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Proteomic response of Escherichia coli to the alkaloid extract of Papaver polychaetum

Proteomic response of Escherichia coli to the alkaloid extract of Papaver polychaetum Ann Microbiol (2010) 60:709–717 DOI 10.1007/s13213-010-0118-0 ORIGINAL ARTICLE Proteomic response of Escherichia coli to the alkaloid extract of Papaver polychaetum Çağakan Ozbalci & Çağlayan Unsal & Dilek Kazan & Berna Sariyar-Akbulut Received: 2 March 2010 /Accepted: 30 July 2010 /Published online: 31 August 2010 Springer-Verlag and the University of Milan 2010 Abstract The cellular response of Escherichia coli exposed synthase, pyridine nucleotide transhydrogenase STHA) and to alkaloids extracted from a biennial endemic plant, regulation. These results provide clues for understanding the Papaver polychaetum, was explored using proteome analy- mechanism of the alkaloid extract-induced stress and cytotox- sis. Following determination of the minimum inhibitory icity on E. coli. The altered proteins can serve as potential concentration of the berberine-containing plant extract as targets for development of innovative therapeutic agents. 1,250 μg/mL, E. coli cells were grown in the presence of . . . 750 μg/mL extract. The response of the bacteria to the Keywords Proteomics Antimicrobial Escherichia coli extract, with berberine found as the major alkaloid, was Papaver polychaetum Berberine analyzed on two-dimensional gels. The differentially expressed proteins in the presence of 750 μg/mL extract were identified by matrix-assisted laser desorption/ionization-time Introduction of flight mass spectrometry. These proteins included those that play vital roles for maintenance such as protein synthesis Widespread bacterial drug resistance to current cheap and (elongation factor-Ts), transport (oligopeptide-binding protein effective first-choice drugs raises the number of untreat- A, uncharacterized amino-acid ABC transporter ATP binding able bacterial infections and adds urgency to the search protein YECC), energy metabolism (alpha-subunit of ATP for new infection-fighting strategies (World Health Orga- nization 2002; Vlieghe et al. 2009). In addition to careful use of existing antimicrobials and better hygiene condi- tions, development of novel therapeuticals is the key to Electronic supplementary material The online version of this article fighting the remarkable adaptability of microorganisms. (doi:10.1007/s13213-010-0118-0) contains supplementary material, which is available to authorized users. The challenge in the search for new antimicrobial classes : : lies in the timely knowledge of the molecular mechanism of Ç. Ozbalci D. Kazan B. Sariyar-Akbulut (*) Department of Bioengineering, Faculty of Engineering, action of the drug and the related bacterial response Marmara University, (Bandow et al. 2003). Göztepe Campus, Kadiköy, Use of plants for medicinal purposes has been practised 34722 Istanbul, Turkey for many centuries (Summer 2000; Hanson 2005; van Wyk e-mail: berna.akbulut@marmara.edu.tr and Wink 2005). Due to their unmatched availability of Ç. Unsal chemical diversity, plant extracts, either as pure compounds Department of Pharmacognosy, Faculty of Pharmacy, or as standardized extracts, provide unlimited opportunities Istanbul University, for new drugs to counter multi-resistant microorganisms 34116 Beyazıt, Istanbul, Turkey (Cos et al. 2006). Papaver polychaetum, belonging to the D. Kazan plant kingdom Papaveraceae, is a biennial endemic species TUBITAK-MAM, (southern Turkey) possessing antimicrobial activity (Ünsal Research Institute for Genetic Engineering and Biotechnology, et al. 2007, 2009). The alkaloid found in P. polychaetum is PK: 21, berberine (Sariyar 2002) that is commonly used for various 41470 Gebze-Kocaeli, Turkey 710 Ann Microbiol (2010) 60:709–717 medicinal purposes and is effective against a broad range of was partitioned between CHCl -H O (1:1) to afford a CHCl - 3 2 3 bacteria, protozoa, fungi and viruses. Evidence from soluble fraction (Fr. A, 1,383 mg) and dried over anhydrous piecewise descriptions of individual interactions indicated Na SO (Chen et al. 1999). TLC examination of Fr. A in 2 4 that the effect of berberine is on nucleic acids since it different solvent systems revealed the presence of only one intercalates into DNA molecules (Burbaum and Tobal alkaloid. Next, 60 mg of Fr. A were separated on preparative 2002). However, if evaluated by the global analysis of TLC yielding 28 mg of alkaloid. The structure of