Fitoterapia 76 (2005) 67 – 72 www.elsevier.com/locate/fitote
Antibacterial activity of seed proteins of Robinia pseudoacacia T. Talas-Og˘rasSa,*, Z. ˙Ipekc¸ia, K. Bajrovic¸a,b, N. GfzqkVrmVzVa,c a
TU¨BI˙TAK, Research Institute for Genetic Engineering and Biotechnology (RIGEB), P.O Box. 21 41470 Gebze-Kocaeli, Turkey b Institute for Genetic Engineering and Biotechnology 71 000 Sarajevo, Bosnia-Herzegovina c Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, 34459 Vezneciler-Istanbul, Turkey Received 22 January 2004; accepted in revised form 26 October 2004 Available online 8 December 2004
Abstract A low molecular weight cationic peptide was isolated from Robinia pseudoacacia seed and tested in vitro against seven bacteria (Corynebacterium michiganense, Staphylococcus aureus, Bacillus subtilis, Erwinia carotovora subsp. carotovora, Pseudomonas syringae pv syringae, Xanthomonas campestris pv campestris, and Escherichia coli). The peptide inhibited the growth of the tested strains. The effective concentrations required for 50% inhibition of bacterial growth ranged between 20 and 120 Ag ml 1 protein. S. aureus was found to be the most sensitive strain, however, E. coli was not affected much when compared with others. Reduction of antibacterial activity of the peptide with CaCl2 addition into the growth medium was also observed. D 2004 Elsevier B.V. All rights reserved. Keywords: Peptides; Antibacterial activity; Robinia pseudoacacia
1. Introduction Recently, a number of studies concerning the search for new antimicrobial agents from plants and antimicrobial screening of the extracts have been performed [1]. Antimicrobial * Corresponding author. Tel.: +90 262 641 2300x4024; fax: +90 262 646 2939. E-mail address:
[email protected] (T. Talas-Og˘rasS). 0367-326X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2004.10.020
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peptides (AMPs) constitute a heterogenous class of low molecular weight proteins, which are recognized as important components of innate defense system of both animals and plants and they directly interfere with the growth, multiplication and spread of microbial organisms [2,3]. Plants produce a wide array of defense protein in their different tissues to anticipate and cope with the attacks of microbial pathogens. Up to date, several classes of proteins with antibacterial and/or antifungal properties have been isolated [4]. Some of these proteins are classified as thionins, lipid transfer proteins, plant defensins, chitinases, 2S albumins, and ribosome inactivating proteins [5,6]. Here, the small seed proteins of Robinia pseudoacacia L. Rozynskiana (Leguminosae) were isolated, and the in vitro antibacterial activity of an effective basic peptide was investigated against microbial strains.
2. Experimental 2.1. Extraction of the seed proteins The seeds of R. pseudoacacia (Black locust) were collected from the campus of our Institute in September. One hundred grams of seeds were ground, and the resulting fine powder was fully homogenized with four volumes of cold extraction buffer (10 mM Na2HPO4, 15 mM NaH2PO4, 100 mM KCl, 1.5 mM EDTA) containing freshly added 1 mM phenylmethylsulfonyl fluoride. The homogenate was stirred for 2 h at 4 8C and then passed through cheesecloth. The extract was clarified by centrifugation for 30 min at 10 000 rpm, and solid ammonium sulphate was added to the supernatant to obtain 30% saturation. After gentle stirring for 2 h at 4 8C, the crude proteins were collected by centrifugation (for 30 min at 10.000 rpm), and the supernatant was adjusted to 70% relative ammonium sulphate saturation. The slurry was stirred for 2 h at 4 8C, and the precipitate was recovered by centrifugation, as explained previously, and the pellet was solubilised in a minimal amount of distilled water. The meal was kept at 80 8C for 10 min to denature some proteins. A final centrifugation (30 min at 10.000 rpm) was performed to discard heat-denatured protein precipitates, and the supernatant was dialyzed extensively against distilled water using a dialysis tubing with a molecular weight cut-off 1 kDa. Protein fractions smaller than 10 kDa were obtained using Centricon YM-10 filter (Millipore). The protein extract of R. pseudoacacia seeds was filtered through YM-10 and applied directly to a DEAE-52 cellulose ion-exchange column (1.010 cm), which had been previously equilibrated with 20 mM Tris–HCl buffer (pH 8.0). The column eluates were collected at a flow rate of 22.5 ml/h, and the elution profile was monitored at 280 nm. Unadsorbed protein fractions were collected with the equilibration buffer, while adsorbed protein materials were eluted by the addition of 0.5 M NaCl in the same buffer. A part of the eluates was used for protein determination and electrophoresis, and the other was filtered through a 0.22 Am syringe filter and used for antimicrobial activity assays. Protein concentration was determined by the method of Bradford [7], using bovine serum albumin as reference protein. Denaturing SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out as described by Laemmli [8], using 15% polyacrylamide slab gel. The gel was stained by Coomassie brilliant blue R-250.
