BLOCH JÚNIOR, C.

COSTA, F. C.

FRANCO, O. L.

MARIA NETO, S.

English

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Susceptibility of Human Pathogenic Bacteria to Antimicrobial Peptidesfrom Sesame KernelsFbio Teles Costa,1 Simone Maria Neto,1 Carlos Bloch Jr.,2 Octvio Luiz Franco11Centro de Anlises Protemicas e Bioqumicas, Programa de Ps-GraduaÅ¼o em CiÞncias Genmicas e Biotecnologia,Universidade Catlica de Braslia, SGAN, Quadra 916, Mdulo B, Av. W5 Norte, 70.790-160, Asa Norte, Braslia-DF, Brazil2Laboratrio de Espectrometria de Massa, Embrapa Recursos Genticos e Biotecnologia, Parque EstaÅ¼o Biolgica, Final W5, 70.770-900,Asa Norte, Braslia-DF, BrazilReceived: 1 March 2007 / Accepted: 19 March 2007Abstract. Hospital infection caused by Gram-negative bacteria is a serious and common problem,especially in developing countries. Aiming to reduce these infections, this report focuses on the iden-tification and characterization of novel antimicrobial peptides from sesame (Sesamum indicum) kernelmeals. Thus, sesame flour was extracted and precipitated with ammonium sulfate (100%). After dialysis,a rich fraction was applied to affinity red-Sepharose CL-6B chromatography, followed by reversed-phase high-performance liquid chromatography. Mass spectrometry analysis indicated the presence of amajor peptide with molecular mass of 5.8 kDa in both cultivars. The bactericidal activities of anti-microbial peptides were evaluated against several human pathogens that had been effective only againstKlebsiella sp., a Gram-negative bacterium responsible for human urinary infection. These data indicatethe biotechnological potential of sesame peptides as an alternative method for hospital infection controland also the decrease of bacterial resistance to synthetic antibiotics.Sesame (Sesamum indicum L.) is an older oleaginouskernel that has been commonly used by several nationsas a source of food and medicines [1–3]. Includingsesame, all living organisms are able to develop self-defense mechanisms against pathogen attack. Amongthese defenses, were included important compoundssuch as phenols, secondary compounds, and antimicro-bial peptides (AMPs) [4]. Antimicrobial peptides havebeen isolated from insects [5, 6], mammals [7], micro-organisms [8], and several plant tissues such as leaves,flowers, and seeds [4]. In the large AMP class, severalfamilies are included, such as defensins [9], 2S albumins[10], vicilins [11], and several others. All of them con-tribute to plant innate host defense and represent animportant source for antibiotics. Most of these peptidesshow the ability to bind on lipids of the cell surface, toinduce cell permeability, and to cause enhanced lethalityto pathogenic bacteria [12–14].On the other side, an uncontrolled increase ofpathogens with antibiotic resistance, including severalGram-negative bacteria such as Escherichia coli,Klebsiella sp., and Proteus sp., is the cause of a seriousproblem known as hospital infection [15]. These lethalinfections, commonly observed in developing countries,have led to a search for new strategies to control theseinfectious agents. A primary search has been focused onAMPs, since these peptides have shown high activityagainst pathogens but low toxicity against mammaliancells [14, 16]. In this report, cationic antimicrobialpeptides from S. indicum kernels of black and whitecultivars (AMP-SiB and AMP-SiW) were purified andcharacterized, and their ability to control human patho-genic Gram-negative was evaluated.Materials and MethodsExtraction and Isolation of S. indicum Kernels Peptides. Kernels oftwo sesame (S. indicum) cultivars (black and white) were evaluated.Seeds were washed with sanitary water (30% solution) for 5 minCorrespondence to: O. L. Franco; email: ocfranco@pos.ucb.brCURRENT MICROBIOLOGY Vol. 55 (2007), pp. 162–166DOI: 10.1007/s00284-007-0131-0 CurrentMicrobiologyAn International Journalª Springer Science+Business Media, LLC 2007followed by distilled water for another 5 min. Sesame kernel mealswere extracted with a solution containing 0.6M NaCl and 