following Bizerra et al. [4]. The primerairs for ERG11 were: forward, 5′-ATGGCTATTGTTGATACTGC-3′

nd reverse, 5′-GCATTGTAAATGAATTCGTG-3′; for gene MDR1ere: forward, 5′-CCCAGAAGTTTTCATTCCA-3′ and reverse,

′-CCCCAAGCAACAGGATAAT-3′; and for gene ACT1 were:orward, 5′-ATGGACGGGGGTATGTTTCA-3′ and reverse, 5′-ACATAAGTAATTTCCAATGTG-3′. The 2−��CT method was used

o calculate relative changes in gene expression in real-timeuantitative PCR experiments [18].

.8. In silico analyses and molecular modeling

Initially, PSI-BLAST was used in order to find best templates foromology modeling, but no results were observed. Antimicrobialeptide Database (APD) server [37] was utilized for discovery tem-lates in antifungal database. Antifungal peptide in pdb databasejr3 peptide [34] showed 18% identity and conserved of its cysteineesidues. Fifty theoretical three-dimensional peptide structuresere constructed by Modeler v.9.8 using the template above [10].

he final model was evaluated by (a) examining geometry, stere-chemistry, and energy distributions, (b) analyzing packing andolvent exposure characteristics using PROSA II and (c) stereochem-cal quality using PROCHECK [39]. In addition, RMSD was calculatedy overlap of C� traces and backbones onto the template structurehrough the program 3DSS [30]. The peptide structures were visu-lized and analyzed on SPDB viewer v.3.7 and Delano Scientific’sYMOL http://pymol.sourceforge.net/.

. Results

.1. Peptide isolation and purification

Fig. 1a shows the chromatographic profile of peptides purifiedrom T. natans fruits. All the fractions were collected as individ-al peak. The antifungal activity of each fraction (10 �g mL−1) wasested against C. tropicalis strain NCIM 3110. Only fraction 3 showedctivity against C. tropicalis. In order to determine the purity of thesolated fraction-3, it was again applied to the same column forechromatography with a modification of gradient profile. For elu-ion, a gradient of solvent B was used as 0–15% for 0–45 min and5–100% for 45–60 min. A single peak was observed by monitoringhe eluted fraction at 215 nm. Pure peptide (≥95% after rechro-

atography) was obtained at a yield of ∼7.625 mg from 500 g ofruits.

.2. Mass spectrometric study and peptide sequencing

MALDI TOF-MS analysis of fraction 3 showed that the monoiso-opic molecular mass of the peptide was 1230.237 (Fig. 1b). Further,andem mass spectrometry (MS/MS) provides a means for frag-

fruits of T. natans (a). Diagonal line indicates a gradient of solvent B; the mobilephase and other conditions are described in the text. MALDI TOF Mass Spectrum ofantifungal peptide (Fr-3) (b).

menting mass selected precursor peptide ions and measuring themass to charge (m/z) ratio of any daughter ions. This process isuseful because it produces two principal classes of fragment ions,b and y type ions respectively [1]. Manual interpretation of peptidespectra for the purpose of protein identification, a process usuallyreferred to as de novo sequencing, is informative about the pri-mary sequence of the peptide. After considering the b and y typeions from the spectrum, the sequence “LMCTHPLDCSN” was builtas shown at the top of Fig. 2. After sequencing, the peptide wasnamed Tn-AFP1.

3.3. Antifungal assay and biofilm inhibition

The antifungal susceptibility assay showed that the strain wassusceptible to peptide Tn-AFP1. The MIC value of Tn-AFP1 againstC. tropicalis was 32 �g mL−1. Biofilm formation by C. tropicalis onpolystyrene surface was examined by inverted microscope (Fig. 3).The control well without peptide shows aggregation of intensehypal budding that forms a mat-like structure. In the well contain-ing 4 �g mL−1, distinct microcolonies of yeast could be seen, andat a concentration of 8 �g mL−1 and 16 �g mL−1, few filamentouscells were visualized on the surface of the strip with sessile cells,but the number of sessile cells was decreasing. Interestingly, at 32and 64 �g mL−1, the numbers of cells were decreased drasticallyand their morphological changes (shrinkage) were observed. At aconcentration of 64 �g mL−1, almost all the cells had died. Over-all, these results indicate that Tn-AFP1 inhibits biofilm formationof C. tropicalis in a dose-dependent manner on a polystyrene sur-face, which is similar to that on catheter surfaces. The level of XTT

activity of biofilms formed by C. tropicalis was decreased graduallywith increasing the concentration of Tn-AFP1 and 50% inhibitionobserved at 16 �g mL−1 concentration (Fig. 5a). In order to deter-mine the expression of the genes ERG11 and MDR1, responsible