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2.2. Microbial strains Antibacterial activity was assayed against three Gram(+) (Corynebacterium michiganense CM 102, Staphylococcus aureus ATCC 6538, and Bacillus subtilis ATCC 6633) and four Gram( ) bacterial strains (Erwinia carotovora subsp. carotovora NCPPB 929, Pseudomonas syringae pv. syringae NCPPB 1075, Xanthomonas campestris pv. campestris NCPPB 528, and Escherichia coli strain HB 101). The microbial strains used were from stock cultures of RIGEB and grown in liquid Luria–Bertani (LB) medium. 2.3. Antimicrobial activity assay The antibacterial activity of the total seed extract and the column eluated protein fractions was determined by paper disk diffusion [9] and microplate-based turbidity measurement assays [10]. The microplate was incubated at 30–37 8C for 24 h, and microbial growth was quantified by measuring culture turbidity at 595 nm using an automatic microplate reader Model 3550 (Bio-Rad). All assays were performed in triplicate. The growth medium for antibacterial assay was also supplemented with 5 mM CaCl2.
3. Results and discussion In this study, a small heat-stable cationic peptide of the seed of R. pseudoacacia was isolated, and antibacterial activity was investigated against seven bacterial strains. Purification of the small cationic peptide of R. pseudoacacia seed entailed the following steps. The crude extract of the seed was precipitated between 30% and 70% relative saturation with ammonium sulphate, denaturation of the high molecular weight proteins at 80 8C for 10 min, dialysis, and YM-10 filtration. The overall purification yield of the proteins is summarized in Table 1. The heat stable low molecular weight proteins were applied to anion exchange chromatography on DEAE-52 cellulose column. Basic protein fractions (peak B1) were previously eluted as unbound material through the column in the void volume (Fig. 1). The column was washed with 0.5 M NaCl addition to the elution buffer, and bound fractions (peak B2) were also eluted. The protein fractions of the seed were collected and used for antibacterial activity assay. Table 1 Stages of purification processes of the R. pseudoacacia seed proteins Protein isolation stage
Total protein (mg)a
Protein recovery (%)
Crude seed extract (NH4)2SO4 precipitation (70%) 80 8C denaturation Dialysis YM-10 filtration
864 174 120 109 38.7
100 20.1 13.8 12.6 4.4
a
Protein amount was determined by the method of Bradford.
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Fig. 1. Elution profile of the crude seed extract of R. pseudoacacia seeds from a DEAE-cellulose anion-exchange column. The column had previously been equilibrated with the starting buffer (20 mM Tris–HCl, pH 8.0) and washed with the same buffer. The bound protein fractions were eluted with 0.5 M NaCl in the starting buffer and indicated with the arrow. The flow rate was 22.5 ml/h, and 1.5 ml fractions were collected.