0.1%HCl, in aproportion of 1:3 (w/v), for 6 h at 4C. This crude extract wascentrifuged at 4000g for 2 h at 4C. Proteins in the supernatant wereprecipitated with (NH4)2SO4 at 100% saturation with constant stirring.After centrifugation (under equal conditions), the precipitate wasresuspended and dialyzed (3.0 kDa cut off) against distilled water. Arich fraction was lyophilized and 5 mg of soluble protein was appliedonto affinity red-Sepharose CL-6B chromatography, equilibrated withbuffer A (0.1M Tris-HCl, pH 7.0, containing 0.05M CaCl2). Nonretainedproteins (NRP) were displaced with the same buffer. Retained proteins(RP) were eluted with buffer B (0.1M Tris-HCl, pH 7.0, containing 3.0MNaCl). Protein elution was analyzed at 280 nm. RP was dialyzed,lyophilized, resuspended with 0.1% trifluoroacetic acid (TFA), andapplied to analytical reversed-phase high-performance liquidchromatography (HPLC) (Vydac C-18TP 522) where the retainedfraction was eluted using a linear gradient of acetonitrile (0–100%) at aflow rate of 1.0 mL/min.Molecular Mass Analyses. Protein fractions were analyzed by 12%sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according Laemmli [17]. Gels were silver stained andbromophenol blue was used as the tracking dye. AMP-Si was alsosubmitted to mass spectrometry analyses according Franco et al. [9].Bioassays. Klebsiella sp., Proteus sp., and E. coli were challengedby use of 1.0 mL of Luria–Bertani (LB) broth (10 g/L NaCl, 5 g/Lyeast extract, and 45 g/L bactopeptone). The bacteria were grown inLB broth for 18–24 h at 37C before starting peptide evaluation.Distilled water was used as the negative control and 40 lg/mLchloranphenicol was used as the positive control. The proteinfractions eluted from reversed-phase chromatography wereincubated with Gram-negative bacteria on LB broth for 4 h at37C at standard concentrations of 10, 25, 50, and 100 lg/mL.Evaluation of bacteria growth was done by spectrophotometricanalyses at 595 nm, for each experimental hour. Each experimentwas carried out in triplicate.ResultsPurification of AMP-Sis. In order to identify novelantimicrobial peptides in sesame kernels, crude extractsfrom both cultivars were challenged against humanpathogenic bacteria. After enrichment, these fractionswere applied to affinity Red-Shepharose CL-6Bchromatography (Fig. 1A), which is commonly utilizedto purify cationic and hydrophobic peptides [10, 18, 19].Only retained protein fractions from white and blackcultivars were capable of reducing Proteus sp. andKlebsiella sp. growths (data not shown). After dialyses,RP was submitted to a reversed-phase HPLC (Figs. 1Band 1C). The white sesame cultivar showed a majorpeak at 18% acetonitrile, whereas the black sesamecultivar presented antibacterial proteins eluted in amajor peak of 23% acetonitrile.Molecular Mass Analysis. The SDS-PAGE analyses ofHPLC major peaks showed single bands for bothcultivars that correspond to proteins with 5 kDa(data not shown). This result was confirmed by massFig. 1. A Red-Sepharose chromatographic profiles of white (dottedline) and black (solid line) sesame kernels. NRP corresponds to thenonretained peak and RP to the retained one. Black arrows indicate asingle elution step of 3.0M NaCl. Therefore, white and black RPswere applied to a reversed-phase HPLC-generating chromatographic -profiles (B) and (C), respectively. Diagonal lines indicate acetoni-trile gradient elution (0–100%).F. T. Costa et al.: Sesame Antimicrobial Peptides 163spectrometric analysis, where was observed twopeptides of 5799.69 and 5800.74 Daltons, respectively,from black and white cultivars (Figs. 2A and 2B)were observed, denoted AMP-SiB and AMP-SiW,respectively.Biological Activity. Crude extract was evaluatedagainst the three Gram-negative bacteria previouslydescribed, at a standard concentration of 100 lg/mL.However, this extract was only active against Klebsiellasp., being capable of reducing their development by50% (data not shown). Moreover, purified peptidesobtained from HPLC fractions were compared againstGram-negative bacteria. AMP-SiW