Food Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts Ann-Li Yong b, Keng-Fei Ooh b, Hean-Chooi Ong c, Tsun-Thai Chai a,b, Fai-Chu Wong a,b,⇑ a
Centre for Biodiversity Research, Universiti Tunku Abdul Rahman, 31900 Kampar, Malaysia Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, 31900 Kampar, Malaysia c Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia b
a r t i c l e
i n f o
Article history: Received 14 October 2014 Received in revised form 14 November 2014 Accepted 18 November 2014 Available online xxxx Keywords: Callicarpa formosana Escherichia coli Melastoma candidum Natural products Protein expression Scanning electron microscope Scutellaria barbata Staphylococcus aureus
a b s t r a c t In this paper, we investigated the antibacterial mechanism and potential therapeutic targets of three antibacterial medicinal plants. Upon treatment with the plant extracts, bacterial proteins were extracted and resolved using denaturing gel electrophoresis. Differentially-expressed bacterial proteins were excised from the gels and subjected to sequence analysis by MALDI TOF–TOF mass spectrometry. From our study, seven differentially expressed bacterial proteins (triacylglycerol lipase, N-acetylmuramoylL-alanine amidase, flagellin, outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin) were identified. Additionally, scanning electron microscope study indicated morphological damages induced on bacterial cell surfaces. To the best of our knowledge, this represents the first time these bacterial proteins are being reported, following treatments with the antibacterial plant extracts. Further studies in this direction could lead to the detailed understanding of their inhibition mechanism and discovery of target-specific antibacterial agents. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Medicinal plants are rich reservoirs of phenolic compounds, and different parts of the plants have been consumed traditionally for a variety of medicinal applications (Jaberian, Piri, & Nazari, 2013; Wong, Li, Cheng, & Chen, 2006). For the past few decades, the studies on medicinal plants have grown dramatically, and their usages have increased worldwide, partly encouraged by the great commercial potentials of plant-derived therapeutic agents (Cragg, Grothaus, & Newman, 2014). The potentials of plant-derived therapeutic agents are further encouraged by the fact that a significant number of currently available pharmaceutical drugs could be traced back to their origins in plants. Well-known examples include vinblastine (an alkaloid drug) derived originally from Catharanthus roseus (Madagascar periwinkle) and topotecan (a camptothecin analog) derived from Camptotheca acuminate (Asian happy tree) (Facchini & De Luca, 2008; Lorence & Nessler, 2004). In fact, it was estimated that nearly half of modern pharmaceutical drugs are derived from plants (Newman & Cragg, 2007). Additionally,
⇑ Corresponding author at: Centre for Biodiversity Research, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900 Kampar, Malaysia. Tel.: +605 468 8888x4521; fax: +605 4661676. E-mail address: [email protected] (F.-C. Wong).
increasing plant-based food supplements have been marketed to cater for the strong consumer demands. Example includes the cranberry supplement, which was reported to be effective in treating urinary tract infection (Caillet, Côté, Sylvain, & Lacroix, 2012; Wu, Qiu, Bushway, & Harper, 2008). With the increasing occurrences of multi-drug resistant bacteria strains, the world is currently facing an urgent need to find new antibiotic derivatives (Gardete & Tomasz, 2014). Accumulated evidences are pointing toward the effectiveness of plant-derived compounds in inhibiting multi-drug resistant bacterial strains such as methicillin-resistant Staphylococcus aureus (Gyawali & Ibrahim, 2014). Previously, our laboratory and others had reported the minimum inhibitory concentrations (MIC) of three antibacterial medicinal plants, namely Callicarpa formosana, Melastoma candidum, and Scutellaria barbata (Wang, Hsu, & Liao, 2008; Wong, Yong, Ong, & Chaia, 2013). However, the exact bacterial inhibition mechanism of these aforementioned plant extracts remained to be elucidated. For instance, it is still unclear which bacterial pathways or enzymes are being targeted by these antibacterial plant extracts. Additionally, no information is available regarding their effects on the structural integrity of bacterial cell membranes. In this paper, we evaluated the antibacterial mechanism of these aforementioned edible medicinal plants, using proteomic
http://dx.doi.org/10.1016/j.foodchem.2014.11.103 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yong, A.-L., et al. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.103
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analysis and scanning electron microscope (SEM). Upon treatments with antibacterial plant extracts, differentially-expressed bacterial proteins were first resolved using denaturing gel electrophoresis, followed by sequence determination with MALDI TOF–TOF mass spectrometry. Additionally, we investigated on how the antibacterial plant extracts affected the bacteria physically, through viewing of the bacterial membrane surfaces with SEM. Lastly, gas chromatography–mass spectrometry (GC–MS) was also applied to investigate the phytochemical contents of the medicinal plant extracts. Through the combination of these techniques, we aim to identify potential phytochemical-mediated antibacterial therapeutic targets, along with better understanding of their mechanisms. 