this alkaloid changes in the protein expression profiles, the cellular was identified as berberine by comparing its physical and response to either pure berberine or berberine in an extract spectral data and TLC R values with an authentic sample. will be expected to be of different complexities. Proteins constitute the vast majority of drug targets against Stress treatment with the alkaloid extract and survival test which pharmaceutical drug design processes are initiated. In this respect, proteomics is gaining widespread use in drug Minimum inhibitory concentration (MIC) determination discovery and drug development programs allowing the dynamic study of interrelationships between proteins in Minimum inhibitory concentration (MIC) was defined as the microbial systems following drug treatment. This, in turn, lowest concentration of P. polychaetum alkaloid extract which contributes important insight for understanding the mechanistic completely inhibited E. coli growth at 37°C after incubation basis for drug action (Yoshida et al. 2001). There are a couple for 18–24 h (Amsterdam 1996). The broth dilution method of reports concerning the antimicrobial effects of berberine, (microdilution) was used to determine MIC. Serial two-fold but we are unaware of any published studies on proteomic dilutions of the plant extract and berberine dissolved in analysis of bacteria exposed to berberine or berberine DMSO were prepared in sterile 96-well U-bottomed, micro- containing plant extracts. titer plates; 50 μL of the cell culture (10 /mL) was also In this study, Escherichia coli served as a model to deposited in each well. MIC was evaluated with reference to investigate the physiological changes caused by P. polychaetum control cells, mixed with DMSO or fresh growth media. alkaloid extract. E. coli is the most widely studied bacterium in the world, and has an extreme importance as a model organism Cell culture conditions in many research fields due to its rapid growth rate and simple nutritional requirements (Ingledew and Poole 1984). In Escherichia coli cells (50 μL), grown to 0.7 at OD 600 nm additiontothese,incontrasttomanyother organisms, most were used to inoculate 50 mL of fresh media in 250-mL of its gene products have been functionally assigned making it erlenmeyer flasks. Cells were incubated at 37°C and 180 rpm. ideal for proteomic studies (Nandi et al. 2004;Herrmannand Final concentration of the extract or berberine in growth Ruppert 2006). The method used in this work will facilitate the media was 750 μg/mL. In addition to the control group, identification of target proteins involved in key biological culture with DMSO supplement was maintained to evaluate processes in E. coli that may serve as potential drug targets. the differences caused by the solvent. To keep growth volume constant, water was added to the control culture. Cell growth was monitored spectrophotometrically at OD 600 nm. Materials and methods Viable cell count was determined by plating samples of liquid cultures. Bacterial strain and growth conditions Sample preparation: protein extraction Escherichia coli ATCC 29425 was routinely cultured and maintained in Luria-Bertani (LB) broth (per liter: 10 g Following 18 h of incubation, cells were collected by tryptone, 5 g yeast extract, 10 g NaCl) or on LB agar plates centrifugation at 4°C and 6,000 rpm for 20 min. Super- (Cho et al. 2007) at 37°C for 18 h. natants were discarded and the cells were washed twice with 50 mM Tris buffer (pH 7.8). ProteoPrep® Sample Plant material, extraction and analysis of the alkaloids Extraction Kit (SIGMA PROT-TOT) was used for the extraction of cellular proteins using the protocol provided Papaver polychaetum was collected from İçel-Arslanköy by the manufacturer. Based on cell yield, 150–250 mg of (southern part of Turkey) in August 2005 (altitude 2,100 m). cell pellets were resuspended in 1.5 ml of extraction buffer Voucher specimens were identified by N. Sadıkoğlu and are and incubated for 15 min at room temperature. Liquid deposited in the Herbarium of the Faculty of Pharmacy, nitrogen was used for multiple cycles of freeze–thaw for cell Istanbul University (ISTE 83225). Aerial parts of P. poly- lysis without oxidation. Suspensions containing whole cell chaetum (300 g) were extracted with MeOH and the extract proteins were centrifuged at 15,000 g for 10 min at 15°C. was concentrated under reduced pressure. The MeOH extract Supernatants containing the cellular proteins were decanted Ann Microbiol (2010) 60:709–717 711 into