and AMP-SiB werecapable of inhibiting 25% of Klebsiella sp. growth(Figs. 3A and 3B) at concentration of 100 lg/mL.Nevertheless, at lower concentrations (10, 25, and 50lg/mL) no activity was observed. Furthermore, nodeleterious activity of purified peptides was observedagainst Proteus sp. and E. coli (data not shown).DiscussionIn spite of the sesame peptides reported here beingunpublished, similar biochemical properties have beenfound in other bactericidal peptides. Among them wecould observe similarities in the acetonitrile concentra-tions elution, as in the isolation of antimicrobial peptidesfrom Japanese bamboo shoots (Phyllostachys pubens-cens) [20], bulbs of the tulip (Tulipa gesneriana L.) [21],buckwheat seeds (Fagopyrum esculentum M.) [22],passion fruit seeds (Passiflora edulis) [10], cowpeaseeds (Vigna unguiculata) [9–19], macadamia nut ker-nels (Macadamia integrifolia) [11], and radish seeds(Raphanus sativus L.) [23]. Also, molecular massesFig. 2. Matrix assisted laser desorption/ionization time-flight spectraof AMP-Sis from white (A) and (B) black cultivars.Fig. 3. Antimicrobial activities of AMP-Sis from white (AMP-SiW)(A) and black (AMP-SiB) (B) sesame kernels are indicated by blacktriangles. Black squares correspond to the negative control (distilledwater) and black circles correspond to the positive control (chloran-phenicol, 40 lg/mL). Vertical bars correspond to the standard devia-tion. Each assay was carried out in triplicate.164 CURRENT MICROBIOLOGY Vol. 55 (2007)were similar when compared to Cp-thionins fromV. unguiculata seeds [9, 19] and 2S albumins fromP. edulis [10]. Both classes show antimicrobial proper-ties. AMP-Sis masses are similar to molecular weightsobserved in different antimicrobial peptide classes. Forthis reason, at this moment and using this parameter, it isimpossible to classify them correctly. Further sequenc-ing experiments will be conduced in order to elucidatethis specific property.Furthermore, AMP-SiB and AMP-SiW showedinhibitory activity against Klebsiella sp. Similar resultswere obtained by using antimicrobial peptides fromdifferent plant sources, such as cowpea seeds [9]. In thisstudy, it was shown that cowpea peptide was activeagainst both Gram-positive and Gram-negative bacteria[9]. Otherwise, several AMPs showed inhibitory activityagainst a wide variety of pathogenic micro-organisms,which included fungi, bacteria, and protozoa, indicatingthe enormous biotechnological potential of AMPs [10,13, 21–23]. Moreover, it was observed that the expres-sion of an antimicrobial peptide from motherwort(Leonurus japonicus Houtt) was able to promote diseaseresistance in tobacco [24].Hence, our results showed severe growth inhibitionof the human pathogen Klebsiella sp., a bacteriumresponsible for urinary and lung infections in weakenedhospital patients. These data suggest that the develop-ment of new drugs from AMPs might be successful forthe treatment of several infections, reducing hospitalcosts and also human mortality. The results observedhere are quite encouraging, when compared with otherAMPs with activities against Gram-negative bacteria. Inaddition, this research indicated the remarkable potentialof the unexploited genetic resources of plant biodiver-sity. The discovery of valuable genes in plant biodi-versity might also be an alternative for countries indevelopment which have biotechnological research buthave difficulties accessing novel drugs due to elevatedcosts and also patent restrictions.ACKNOWLEDGMENTSWe would thank Dra. Silvia E. T. Bittencourt and the entire Centro deAnlises Protemicas e Bioqumicas (CAPB) team for technicalassistance. This work was supported by CAPES, FAPEMIG, CNPq,and UCB.Literature Cited1. 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Susceptibility of human pathogenic bacteria to antimicrobial peptides from sesame kernels.

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Susceptibility of human pathogenic bacteria to antimicrobial peptides from sesame kernels.

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