2. Material and methods 2.1. Preparation of plant extracts Medicinal plants were collected around Perak state (Malaysia) from April to July of 2012. The plants species were authenticated by Professor Dr. Hean-Chooi Ong at the Institute of Biological Sciences, University of Malaya, Malaysia. These plants were dried in oven at 40 °C until constant weights were achieved. The dried plants were then pulverized using a warring blender, followed by extraction using methanol solvent. The supernatants were collected, filtered and then stored in 20 °C. 2.2. Isolation of bacterial proteins for SDS–PAGE analysis The bacteria strains (Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, S. aureus ATCC 6538) were cultured overnight in Luria–bertani (LB) broth using conical flasks in an orbital shaker at 37 °C and 200 rpm. Bacterial cultures were adjusted to 0.5–1 McFarland standard before used in subsequent experiments. The bacterial cultures were then treated with optimal concentrations (2–6 mg/ml) of the filtered plant extracts, and sterile water is used as the negative control. After the addition of plant extracts, the bacterial cultures were keep in an orbital shaker (37 °C, 200 rpm). At the designated time intervals, 20 ml of bacterial culture was collected and centrifuged at 9000 rpm for 10 min to separate the bacterial cell pellet from the supernatant. The pellet was then subjected to treatment with Bacterial Proteins Extraction Reagent (Thermo Scientific), and the supernatant was subjected to ammonium sulphate precipitation. The precipitated proteins were collected by centrifugation at 9000 rpm for 10 min. 2.3. SDS–PAGE gel analysis and identification of differentially expressed bacterial proteins SDS–PAGE gel electrophoresis was carried out using 12% resolving gel [1.5 M Tris–HCl pH 8.8, 10% SDS (Fisher Scientific), 40% bisacrylamide (Bio Basic Canada)], and 4% stacking gel [0.5 M Tris–HCl pH 6.9, 10% SDS, 40% bis-acrylamide]. The bacterial proteins were treated with dithiothreitol (DTT) and boiled, before loading into the wells. Gel electrophoresis was performed with constant electric current of 135 mV, until the bromophenol blue (BPB) reached the bottom of the gel plate. The protein gel was then stained with Coomassie Brilliant Blue R-250, and the molecular weights of the bacterial proteins were determined using commercial protein markers (Spectra Multicolor Broad Range Protein Ladder, Thermo Scientific). Differentially expressed protein bands were excised from the gel using sterile razor blades and analysed by MALDI TOF–TOF mass spectrometry (4800 Proteomics Analyzer, AB Sciex) (Proteomics International, Perth, Australia). The spectra were analysed using Mascot sequence matching software (Matrix Science) with Ludwig NR Database to identify the proteins of interest.
2.4. Investigation of morphological changes on bacteria membranes via scanning electron microscopy (SEM) The bacteria strains were cultured overnight in an orbital shaker set at 37 °C and 200 rpm. Next, bacterial cultures were adjusted to 0.5–1 McFarland standard and treated with antibacterial plant extracts (0.6–5.0 mg/ml) or sterile water (control) for 2 h. Treated and non-treated bacteria cultures were incubated with 2.5% glutaraldehyde overnight. Following three washes with PBS, the samples were hydrated with ascending concentrations of ethanol (25% for 5 min, 50% for 10 min, 75% for 10 min and three changes of 100% ethanol for 10 min each). The samples were then dried through freeze drying. Subsequently, the dried samples were adhered onto double-sided adhesive conductive carbon tape (which was mounted on a copper stage). The samples were then coated with platinum (JEOL JFC-1600 Auto Fine Coater) before viewing by SEM. 2.5. Gas chromatography–mass spectrometry (GC–MS) analysis of TCL spots Thin layer chromatography (TLC) was performed as previously described (Eloff, 2004). TLC spots were analysed by gas-chromatography equipped with mass spectrometry (GCMS-QP2010 Plus, Shidmadzu). Column (BPX-5, SGE Analytical Science) temperature was initially set at 110 °C and increased to 280 °C (increased at a rate of 5–10 °C/min). The flow rate of the carrier gas (helium) was set to 1 ml/min. The total running time of the gas chromatography was 36 min. The mass spectra were obtained from the range of m/e 40–700. The identities of the samples were determined by comparing the mass spectra with NIST Gas Chromatography Library Database. 3. Results and discussion 3.1. Identification of differentially expressed bacterial proteins Through proteomic study, our objective was to identify differentially expressed bacterial proteins, upon treatment with antibacterial plant extracts. In this approach, we focused on three bacterial strains (E. coli, P. aeruginosa, S. aureus) treated with three medicinal plant extracts (C. formosana, M. candidum, S. barbata). Bacterial cellular proteins were extracted using Bacterial Protein Extraction Reagents (Thermo Scientific) and ammonium sulphate precipitation. In our results, a total of seven differentially expressed bacterial proteins were identified (Fig. 1). Differentially expressed bacterial proteins were excised and subjected to sequence analysis by mass spectrometry. The identified proteins and their characteristics were summarised and reported in Table 1. Protein bands a, b (Fig. 1A) and c (Fig. 1B) were extracted from bacterial culture supernatants, using ammonium sulphate precipitation method. Based on MALDI TOF–TOF protein sequencing results, protein bands a to c were determined as triacylglycerol lipase, N-acetylmuramoyl-L-alanine amidase and flagellin, respectively. Protein bands d, e (Fig. 1C) and f, g (Fig. 1D) were extracted using Bacterial Protein Extraction Reagents and identified as outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin, respectively. Among the seven differentially-expressed proteins identified, two of them (bands f and g) are pertaining to the bacterial protein translational machinery. The 30S ribosomal protein S1 (band f) is an essential component critically involved in the translational pathway (Lafontaine & Tollervey, 2001). Interestingly, the 60 kDa chaperonin (band g) was previously reported with functional roles to stimulate proper folding of newly synthesized bacterial polypeptides, as well as to promote refolding of denatured proteins
Please cite this article in press as: Yong, A.-L., et al. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.103
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Fig. 1. (A) Protein expression profile of S. aureus following exposure to C. formosana extract. (B) Protein expression profile of E. coli following exposure to S. barbata extract. Protein expression profiles of (C) E. coli and (D) P. aeruginosa, following exposure to M. candidum extract. ( ) and (+) indicated the absence or presence of plant extract in bacterial culture medium, respectively. Labels (a–g) indicated differentially expressed protein bands selected for MALDI TOF–TOF sequence analysis.