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1744 S.M. Mandal et al. / Peptides 32 (2011) 1741–1747

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.1 s. Sequence of the peptide was determined manually.

or C. tropicalis biofilm formation, a real-time PCR experiment waserformed. The analysis of expression revealed that both genes

ere down-regulated in the sessile cells treated with Tn-AFP1 com-ared with control cells that were grown without Tn-AFP1. MDR1nd ERG11 down-regulation was 1.5 and 1.0 fold respectively inomparison to control (Fig. 4).

ig. 3. Biofilm formation by C. tropicalis on polystyrene plate in the presence and absenaken from the well containing different concentrations peptide as 0 �g mL−1 (a); 4 �g mhat Tn-AFP1 inhibits the biofilm formation of C. tropicalis in a concentration-dependent m

to fragment peptide with collision energy of 30 eV. The desolvation and cone gas the scan range of 40–1400 (m/z), the scan time was 0.5 s and inter-scan delay was

3.4. Evaluation of red blood cell lysis

The membrane-damaging property of Tn-AFP1 was analyzedby the quantification of released hemoglobin. After 30 min and60 min incubation with Tn-AFP1 (32 �g mL−1), it displayed alower membrane-damaging effect causing a significantly lower

ce of Tn-AFP1. Picture was captured using an inverted microscope. Pictures wereL−1 (b); 8 �g mL−1 (c); 16 �g mL−1 (d); 32 �g mL−1 (e) and 64 �g mL−1 (f). It reveals

anner.

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S.M. Mandal et al. / Peptides

Fig. 4. Agarose (1%) gel electrophoresis analysis of PCR amplification pattern ofACT1, MDR1 and ERG11 genes in C. tropicalis after being grown with Tn-AFP1,16 �g mL−1 (Lane 2) and without Tn-AFP1 (Lane 1) for 18 h (a). Quantitative realtime PCR analysis of MDR1 and ERG11 genes in C. tropicalis after being grown withTba

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n-AFP1 (16 �g mL−1) and without Tn-AFP1 for 18 h. MDR1 and ERG11 representlack color for treated peptide and gray color for control cells in histogram (b). Datare the mean of triplicates ± S.D.

emoglobin release than the (+)ve control (Fig. 5B). No significantifference in hemolysis could be detected between Tn-AFP1 andve control after 30 min of incubation, whereas extending the incu-ation time of Tn-AFP1 to 60 min did show the 10% hemolysis in

ig. 5. Quantification of biofilm formation and hemolytic activity in the presence of Tn-after 48 h incubation) with increasing the Tn-AFP1 concentration (a), and hemolytic asshe mean of triplicates ± S.D.

ig. 6. Tridimensional structural model of antifungal peptide purified here. Structure wiscussed in main text.

32 (2011) 1741–1747 1745

respect to (+)ve control. It revealed that Tn-AFP1 has no adverseeffect on red blood cells at concentration of 32 �g mL−1.

3.5. Molecular modeling analyses

The three-dimensional model of antifungal peptide showed 18%of identity with 2jr3, an antifungal peptide purified from Chi-nese soft-shelled turtle eggshell matrix whose three dimensionalstructures was resolved by NMR (Fig. 6). The peptide templatealso showed a dual role in the process to stabilize a thin film ofmetastable vaterite and strong antimicrobial activity against Gram-negative bacteria. The validation of the 3D model of our antifungalpeptide by Ramachandran plot showed that the model presented50% of the amino acid residues in physically acceptable regionsand 50% of the amino acid residues in physically allowed-for loopstructure formation in relation to torsion angles phi and psi. Thez-score value in PROSA II program can be used to check whetherthe input structure is within the range of scores typically found fornative proteins of similar size. The z-score value was −0.86, com-pared with NMR structures’ antifungal peptides of similar length(z-score −0.32 to −1.29) [35,36]. The value of root main square

deviation (RMSD) for antifungal peptide was 1.88 A at polypeptidelength chain target versus template. This value indicated that themodel presented modification was accepted in Ramachandran plot.The three-dimensional inhibitor model showed that the structural