clean tubes and tributylphospine was added to a final modifications of the protocol described by Hooven and concentration of 5 mM as a reducing agent. The mixture Baird (2008). Peptides formed by tryptic cleavage reactions was incubated 1 h at room temperature. Proteins were were mixed with 1 μl saturated alpha-cyano-4 hydroxycin- alkylated with 15 mM iodoacetamide for 1.5 h at room namic acid matrix solution (0.5 mg/mL alpha-cyano-4 temperature. Protein samples were centrifuged at 20,000 g hydroxycinnamic acid diluted with 0.1% trifluoroacetic for 5 min at room temperature to prevent insoluble material acid in 49.5% ethanol and 49.5% acetonitrile), and 1 μLof contamination and the supernatants were aliquoted and the resulting peptide–matrix mixture was spotted onto the stored at −80°C for further applications. MALDI target plate. Alcohol dehydrogenase was used as Protein concentration was estimated by the Bradford the calibration standard and Glu-fibrinopeptide was used as protein assay (Bradford 1976). the standard. Mass spectrometric analysis of the peptides was performed on a MALDI-time-of-flight (TOF) micro LR Two-dimensional gel electrophoresis (Waters, Manchester, UK), equipped with a pulsed nitrogen laser (k=337 nm). The instrument operated in positive ion Non-equilibrium pH gradient electrophoresis (NEpHGE) reflectron mode with the source voltage set to 15,000 V. The technique (Klose and Kobalz 1985) has been used for the pulse voltage was optimized at 3,250 V, the detector and two-dimensional separation of bacterial proteins. Samples reflectron voltages were set to 1,850 and 500 V, respectively. were separated in the first dimension on capillary rods Measurements were performed in the mass range m/z 500– using polyacrylamide gels containing 9 M urea, 3.5% 3,000 with a suppression mass gate set to m/z 500 to prevent acrylamide, 0.3% piperazine diacrylamide and 4% ampho- detector saturation from matrix cluster peaks and an extraction lyte mixture (pH 2–11). Next, 300 and 60 μg protein- delay of 600 ns. All spectra were processed and analyzed containing samples were loaded onto the anodic side of the using the MassLynx 4.0 software (Waters, Milford, MA, tubular gels for colloidal coomasie brilliant blue (CBB)- USA). MASCOT search engine (Matrix Science, London, G250 and silver staining, respectively. Optimized running UK, http:www.matrixscience.com) was employed to assign voltages were 100 V, 60 min; 200 V, 60 min; 400 V, monoisotopic peptide masses. Peptide mass tolerance was set 990 min; 600 V, 60 min; and 1,000 V, 30 min. At the end of at 1 Da. Maximum number of missed cleavages was set to 1 each run, gels were incubated 10 min at room temperature with trypsin as the protease. Taxonomy to be searched was in 1% DTT solution. The second dimensional separation selected as E. coli to increase the specificity of the results. was performed using 12% acrylamide gels (Laemmli 1970) at 120 mA for 15 min and 150 mA for 135 min. Protein Statistical analysis spots were visualized either with silver or colloidal CBB G- Experiments were performed at least five times. All data 250 staining prior to mass spectrometric analysis. Stained gels were scanned and the ProgenesisSameSpots software were plotted as the mean ± standard deviations, and (free trial version) was used to detect and match spots. statistically significant differences were determined using the t test (MS-office Excel). The gels have been compared In-gel tryptic digestion of proteins in CBB-G250 stained using the tools in Progenensis software. Following scan- gels ning, the images have been automatically aligned and samespots have been detected with background subtraction, The entire gel slab was rinsed with HPLC grade water and normalization and matching. The lists of spots statistically the spots of interest were excised with a clean scalpel. For ordered by p value from the one-way ANOVA analysis destaining, these spots were transferred to clean tubes and were then viewed. By going through the spot rank table, top incubated for 30 min in 100 μL 1:1 (v/v) 100 mM ranked spots have been accepted for further study. ammonium bicarbonate/acetonitrile mixture with occasional vortexing. Gel pieces were shrunk by dehydration in acetonitrile. Dried gel pieces were then swollen with Results sufficient trypsin containing buffer (6 ng/μL trypsin in 50 mM ammonnium carbonate) for 30 min on ice. Gels Survival of E. coli under the alkaloid extract stress were further incubated at 37°C overnight to have sufficient peptide