Table 1 Summary of identified differentially-expressed bacterial proteins, using MALDI TOF–TOF MS. Protein bands (a–g) are labelled corresponding to those in Fig. 1. Protein band
Protein size (in aa)
No. of peptide sequence identified
No. of amino residue identified
Sequence covered (%)
Annotation
a b c d e f g
681 619 486 350 99 559 548
1 4 5 7 1 8 5
11 43 73 75 14 126 71
1.61 6.95 15.02 21.43 14.14 22.54 12.96
Triacylglycerol lipase N-acetylmuramoyl-L-alanine amidase Flagellin Outer membrane protein A Stringent starvation protein A 30S ribosomal protein S1 60 kDa chaperonin
under stress conditions (Kurochkina et al., 2012). Meanwhile, literature search also highlighted on the critical roles of triacylglycerol lipase (band a) and stringent starvation protein A (band e) in bacterial metabolic pathways. Triacylglycerol lipase is a bacterial enzyme involving in hydrolysis of triglycerides into fatty acids and glycerol (Kim, Song, Shin, Hwang, & Sun, 1997), while stringent starvation protein A was reported with ability to promote tolerance of E. coli in acidic environment, such as human digestive tracts (Hansen et al., 2005). On the other hand, proteins pertaining to the integrity of bacterial membranes were also identified. N-acetylmuramoyl-L-alanine amidase (band b) is a bacterial enzyme participating in the biosynthesis of peptidoglycans, an important building component in bacterial cell walls (Machowski, Senzani, Ealand, & Kana, 2014). Meanwhile, flagellin (band c) and outer membrane protein A (band d) are critical bacterial structural proteins. Flagellin is the major component in flagella filaments, which is responsible for bacterial mobility, invasion and contributing to pathogenicity (Ramos, Rumbo, & Sirard, 2004). In our results, the presence of flagellin was detected in the culture supernatants, upon treatment with the plant extract. Based on this observation, it is tempting to speculate that the antibacterial plant extract may somehow degrade and lead to the disintegration of the bacterial flagellin. Interestingly, inhibition of flagellin synthesis had previously been reported in E. coli, upon exposure to carvacrol (a monoterpenoid) (Burt et al., 2007). On the other hand, outer membrane protein A (OmpA) is critical for maintaining bacterial membrane integrity, through physical linkage with peptidoglycan layer. Previous study had also reported on the significance of OmpA in the bacterial conjugation process (Ried & Henning, 1987). Although the exact mechanism
remains to be determined, further works in this direction could potentially lead to the discovery of flagellin-specific and OmpAspecific antibacterial compounds. 3.2. Investigation of morphological changes on bacterial membranes via scanning electron microscopy (SEM) In order to have a better understanding of the antibacterial mechanism, plant extracts were further studied for their effects on the bacterial cell surfaces. Using SEM, we were able to observe the morphological changes in two bacteria strains (S. aureus and E. coli), upon treatment with antibacterial M. candidum extract (Fig. 2). In S. aureus, the untreated bacterial cells appeared grape-shaped, and the cells surfaces were intact with no damage observed (Fig. 2A). However, in the samples treated with M. candidum extract, the bacterial cells did not retain the grape-shaped characteristic (Fig. 2B). Moreover, uneven fragments were observed, indicating the damages induced on bacterial cell membranes. In E. coli, the untreated bacterial cells appeared to be in perfect rod-shaped, with no damage observed on the cell surfaces (Fig. 2C). Yet, in bacterial cells treated with M. candidum extract, we observed the formation of irregular fragments, and the cells did not retain much of its rod-shaped (Fig. 2D). Moreover, the treated bacterial cell surfaces were uneven, and the cells appeared damaged. Through the use of SEM, we observed that the bacterial membrane surfaces were damaged, upon treatment with M. candidum extract. Interestingly, it was previously reported by others that both bacterial membranes and cytoplasm were easily affected by passive diffusion of phenolic acids, with subsequent disruption of
Please cite this article in press as: Yong, A.-L., et al. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.103
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Fig. 2. Scanning electron microscope recordings of bacterial cell surfaces: (A) S. aureus (control) and (B) S. aureus (treated with M. candidum extract); (C) E. coli (control) and (D) E. coli (treated with M. candidum extract).
the M. candidum extract. It is interesting and potentially rewarding to persuade further the exact mechanism, as well as the bioactive compounds involved, in this M. candidum-induced bacterial membrane disruption. 3.3. GC–MS analysis
Fig. 3. GC analysis of the methanol fraction of C. formosana extract. Major peaks (compounds 1–6) were selected for further analysis with MS.