AFP1. XTT reduction activity was decreased during C. tropicalis biofilm formationay of Tn-AFP1 (32 �g mL−1) for 30 min and 60 min incubation at 37 ◦C (b). Data are

as cartoon represented and stick was used for side chains. Named residues are

Journal Identification = PEP Article Identification = 68423 Date: August 3, 2011 Time: 8:41 pm

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oil fold had the presence of a single disulfide bond (Cys3–Cys9)nd proline residue in loop center (Pro6), which causes a torsionreating wedge structure commonly found in antifungal structuresFig. 6).

. Discussion

Several hundreds of antifungal peptides have been isolatednd identified from a variety of organisms. In this study, Tn-FP1, one novel small antifungal peptide with molecular mass of230 Da was isolated and identified from the fruits of T. natanshat inherently defending million of water born pathogens. Tn-FP1 showed better antifungal activity against C. tropicalis with

nhibitory effect on biofilm formation, compare to several reportedeptides [7]. A number of genetic factors were recognized duringhe biofilm formation of C. tropicalis. Earlier, it was documentedhat the expression of both (MDR1 and ERG11) genes was notedo be higher in the biofilm formation for C. tropicalis [4]. Overex-ression of MDR1, ATP-binding cassettes pump [3] and lanosterol4-alpha-demethylase (ERG11), a member of cytochrome P450amily that functions in ergosterol biosynthesis, has been associ-ted with drug resistance and biofilm-induction [12]. The resultsf this study showed that Tn-AFP1 mainly affects MDR1 genexpression in comparison to ERG11, which might be a cause ofnhibition of biofilm formation in C. tropicalis. Since the expres-ion of genes MDR1 and ERG11 did not change drastically, it seemshat the peptide has multifactorial effects on C. tropicalis biofilmormation.

Peptide structure was previously visualized in several anti-ungal peptide structures from mammalian sources deposited inrotein Data Bank, pdb: 1dfn, 3hj2 and 3gny [15,38]. All of themre higher proteins with clear similarities to human defensin struc-ures. Moreover, they also presented a similar loop conformationith a central proline residue conserved between two cysteines

paced by two variable residues. In the solved structures [15,38], pattern of CXXPXC was observed in this loop. Otherwise, inhe structure provided here a similar pattern of CXXPXXC wasbserved, suggesting the essentiality of this loop for antifungalctivity. Furthermore, we observed a clear structural feature rela-ion between an antifungal plant peptide and human defensins.he peptide reported here showed hydrophilic charged residuesHis5 and Asp8) exposed on the peptide surface, which could act inooperation with hydrophobic residues (Leu1, Thr4 and Leu7) thatay play a key role in the interaction with fungal cell membranes.lthough these membranes show a major hydrophobic composi-

ion, some components could also be hydrophilic. Among themre observed a subclass of neutral glycosphingolipids that containncharged sugar residues such as glucose and galactose. More-ver, the most common neutral glycosphingolipids encounteredn fungi are glucosilceramides (GlcCer), present in membranes ofeveral fungi, such as Pichia pastoris [28], Aspergillus fumigatus [5],andida albicans [20], among others. Therefore, the amphipathicityf fungal target perfectly matches the physical-chemical proper-ies of the peptide presented here.In summary, this is the firsteport of an antifungal peptide from fruits of T. natans againsthe human pathogen, C. tropicalis. Moreover, it seems that thiseptide activity is involved in membrane interaction, since thexpression of enzymes involved in drug resistant and lipid syn-heses are clearly modified. The activity can be improved by usingrug design techniques in order to modify the peptide by substi-

ution or deletion of residues, improving antifungal activity andeducing probable collateral effects. This means that this smalleptide with low disulfide bond contents could be a promising can-idate for clinical use as an antimycotic pharmaceutical in the nearuture.

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32 (2011) 1741–1747

Acknowledgements

LM and OLF are supported by CNPq, CAPES, FAPDF, FAPEMIG andUCB.