recovery (Shevchenko et al. 2007). Initial efforts involved the minimization of the amount of toxic solvent DMSO. Consequently, concentration of the Mass spectroscopy and databank searching extract and berberine in the stock solution was fixed to 9.0 mg/mL. Matrix-assisted laser desorption/ionization mass spectrom- MIC of P. polychaetum alkaloid extract was found as etry (MALDI-MS) analysis was performed with slight 1,250 μg/mL for E. coli. For the same cells, the MIC value 712 Ann Microbiol (2010) 60:709–717 1,E+12 obtained with pure berberine was identical. This was not unexpected since the only alkaloid found in P. polychaetum was berberine. Taking this value as the upper limit, 1,E+09 concentration of P. polychaetum extract was adjusted to 750 μg/mL to investigate the alterations the extract 1,E+06 enforces on E. coli. The results were evaluated with reference to control cells, also following the changes caused by DMSO. 1,E+03 Figure 1 revealed that DMSO significantly retarded cell growth and reduced growth rate. Toxic effects of the 1,E+00 extract and berberine became apparent only after 2 h of 11 14 16 growth. Growth time (hr) Lag phase was clearly longer for the drug-treated cells. Nevertheless, all E. coli cultures grown in the presence of Fig. 2 Number of viable cells; control (vertical shading), DMSO DMSO (w/o drug) entered stationary phase at around the supplemented (dotted shading), and alkaloid extract (diagonal shading) supplemented 10th hour of growth. Growth period in the presence of the drugs was shorter. The OD 600 nm was as low as 1.0 for the drug-treated cells whereas it was 2.2 for the cells grown under the effect of DMSO only. underlying mechanisms of cell death upon exposure to P. polychaetum alkaloid extract. Differences were considered Further analysis was performed to correlate optical density measurements to cell viability. Number of colony as significant when they were due to drug treatment but not forming units in cultures after 11, 14 and 16 h of growth DMSO. Approximately 600 protein spots were detected on have been plotted in Fig. 2. the 2-DE gel with isoelectric focusing from pH 4.5 through During the course of growth between 11 and 16 h, the 8.0. Representative 2-D analytical gel images are shown in Fig. 3. viable cell number remained relatively constant versus the control and the DMSO supplemented cultures. In contrast, The images from P. polychaetum alkaloid extract and berberine-exposed cells were almost identical, showing that the presence of the extract caused a sharp reduction in cell viability after 14 h of growth. the effect of the antimicrobial alkaloid extract and berberine were very similar and that the trace amounts of organic Effect of plant alkaloid extract on global protein expression acids, phenolic compounds or pigments remained in the extract had no significant effect. Silver-stained gel images profiles of E. coli were analyzed to find a total of 28 differentially expressed Comparative proteomic analysis has been used to identify proteins due to drug treatment. target-related proteins which will lead to the elucidation of Spots that exhibited significant increase or decrease in abundance due to drug treatment, but not DMSO, have been indicated in magnified views of control and extract treated cultures on Fig. 4. Identification of differentially expressed proteins Peptide mass fingerprinting using MALDI-TOF was per- formed to identify the proteins that demonstrated altered expression in 2-DE. Peptide mass fingerprints (PMFs) were 0,1 obtained for the selected protein spots and all PMFs were Control searched with MASCOT software in Swissprot database for identification. The result has high confidence if the protein DMSO 0,01 was ranked at the best hit with a significant score and high plant ext. sequence coverage. berberine Finally of the 28 differentially expressed protein spots, 0,001 0 4 8 12 16 20 10 of them showed >2-fold increase and, out of these 10 Time (hrs) spots, for 9 of them significant matches were obtained from the protein database. Results of the selected proteins are Fig. 1 Growth profiles of E. coli in the presence of DMSO, berberine and, alkaloid extract with reference to control culture presented in Table 1. OD600nm Colony forming units Ann Microbiol (2010) 60:709–717 713 Fig. 3 Proteome maps of cultures in the presence of DMSO (b), 750 μg/mL plant extract (c), and 750 μg/mL berberine (d) with reference to control culture (a). The images are representative of five replicate gels Table 1 shows MALDI-TOF