the bacterial membrane integrity (Campos et al., 2009). As observed in Fig. 2, compared to the untreated samples, bacterial cell membranes were significantly affected, following exposure to
In order to have a better understanding of the phytochemical contents, we also analysed the plant extract using GC–MS. From the GC spectrum with C. formosana methanolic extract, six major peaks were identified (Fig. 3). Peaks 1 to 6 were selected and subjected to MS analysis. The identities and characteristics of these peaks were determined and summarised (Table 2). Literature search indicted that Compound 2 (phenol, dimethyl) (Velmurugan, Viji, Babu, Punitha, & Citarasu, 2012), Compound 5 (hexadecanoic acid, methyl ester) (Dhankhar, Kumar, Ruhil, Balhara, & Chhillar, 2012), and a synthetic derivative of Compound 3 (1-propanone) (Chitra et al., 2011; Gul, Sahin, Gul, Ozturk, & Yerdelen, 2005) have previously been reported by other groups with bacterial inhibition activities. Additionally, the presence of Compound 4 (aminononadecane) was reported in Tamarix boveana, a perennial shrub with bacterial inhibition activities (Saïdana et al.,
Table 2 Major chemical compounds identified from the active fraction of C. formosama methanol extract by GC–MS. Peak number
Retention time (min)
Name of the compounds
Molecular formula
Molecular weight
Quality (%)
1 2 3 4 5 6
9.57 12.42 15.72 18.50 21.30 21.53
2-Heptanamine, 5-methyl Phenol, 2,4-bis(1,1-dimethylethyl) 1-Propanone, 2-amino-1-phenyl Aminononadecane Hexadecanoic acid, 15-methyl, methyl ester Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy, methyl ester
C8H19N C14H22O C9H11NO C19H41N C18H36O2 C18H28O3
129 206 149 283 284 292
81 91 80 83 86 79
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2008). It is possible that some of the identified compounds, either alone or work in a synergistic manner, contributed toward the observed bacterial inhibition activities. However, we could not rule out the possible functional roles of some other unidentified minor phytochemicals. In conclusion, seven differentially-expressed bacterial protein targets were identified in this study. These seven proteins could further be classified into bacterial pathways pertaining with protein translation, membrane integrity and metabolism. Moreover, our SEM study also demonstrated the damages which were induced onto the bacterial membrane surfaces, upon treatments with the antibacterial plant extract. Further works in this direction could potentially lead to the discovery of pathway-specific antibacterial agents, targeting bacterial protein translational machinery and cellular membranes. Acknowledgements We would like to extend our gratitude to funding supports from UTAR Research Fund (UTARRF) and Malaysia Toray Science Foundation (MTSF). References Burt, S. A., Van Der Zee, R., Koets, A. P., De Graaff, A. M., Van Knapen, F., Gaastra, W., et al. (2007). Carvacrol induces heat shock protein 60 and inhibits synthesis of flagellin in Escherichia coli O157:H7. Applied and Environmental Microbiology, 73(14), 4484–4490. Caillet, S., Côté, J., Sylvain, J. F., & Lacroix, M. (2012). Antimicrobial effects of fractions from cranberry products on the growth of seven pathogenic bacteria. Food Control, 23(2), 419–428. Campos, F. M., Couto, J. A., Figueiredo, A. R., Tóth, I. V., Rangel, A. O. S. S., & Hogg, T. A. (2009). Cell membrane damage induced by phenolic acids on wine lactic acid bacteria. International Journal of Food Microbiology, 135(2), 144–151. Chitra, S., Paul, N., Muthusubramanian, S., Manisankar, P., Yogeeswari, P., & Sriram, D. (2011). A facile synthesis of carbocycle-fused mono and bis-1,2,3selenadiazoles and their antimicrobial and antimycobacterial studies. European Journal of Medicinal Chemistry, 46(11), 5465–5472. Cragg, G. M., Grothaus, P. G., & Newman, D. J. (2014). New horizons for old drugs and drug leads. Journal of Natural Products, 77(3), 703–723. Dhankhar, S., Kumar, M., Ruhil, S., Balhara, M., & Chhillar, A. K. (2012). Analysis toward innovative herbal antibacterial & antifungal drugs. Recent Patents on Anti-Infective Drug Discovery, 7(3), 242–248. Eloff, J. N. (2004). Quantification the bioactivity of plant extracts during screening and bioassay guided fractionation. Phytomedicine, 11(4), 370–371. Facchini, P. J., & De Luca, V. (2008). Opium poppy and Madagascar periwinkle: Model non-model systems to investigate alkaloid biosynthesis in plants. The Plant Journal, 54(4), 763–784.
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Gardete, S., & Tomasz, A. (2014). Mechanisms of vancomycin resistance in Staphylococcus aureus. Journal of Clinical Investigation, 124(7), 2836–2840. Gul, H. I., Sahin, F., Gul, M., Ozturk, S., & Yerdelen, K. O. (2005). Evaluation of antimicrobial activities of several Mannich bases and their derivatives. Archiv der Pharmazie, 338(7), 335–338. Gyawali, R., & Ibrahim, S. A. (2014). Natural products as antimicrobial agents. Food Control, 46, 412–429. Hansen, A. M., Gu, Y., Li, M., Andrykovitch, M., Waugh, D. S., Jin, D. J., et al. (2005). Structural basis for the function of stringent starvation protein A as a transcription factor. Journal of Biological Chemistry, 280(17), 17380–17391. Jaberian, H., Piri, K., & Nazari, J. (2013). Phytochemical composition and in vitro antimicrobial and antioxidant activities of some medicinal plants. Food Chemistry, 136(1), 237–244. Kim, K. K., Song, H. K., Shin, D. H., Hwang, K. Y., & Sun, S. W. (1997). The crystal structure of a triacylglycerol lipase from Pseudomonas cepacia reveals a highly open conformation in the absence of a bound inhibitor. Structure, 5(2), 