References

[1] Andersen JS, Svensson B, Roepstorff P. Electrospray ionization and matrix-assisted laser desorption ionization spectrometry: powerful analytical toolsin recombinant protein chemistry. Nat Biotechnol 1998;14:449–57.

[2] Balkovec J. Lipopeptide antifungal agents. Expert Opin Invest Drugs1994;3:65–82.

[3] Barchiesi F, Calabrese D, Sanglard D, Falconi Di Francesco L, Caselli F, GianniniD, et al. Experimental induction of fluconazole resistance in Candida tropicalisATCC 750. Antimicrob Agents Chemother 2000;44:1578–84.

[4] Bizerra FC, Nakamura CV, De Poersch C, Estivalet Svidzinski TI, Borsato Que-sada RM, Goldenberg S, et al. Characteristics of biofilm formation by Candidatropicalis and antifungal resistance. FEMS Yeast Res 2008;8:442–50.

[5] Boas MH, Egge H, Pohlentz G, Hartmann R, Bergter EB. Structural determina-tion of N-2′-hydroxyoctadecenoyl-1-O-betad-glucopyranosyl-9-methyl-4,8-sphingadienine from species of Aspergillus. Chem Phys Lipids 1994;70:11–9.

[6] Broekaert WF, Van Parijs J, Leyns F, Joos H, Peumans WJ. A chitin-bindinglectin from stinging nettle rhizomes with antifungal properties. Science1989;245:1100–2.

[7] Burrows LL, Stark M, Chan C, Glukhov E, Sinnadurai1 S, Deber CM. Activ-ity of novel non-amphipathic cationic antimicrobial peptides against Candidaspecies. J Antimicrob Chemother 2006;57:899–907.

[8] Dangl JL, Jones JD. Plant pathogens and integrated defense responses to infec-tion. Nature 2001;411:826–33.

[9] De Lucca AJ, Walsh TJ. Antifungal peptides: origin, activity, and therapeuticpotential. Rev Iberoam Micol 2000;17:116–20.

10] Eswar N, Marti-Renom MA, Webb B, Madhusudhan MS, Eramian D, Shen M,et al. Comparative protein structure modeling with MODELLER. Current pro-tocols in bioinformatics. John Wiley & Sons, Inc.; 2006. Supplement 15, pp.5.6.1–30.

11] Fehlbaum P, Bulet P, Michaut L, Lagueux M, Broekaert WF, Hertu C, et al. Insectimmunity. Septic injury of drosophila induces the synthesis of a potent antifun-gal peptide with sequence similarity to plant antifungal peptides. J Biol Chem1995;269:33159–67.

12] Garcia-Sanchez S, Aubert S, Iraqui I, Janbon G, Ghigo JM, d’Enfert C. Candidaalbicans biofilms: a developmental State Associated With Specific and StableGene Expression Patterns. Eukaryot Cell 2004;3:536–45.

13] Gauri SS, Mandal SM, Pati BR, Dey S. Purification and structural characteriza-tion of a novel antibacterial peptide from Bellamya bengalensis: activity againstampicillin and chloramphenicol resistant Staphylococcus epidermidis. Peptides2011;32:691–6.

14] Gozia O, Ciopraga JT, Bentia M, Lungu I, Zamfirescu R, Tudor R, et al. Anti-fungal properties of lectin and new chitinases from potato tuber. FEBS Lett1995;370:245–9.

15] Hill CP, Yee J, Selsted ME, Eisenberg D. Crystal structure of defensin HNP-3,an amphiphilic dimer: mechanisms of membrane permeabilization. Science1991;251:1481–5.

16] Karg S. The water chestnut (Trapa natans L.) as a food resource during the 4thto 1st millennia BC at Lake Federsee, Bad Buchau (southern Germany). EnvironArchaeol 2006;11:125–30.

17] Law D, Moore CB, Joseph LA, Keaney MG, Denning DW. High incidenceof antifungal drug resistance in Candida tropicalis. Int J Antimicrob Agents1996;7:241–5.

18] Livak KJ, Schmittgen TD. Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-Delta Delta C (T)) method. Methods2001;4:402–8.

19] Mandal SM, Dey S, Mandal M, Neto SM, Franco OL. Identification and struc-tural insights of three novel antimicrobial peptides isolated from green coconutwater. Peptides 2009;30:633–7.