In vitro antibacterial susceptibility tests of the total seed extract and the protein fractions were assayed against seven bacterial strains. The cationic protein fractions from peak one (B1) showed significant antibacterial activity against most of the tested bacteria, while insignificant antibacterial activity was detected with the acidic protein fractions of the second peak (B2). The heat stable cationic protein fractions of the R. pseudoacacia seed exhibited different antibacterial activity profiles on the growth of the tested pathogenic bacteria, but the highest activity against most of the bacteria was obtained with fraction number 14, which was named as Rp-AMP1 (the small cationic AMP of the R. pseudoacacia seed). The antibacterial activity of Rp-AMP1 against S. aureus was shown by paper disk diffusion assay. Protein concentrations required for 50% inhibition (IC50) of bacterial growth were determined using the microplate-based turbidity assay. Rp-AMP1 showed inhibitory activity against all bacterial strains. In the low-ionic-strength medium, IC50 values ranged from 20 to 120 Ag/ml peptide for Gram(+) and Gram( ) bacteria. S. aureus seemed to be the most sensitive bacterial strain, with a minimum IC50 value of 20. The inhibitory effect of total seed extract of R. pseudoacacia against the bacterial strains was also examined, and it was not effective as Rp-AMP1 (data not shown). Several small basic peptides have been reported to be strongly inhibited by cations, especially calcium [11,12]. The antagonistic effect of calcium ion on antibacterial activity of Rp-AMP1 was examined by adding CaCl2 to the growth medium. The addition of 5 mM CaCl2 to the growth medium significantly reduced the antibacterial activity of RpAMP1 at least sixfold (Table 2). This phenomenon has been identified for a number of small antimicrobial peptides [13,11]. The molecular weight estimation of the Rp-AMP1 was determined by 15% SDS–PAGE (Fig. 2). After treatment with disulfide-reducing agents, Rp-AMP1 gave a approximately 5 kDa protein band, which is similar to the molecular weight of known AMPs [14,6]. Its low molecular mass, coupled with its high antibacterial potency, should make it a strong candidate for the exploitation of its biological activities. The bioassays showed that the cationic protein fraction of the R. pseudoacacia seed affected on a relatively broad spectrum
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Table 2 Antibacterial activity of Rp-AMP1 Microorganism
E. carotovora P. syringae X. campestris E. coli C. michiganense S. aureus B. subtilis a b c
IC50 (Ag/ml)a Medium Ib
Medium IIc
80 100 80 120 30 20 40
N200 N200 N200 N200 180 180 N200
Protein concentrations required for 50% growth inhibition after 24 h of incubation. Half strength LB growth medium. Medium I supplemented with 5 mM CaCl2.
of human and plant pathogenic bacteria. On the other hand, the total seed extract of the R. pseudoacacia did not show significant antibacterial activity against the tested bacteria. The seed extracts of some plant species showed higher antibacterial activity than did other parts of the plant [15]. In some studies, potent antimicrobial proteins and extracts have been purified from different seeds [14,16]. Consequently, it is also believed that seed proteins play an important role in the protection of seeds against microbial invaders. In vitro antimicrobial screening permits the selection of plant extract with useful properties to be used for further biochemical and pharmacological studies. Up to now, several types of antimicrobial peptides have been isolated, and their structures have been characterized. Some examples of cysteine-rich, highly basic antimicrobial peptides are toxic to either Gram(+) or Gram( ) bacteria, fungi, and yeast [17]. AMPs have potential to combat diseases caused by pathogens that are becoming increasingly resistant to traditional antibiotics. Different kinds of antimicrobial peptides of plants and animals evolved as weapons directed against a particular group of infectious agents [18].
Fig. 2. Fifteen percent SDS–PAGE of total seed extract (lane 2) of R. pseudoacacia and Rp-AMP1 (lane 1, 15 Ag peptide). Lane 3 shows molecular marker proteins, from top to downward: trypsinogen (24.000 kDa), alactalbumin (14.2 kDa), and aprotinin (6.500 kDa).
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The mechanism of action of the antimicrobial peptides has not been elucidated in detail yet, but a possible mechanism can be suggested. Some peptide molecules formed a channel on cell membrane and the cell died of the outflowing of cellular contents. This mechanism is different from that of antibiotics [5]. Herein, a chromatographic procedure was described to purify a cationic, small, heat stable peptide from the seeds of R. pseudoacacia. The data support evidence for the existence of a potent antibacterial peptide in R. pseudoacacia seed, which inhibits the growth of some important pathogenic bacteria in vitro. The isolation and antibacterial screening results from our study could provide a guideline in selecting plants for isolating compounds with antibacterial activity. The detailed analysis of the peptide should be clarified by amino acid analysis and protein database searches to find out identity with the known antimicrobial peptides, and further studies are being conducted to elucidate the properties of the peptide for antimicrobial activity.
Acknowledgements This work was in part supported by the project No. 5003301 TUBITAK, Research Institute for Genetic Engineering and Biotechnology.
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