173–185. Kurochkina, L. P., Semenyuk, P. I., Orlov, V. N., Robben, J., Sykilinda, N. N., & Mesyanzhinov, V. V. (2012). Expression and functional characterization of the first bacteriophage-encoded chaperonin. Journal of Virology, 86(18), 10103–10111. Lafontaine, D. L. J., & Tollervey, D. (2001). The function and synthesis of ribosomes. Nature Reviews Molecular Cell Biology, 2(7), 514–520. Lorence, A., & Nessler, C. L. (2004). Camptothecin, over four decades of surprising findings. Phytochemistry, 65(20), 2735–2749. Machowski, E. E., Senzani, S., Ealand, C., & Kana, B. D. (2014). Comparative genomics for mycobacterial peptidoglycan remodelling enzymes reveals extensive genetic multiplicity. BMC Microbiology, 14(1). Newman, D. J., & Cragg, G. M. (2007). Natural products as sources of new drugs over the last 25 years. Journal of Natural Products, 70(3), 461–477. Ramos, H. C., Rumbo, M., & Sirard, J. C. (2004). Bacterial flagellins: Mediators of pathogenicity and host immune responses in mucosa. Trends in Microbiology, 12(11), 509–517. Ried, G., & Henning, U. (1987). A unique amino acid substitution in the outer membrane protein OmpA causes conjugation deficiency in Escherichia coli K-12. FEBS Letters, 223(2), 387–390. Saïdana, D., Mahjoub, M. A., Boussaada, O., Chriaa, J., Chéraif, I., Daami, M., et al. (2008). Chemical composition and antimicrobial activity of volatile compounds of Tamarix boveana (Tamaricaceae). Microbiological Research, 163(4), 445–455. Velmurugan, S., Viji, V. T., Babu, M. M., Punitha, M. J., & Citarasu, T. (2012). Antimicrobial effect of Calotropis procera active principles against aquatic microbial pathogens isolated from shrimp and fishes. Asian Pacific Journal of Tropical Biomedicine, 2(2 Suppl.), S812–S817. Wang, Y. C., Hsu, H. W., & Liao, W. L. (2008). Antibacterial activity of Melastoma candidum D. Don. LWT – Food Science and Technology, 41(10), 1793–1798. Wong, C. C., Li, H. B., Cheng, K. W., & Chen, F. (2006). A systematic survey of antioxidant activity of 30 Chinese medicinal plants using the ferric reducing antioxidant power assay. Food Chemistry, 97(4), 705–711. Wong, F. C., Yong, A. L., Ong, H. C., & Chaia, T. T. (2013). Evaluation of the antibacterial activities of selected medicinal plants and determination of their phenolic constituents. ScienceAsia, 39(6), 591–595. Wu, V. C. H., Qiu, X., Bushway, A., & Harper, L. (2008). Antibacterial effects of American cranberry (Vaccinium macrocarpon) concentrate on foodborne pathogens. LWT – Food Science and Technology, 41(10), 1834–1841.
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Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts Ann-Li Yong b, Keng-Fei Ooh b, Hean-Chooi Ong c, Tsun-Thai Chai a,b, Fai-Chu Wong a,b,⇑ a
Centre for Biodiversity Research, Universiti Tunku Abdul Rahman, 31900 Kampar, Malaysia Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, 31900 Kampar, Malaysia c Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia b
a r t i c l e
i n f o
Article history: Received 14 October 2014 Received in revised form 14 November 2014 Accepted 18 November 2014 Available online xxxx Keywords: Callicarpa formosana Escherichia coli Melastoma candidum Natural products Protein expression Scanning electron microscope Scutellaria barbata Staphylococcus aureus
a b s t r a c t In this paper, we investigated the antibacterial mechanism and potential therapeutic targets of three antibacterial medicinal plants. Upon treatment with the plant extracts, bacterial proteins were extracted and resolved using denaturing gel electrophoresis. Differentially-expressed bacterial proteins were excised from the gels and subjected to sequence analysis by MALDI TOF–TOF mass spectrometry. From our study, seven differentially expressed bacterial proteins (triacylglycerol lipase, N-acetylmuramoylL-alanine amidase, flagellin, outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin) were identified. Additionally, scanning electron microscope study indicated morphological damages induced on bacterial cell surfaces. To the best of our knowledge, this represents the first time these bacterial proteins are being reported, following treatments with the antibacterial plant extracts. Further studies in this direction could lead to the detailed understanding of their inhibition mechanism and discovery of target-specific antibacterial agents. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Medicinal plants are rich reservoirs of phenolic compounds, and different parts of the plants have been consumed traditionally for a variety of medicinal applications (Jaberian, Piri, & Nazari, 2013; Wong, Li, Cheng, & Chen, 2006). For the past few decades, the studies on medicinal plants have grown dramatically, and their usages have increased worldwide, partly encouraged by the great commercial potentials of plant-derived therapeutic agents (Cragg, Grothaus, & Newman, 2014). The potentials of plant-derived therapeutic agents are further encouraged by the fact that a significant number of currently available pharmaceutical drugs could be traced back to their origins in plants. Well-known examples include vinblastine (an alkaloid drug) derived originally from Catharanthus roseus (Madagascar periwinkle) and topotecan (a camptothecin analog) derived from Camptotheca acuminate (Asian happy tree) (Facchini & De Luca, 2008; Lorence & Nessler, 2004). In fact, it was estimated that nearly half of modern pharmaceutical drugs are derived from plants (Newman & Cragg, 2007). Additionally,
⇑ Corresponding author at: Centre for Biodiversity Research, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900 Kampar, Malaysia. Tel.: +605 468 8888x4521; fax: +605 4661676. E-mail address: [email protected] (F.-C. Wong).