20] Matsubara T, Hayashi A, Banno Y, Morita T, Nozawa Y. Cerebroside of the dimor-phic human pathogen, Candida albicans. Chem Phys Lipids 1987;43:1–12.

21] McNeil MM, Nash SL, Hajjeh RA, Phelan MA, Conn LA, Plikaytis BD, et al.Trends in morta mycotic diseases in United States, 1980–1997. Clin Infect Dis2001;33:641–7.

22] National Committee for Clinical Laboratory Standards. Reference method forbroth dilution antifungal susceptibility testing of yeasts. Approved standard,NCCLS document M27-A. Wayne, PA: National Committee for Clinical Labora-tory Standards; 1997.

23] Powderly WG, Mayer KH, Perfect JR. Diagnosis and treatment of oropharingealcandidiasis in patients infected with HIV: a critical reassessment. AIDS Res HumRetroviruses 1999;15:1405–12.

24] Ramage G, VandeWalle K, Wickes BL, López-Ribot JL. Standardized method forin vitro antifungal susceptibility testing of Candida albicans biofilms. AntimicrobAgents Chemother 2001;45:2475–9.

25] Ribeiro SM, Almeida RG, Pereira CAA, Moreira JS, Pinto MFS, Oliveira AC, et al.Identification of a Passiflora alata Curtis dimeric peptide showing identity with2S albumins. Peptides 2011;32:868–74.

26] Riesselman MH, Hazen KC, Cutler J. Determination of antifungal MICs by a rapidsusceptibility assay. J Clin Microbiol 2000;38:333–40.

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[glass, mechanistic insights from enantiomeric human defensins. J Biol Chem2009;284:29180–92.

S.M. Mandal et al. / Pep

27] Rippon JW. Medical mycology. 3rd ed. Philadelphia, USA: WB Saunders Co.;1988.

28] Sakaki T, Zahringer U, Warnecke DC, Fahl A, Knogge W, Heinz E. Sterol glyco-sides and cerebrosides accumulate in Pichia pastoris. Rhynchosporium secalisand other fungi under normal conditions or under heat shock and ethanolstress. Yeast 2001;18:679–95.

29] Shin JH, Kee SJ, Shin MG, Kim SH, Shin DH, Lee SK, et al. Biofilm production byisolates of Candida species recovered from nonneutropenic patients: compar-ison of bloodstream isolates with isolates from other sources. J Clin Microbiol2002;40:1244–8.

30] Sumathi K, Ananthalakshmi P, Roshan MN, Sekar K. 3dSS: 3D structural super-position. Nucleic Acids Res 2006;34:W128–32.

31] Thevissen K, Kristensen HH, Thomma BPHJ, Cammue BPA, Franc ois SEJA. Ther-apeutic potential of antifungal plant and insect defensins. Drug Discov Today

2007;12:966–71.

32] Thomma BPHJ, Cammue BPA, Thevissen KC. Plant defensins. Planta2002;216:193–202.

33] Thomma BPHJ, Cammue BPA, Thevissen KC. Mode of action of plant defensinssuggests therapeutic potential. Curr Drug Targets Infect Disord 2003;3:1–8.

[

32 (2011) 1741–1747 1747

34] Vivekanandan S, Lakshminarayanan R, Jois SDS, Perumal SR, Banerjee Y, Chi-Jin EO, et al. Structure, self-assembly, and dual role of a beta-defensin-likepeptide from the Chinese soft-shelled turtle eggshell matrix. J Am Chem Soc2008;130:4660–8.

35] Wang G, Li X. Correlation of three-dimensional structures with the antibac-terial activity of a group of peptides designed based on a nontoxic bacterialmembrane anchor. J Biol Chem 2005;280:5803–11.

36] Wang G, Li X, Peterkofsky A. NMR studies of aurein 1.2 analogs. Biochim BiophysActa 2006;1758:1203–14.

37] Wang Z, Wang G. APD: the Antimicrobial Peptide Database. Nucleic Acids Res2004;32:590–2.

38] Wei G, de Leeuw E, Pazgier M, Yuan W, Zou G, Wang J, et al. Through the looking

39] Wiederstein M, Sippl MJ. ProSA-web: interactive web service for the recogni-tion of errors in three-dimensional structures of proteins. Nucleic Acids Res2007;35:W407–10.