increasing plant-based food supplements have been marketed to cater for the strong consumer demands. Example includes the cranberry supplement, which was reported to be effective in treating urinary tract infection (Caillet, Côté, Sylvain, & Lacroix, 2012; Wu, Qiu, Bushway, & Harper, 2008). With the increasing occurrences of multi-drug resistant bacteria strains, the world is currently facing an urgent need to find new antibiotic derivatives (Gardete & Tomasz, 2014). Accumulated evidences are pointing toward the effectiveness of plant-derived compounds in inhibiting multi-drug resistant bacterial strains such as methicillin-resistant Staphylococcus aureus (Gyawali & Ibrahim, 2014). Previously, our laboratory and others had reported the minimum inhibitory concentrations (MIC) of three antibacterial medicinal plants, namely Callicarpa formosana, Melastoma candidum, and Scutellaria barbata (Wang, Hsu, & Liao, 2008; Wong, Yong, Ong, & Chaia, 2013). However, the exact bacterial inhibition mechanism of these aforementioned plant extracts remained to be elucidated. For instance, it is still unclear which bacterial pathways or enzymes are being targeted by these antibacterial plant extracts. Additionally, no information is available regarding their effects on the structural integrity of bacterial cell membranes. In this paper, we evaluated the antibacterial mechanism of these aforementioned edible medicinal plants, using proteomic
http://dx.doi.org/10.1016/j.foodchem.2014.11.103 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yong, A.-L., et al. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.103
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analysis and scanning electron microscope (SEM). Upon treatments with antibacterial plant extracts, differentially-expressed bacterial proteins were first resolved using denaturing gel electrophoresis, followed by sequence determination with MALDI TOF–TOF mass spectrometry. Additionally, we investigated on how the antibacterial plant extracts affected the bacteria physically, through viewing of the bacterial membrane surfaces with SEM. Lastly, gas chromatography–mass spectrometry (GC–MS) was also applied to investigate the phytochemical contents of the medicinal plant extracts. Through the combination of these techniques, we aim to identify potential phytochemical-mediated antibacterial therapeutic targets, along with better understanding of their mechanisms. 2. Material and methods 2.1. Preparation of plant extracts Medicinal plants were collected around Perak state (Malaysia) from April to July of 2012. The plants species were authenticated by Professor Dr. Hean-Chooi Ong at the Institute of Biological Sciences, University of Malaya, Malaysia. These plants were dried in oven at 40 °C until constant weights were achieved. The dried plants were then pulverized using a warring blender, followed by extraction using methanol solvent. The supernatants were collected, filtered and then stored in 20 °C. 2.2. Isolation of bacterial proteins for SDS–PAGE analysis The bacteria strains (Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, S. aureus ATCC 6538) were cultured overnight in Luria–bertani (LB) broth using conical flasks in an orbital shaker at 37 °C and 200 rpm. Bacterial cultures were adjusted to 0.5–1 McFarland standard before used in subsequent experiments. The bacterial cultures were then treated with optimal concentrations (2–6 mg/ml) of the filtered plant extracts, and sterile water is used as the negative control. After the addition of plant extracts, the bacterial cultures were keep in an orbital shaker (37 °C, 200 rpm). At the designated time intervals, 20 ml of bacterial culture was collected and centrifuged at 9000 rpm for 10 min to separate the bacterial cell pellet from the supernatant. The pellet was then subjected to treatment with Bacterial Proteins Extraction Reagent (Thermo Scientific), and the supernatant was subjected to ammonium sulphate precipitation. The precipitated proteins were collected by centrifugation at 9000 rpm for 10 min. 2.3. SDS–PAGE gel analysis and identification of differentially expressed bacterial proteins SDS–PAGE gel electrophoresis was carried out using 12% resolving gel [1.5 M Tris–HCl pH 8.8, 10% SDS (Fisher Scientific), 40% bisacrylamide (Bio Basic Canada)], and 4% stacking gel [0.5 M Tris–HCl pH 6.9, 10% SDS, 40% bis-acrylamide]. The bacterial proteins were treated with dithiothreitol (DTT) and boiled, before loading into the wells. Gel electrophoresis was performed with constant electric current of 135 mV, until the bromophenol blue (BPB) reached the bottom of the gel plate. The protein gel was then stained with Coomassie Brilliant Blue R-250, and the molecular weights of the bacterial proteins were determined using commercial protein markers (Spectra Multicolor Broad Range Protein Ladder, Thermo Scientific). Differentially expressed protein bands were excised from the gel using sterile razor blades and analysed by MALDI TOF–TOF mass spectrometry (4800 Proteomics Analyzer, AB Sciex) (Proteomics International, Perth, Australia). The spectra were analysed using Mascot sequence matching software (Matrix Science) with Ludwig NR Database to identify the proteins of interest.
2.4. Investigation of morphological changes on bacteria membranes via scanning electron microscopy (SEM) The bacteria strains were cultured overnight in an orbital shaker set at 37 °C and 200 rpm. Next, bacterial cultures were adjusted to 0.5–1 McFarland standard and treated with antibacterial plant extracts (0.6–5.0 mg/ml) or sterile water (control) for 2 h. Treated and non-treated bacteria cultures were incubated with 2.5% glutaraldehyde overnight. Following three washes with PBS, the samples were hydrated with ascending concentrations of ethanol (25% for 5 min, 50% for 10 min, 75% for 10 min and three changes of 100% ethanol for 10 min each). The samples were then dried through freeze drying. Subsequently, the dried samples were adhered onto double-sided adhesive conductive carbon tape (which was mounted on a copper stage). The samples were then coated with platinum (JEOL JFC-1600 Auto Fine Coater) before viewing by SEM. 2.5. Gas chromatography–mass spectrometry (GC–MS) analysis of TCL spots Thin layer chromatography (TLC) was performed as previously described (Eloff, 2004). TLC spots were analysed by gas-chromatography equipped with mass spectrometry (GCMS-QP2010 Plus, Shidmadzu). Column (BPX-5, SGE Analytical Science) temperature was initially set at 110 °C and increased to 280 °C (increased at a rate of 5–10 °C/min). The flow rate of the carrier gas (helium) was set to 1 ml/min. The total running time of the gas chromatography was 36 min. The mass spectra were obtained from the range of m/e 40–700. The identities of the samples were determined by comparing the mass spectra with NIST Gas Chromatography Library Database. 3. Results and discussion 3.1. Identification of differentially expressed bacterial proteins Through proteomic study, our objective was to identify differentially expressed bacterial proteins, upon treatment with antibacterial plant extracts. In this approach, we focused on three bacterial strains (E. coli, P. aeruginosa, S. aureus) treated with three medicinal plant extracts (C. formosana, M. candidum, S. barbata). Bacterial cellular proteins were extracted using Bacterial Protein Extraction Reagents (Thermo Scientific) and ammonium sulphate precipitation. In our results, a total of seven differentially expressed bacterial proteins were identified (Fig. 1). Differentially expressed bacterial proteins were excised and subjected to sequence analysis by mass spectrometry. The identified proteins and their characteristics were summarised and reported in Table 1. Protein bands a, b (Fig. 1A) and c (Fig. 1B) were extracted from bacterial culture supernatants, using ammonium sulphate precipitation method. Based on MALDI TOF–TOF protein sequencing results, protein bands a to c were determined as triacylglycerol lipase, N-acetylmuramoyl-L-alanine amidase and flagellin, respectively. Protein bands d, e (Fig. 1C) and f, g (Fig. 1D) were extracted using Bacterial Protein Extraction Reagents and identified as outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin, respectively. Among the seven differentially-expressed proteins identified, two of them (bands f and g) are pertaining to the bacterial protein translational machinery. The 30S ribosomal protein S1 (band f) is an essential component critically involved in the translational pathway (Lafontaine & Tollervey, 2001). Interestingly, the 60 kDa chaperonin (band g) was previously reported with functional roles to stimulate proper folding of newly synthesized bacterial polypeptides, as well as to promote refolding of denatured proteins
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Fig. 1. (A) Protein expression profile of S. aureus following exposure to C. formosana extract. (B) Protein expression profile of E. coli following exposure to S. barbata extract. Protein expression profiles of (C) E. coli and (D) P. aeruginosa, following exposure to M. candidum extract. ( ) and (+) indicated the absence or presence of plant extract in bacterial culture medium, respectively. Labels (a–g) indicated differentially expressed protein bands selected for MALDI TOF–TOF sequence analysis.
Table 1 Summary of identified differentially-expressed bacterial proteins, using MALDI TOF–TOF MS. Protein bands (a–g) are labelled corresponding to those in Fig. 1. Protein band
Protein size (in aa)
No. of peptide sequence identified
No. of amino residue identified
Sequence covered (%)
Annotation
a b c d e f g
681 619 486 350 99 559 548
1 4 5 7 1 8 5
11 43 73 75 14 126 71
1.61 6.95 15.02 21.43 14.14 22.54 12.96
Triacylglycerol lipase N-acetylmuramoyl-L-alanine amidase Flagellin Outer membrane protein A Stringent starvation protein A 30S ribosomal protein S1 60 kDa chaperonin
under stress conditions (Kurochkina et al., 2012). Meanwhile, literature search also highlighted on the critical roles of triacylglycerol lipase (band a) and stringent starvation protein A (band e) in bacterial metabolic pathways. Triacylglycerol lipase is a bacterial enzyme involving in hydrolysis of triglycerides into fatty acids and glycerol (Kim, Song, Shin, Hwang, & Sun, 1997), while stringent starvation protein A was reported with ability to promote tolerance of E. coli in acidic environment, such as human digestive tracts (Hansen et al., 2005). On the other hand, proteins pertaining to the integrity of bacterial membranes were also identified. N-acetylmuramoyl-L-alanine amidase (band b) is a bacterial enzyme participating in the biosynthesis of peptidoglycans, an important building component in bacterial cell walls (Machowski, Senzani, Ealand, & Kana, 2014). Meanwhile, flagellin (band c) and outer membrane protein A (band d) are critical bacterial structural proteins. Flagellin is the major component in flagella filaments, which is responsible for bacterial mobility, invasion and contributing to pathogenicity (Ramos, Rumbo, & Sirard, 2004). In our results, the presence of flagellin was detected in the culture supernatants, upon treatment with the plant extract. Based on this observation, it is tempting to speculate that the antibacterial plant extract may somehow degrade and lead to the disintegration of the bacterial flagellin. Interestingly, inhibition of flagellin synthesis had previously been reported in E. coli, upon exposure to carvacrol (a monoterpenoid) (Burt et al., 2007). On the other hand, outer membrane protein A (OmpA) is critical for maintaining bacterial membrane integrity, through physical linkage with peptidoglycan layer. Previous study had also reported on the significance of OmpA in the bacterial conjugation process (Ried & Henning, 1987). Although the exact mechanism
remains to be determined, further works in this direction could potentially lead to the discovery of flagellin-specific and OmpAspecific antibacterial compounds. 3.2. Investigation of morphological changes on bacterial membranes via scanning electron microscopy (SEM) In order to have a better understanding of the antibacterial mechanism, plant extracts were further studied for their effects on the bacterial cell surfaces. Using SEM, we were able to observe the morphological changes in two bacteria strains (S. aureus and E. coli), upon treatment with antibacterial M. candidum extract (Fig. 2). In S. aureus, the untreated bacterial cells appeared grape-shaped, and the cells surfaces were intact with no damage observed (Fig. 2A). However, in the samples treated with M. candidum extract, the bacterial cells did not retain the grape-shaped characteristic (Fig. 2B). Moreover, uneven fragments were observed, indicating the damages induced on bacterial cell membranes. In E. coli, the untreated bacterial cells appeared to be in perfect rod-shaped, with no damage observed on the cell surfaces (Fig. 2C). Yet, in bacterial cells treated with M. candidum extract, we observed the formation of irregular fragments, and the cells did not retain much of its rod-shaped (Fig. 2D). Moreover, the treated bacterial cell surfaces were uneven, and the cells appeared damaged. Through the use of SEM, we observed that the bacterial membrane surfaces were damaged, upon treatment with M. candidum extract. Interestingly, it was previously reported by others that both bacterial membranes and cytoplasm were easily affected by passive diffusion of phenolic acids, with subsequent disruption of
Please cite this article in press as: Yong, A.-L., et al. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.103
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Fig. 2. Scanning electron microscope recordings of bacterial cell surfaces: (A) S. aureus (control) and (B) S. aureus (treated with M. candidum extract); (C) E. coli (control) and (D) E. coli (treated with M. candidum extract).
the M. candidum extract. It is interesting and potentially rewarding to persuade further the exact mechanism, as well as the bioactive compounds involved, in this M. candidum-induced bacterial membrane disruption. 3.3. GC–MS analysis
Fig. 3. GC analysis of the methanol fraction of C. formosana extract. Major peaks (compounds 1–6) were selected for further analysis with MS.
the bacterial membrane integrity (Campos et al., 2009). As observed in Fig. 2, compared to the untreated samples, bacterial cell membranes were significantly affected, following exposure to
In order to have a better understanding of the phytochemical contents, we also analysed the plant extract using GC–MS. From the GC spectrum with C. formosana methanolic extract, six major peaks were identified (Fig. 3). Peaks 1 to 6 were selected and subjected to MS analysis. The identities and characteristics of these peaks were determined and summarised (Table 2). Literature search indicted that Compound 2 (phenol, dimethyl) (Velmurugan, Viji, Babu, Punitha, & Citarasu, 2012), Compound 5 (hexadecanoic acid, methyl ester) (Dhankhar, Kumar, Ruhil, Balhara, & Chhillar, 2012), and a synthetic derivative of Compound 3 (1-propanone) (Chitra et al., 2011; Gul, Sahin, Gul, Ozturk, & Yerdelen, 2005) have previously been reported by other groups with bacterial inhibition activities. Additionally, the presence of Compound 4 (aminononadecane) was reported in Tamarix boveana, a perennial shrub with bacterial inhibition activities (Saïdana et al.,
Table 2 Major chemical compounds identified from the active fraction of C. formosama methanol extract by GC–MS. Peak number
Retention time (min)
Name of the compounds
Molecular formula
Molecular weight
Quality (%)
1 2 3 4 5 6
9.57 12.42 15.72 18.50 21.30 21.53
2-Heptanamine, 5-methyl Phenol, 2,4-bis(1,1-dimethylethyl) 1-Propanone, 2-amino-1-phenyl Aminononadecane Hexadecanoic acid, 15-methyl, methyl ester Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy, methyl ester
C8H19N C14H22O C9H11NO C19H41N C18H36O2 C18H28O3
129 206 149 283 284 292
81 91 80 83 86 79
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2008). It is possible that some of the identified compounds, either alone or work in a synergistic manner, contributed toward the observed bacterial inhibition activities. However, we could not rule out the possible functional roles of some other unidentified minor phytochemicals. In conclusion, seven differentially-expressed bacterial protein targets were identified in this study. These seven proteins could further be classified into bacterial pathways pertaining with protein translation, membrane integrity and metabolism. Moreover, our SEM study also demonstrated the damages which were induced onto the bacterial membrane surfaces, upon treatments with the antibacterial plant extract. Further works in this direction could potentially lead to the discovery of pathway-specific antibacterial agents, targeting bacterial protein translational machinery and cellular membranes. Acknowledgements We would like to extend our gratitude to funding supports from UTAR Research Fund (UTARRF) and Malaysia Toray Science Foundation (MTSF). References Burt, S. A., Van Der Zee, R., Koets, A. P., De Graaff, A. M., Van Knapen, F., Gaastra, W., et al. (2007). Carvacrol induces heat shock protein 60 and inhibits synthesis of flagellin in Escherichia coli O157:H7. Applied and Environmental Microbiology, 73(14), 4484–4490. Caillet, S., Côté, J., Sylvain, J. F., & Lacroix, M. (2012). Antimicrobial effects of fractions from cranberry products on the growth of seven pathogenic bacteria. Food Control, 23(2), 419–428. Campos, F. M., Couto, J. A., Figueiredo, A. R., Tóth, I. V., Rangel, A. O. S. S., & Hogg, T. A. (2009). Cell membrane damage induced by phenolic acids on wine lactic acid bacteria. International Journal of Food Microbiology, 135(2), 144–151. Chitra, S., Paul, N., Muthusubramanian, S., Manisankar, P., Yogeeswari, P., & Sriram, D. (2011). A facile synthesis of carbocycle-fused mono and bis-1,2,3selenadiazoles and their antimicrobial and antimycobacterial studies. European Journal of Medicinal Chemistry, 46(11), 5465–5472. Cragg, G. M., Grothaus, P. G., & Newman, D. J. (2014). New horizons for old drugs and drug leads. Journal of Natural Products, 77(3), 703–723. Dhankhar, S., Kumar, M., Ruhil, S., Balhara, M., & Chhillar, A. K. (2012). Analysis toward innovative herbal antibacterial & antifungal drugs. Recent Patents on Anti-Infective Drug Discovery, 7(3), 242–248. Eloff, J. N. (2004). Quantification the bioactivity of plant extracts during screening and bioassay guided fractionation. Phytomedicine, 11(4), 370–371. Facchini, P. J., & De Luca, V. (2008). Opium poppy and Madagascar periwinkle: Model non-model systems to investigate alkaloid biosynthesis in plants. The Plant Journal, 54(4), 763–784.
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