MS analysis of the proteins. that Papaver species also process significant antimicrobial The accession number, theoretical and predicted molecular activities (Ünsal et al. 2007, 2009). The antimicrobial weight and pI, sequence coverage and score of each protein activity of the alkaloid berberine from P. polychaetum has spot are given. been attributed to its specificity for the minor groove of AT- rich duplexes in DNA sequences (Saran et al. 1995; Choi et al. 2001; Sriwilaijareon et al. 2002; Mazzini et al. 2003; Discussion Chen et al. 2004; Qin et al. 2006). In addition to this information, Sun et al. (1988) reported that berberine For decades, plants of the genus Papaver have known to chloride blocked adhesion by reducing the synthesis of accumulate a rich spectrum of different alkaloids (Preininger fimbrial subunits and the expression of assembled fimbriae 1986; Sariyar 2002; Ziegler et al. 2006). These plants have (1988). This could be associated with the antimicrobial been regarded as important sources of narcotic alkaloids, property of berberine since adhesion to the tissue surface is such as morphine, codeine and thebaine (Ziegler et al. the first step for bacteria to establish infection. Unfortu- 2006; Salehi et al. 2007). More recent studies have shown nately, they observed that E. coli growth was reduced by 714 Ann Microbiol (2010) 60:709–717 Fig. 4 Magnified views of the A8 A8 a b proteome map of the plant A7 A7 extract treated culture (a,c) with A10 A9 A10 A9 reference to culture control (b,d) A15 A15 A19 A13 A19 A13 A16 A16 A18 A17 A18 A17 A12 A12 A14 A14 c d A1 A2 A1 A2 A23 A3 A3 A23 A22 A22 A25 A25 A26 A26 A27 A27 A21 A21 A11 A28 A29 A11 A28 A29 A4 A5 A6 A4 A5 A6 A31 A31 only 10% in the presence of 300 μg/mL berberine chloride, pumps. However, it could be as effective as other although 90% of its ability to adhere was lost (Wang et al. antimicrobial agents against E. coli when administered 2008). Stermitz et al. (2000) reported that berberine is together with an MDR inhibitor (Stermitz et al. 2000). pumped out by bacterial multi-drug resistance (MDR) Since information for this antimicrobial alkaloid was Table 1 Identification of differentially expressed protein spots in the presence of P. polychaetum alkaloid extract Spot no./protein description Accession Sequence Protein Theoretical molecular mass Experimental molecular mass number coverage (%) score (kDa)/pI (kDa)/pI Transport and binding Amino acid/peptide 1 OPPA_ECOLI ↓ P23843 22 47 60.9/6.05 66/6.3 10 YECC_ECOLI ↑ P37774 35 43 27.7/8.89 25/6.6 Sugar 6 MALE_ECOLI ↓ P02928 22 66 43.4/5.53 40/5.6 Membrane repair and maintenance 9 BLC_ECOLI ↑ P39281 24 31 19.8/8.81 22/6.6 Protein synthesis 4 EFTS_ECO24 ↓ P02997 28 54 30.5/5.22 34/5.3 Energy metabolism 3 ATPA_ECOLI ↓ P00822 30 103 55.2/5.80 55/5.3 2 STHA_ECO24 ↓ P27306 21 41 51.6/6.09 66/6.4 Regulation DNA synthesis 8 FRMR_ECO24 ↓ A7ZIA5 29 37 10.3/5.84 20/6.6 Replication 5 INTD_ECOLI ↓ P24218 16 30 45.1/9.76 36/5.6 Ann Microbiol (2010) 60:709–717 715 limited, an exhaustive study with the alkaloid extract of P. Upon exposure to P. polychaetum alkaloid extract, polychaetum was conducted for the comparative analysis of proteins from energy metabolism, soluble pyridine nucleo- cells exhibiting diverse phenotypes under drug stress. tide transhydrogenase STHA and alpha-subunit of ATP synthase ATPA, were down-regulated. STHA is proposed Survival of E. coli exposed to P. polychaetum alkaloid to be localized in the cytoplasmic space and involved in the extract conversion of NADPH to NADH. The down-regulation of STHA may be an indication of the repression in the Cell growth in the presence of the extract indicated that the respiratory activities of the cells under stress. The alpha extract or berberine can be considered to be an antimicro- subunit of ATP synthase is a peripheral membrane protein bial agent against E. coli only when available at higher found integrated to the membrane. The ATP synthase complex concentrations than the agents generally regarded as uses the proton gradient across the membrane to drive ATP antimicrobials. MIC is usually reported to be in the order synthesis from ADP and inorganic phosphate. Under fermen- of <50 μg/mL range for effective antimicrobials. tative conditions, it energizes the inner membrane by Due to the operation of MDR pumps, this value is as catalyzing the extrusion of protons at the expense of ATP high as 1,250 μg/mL for the extract. The high MIC value hydrolysis (Futai and Kanazawa 1983). The alpha-subunit has has not hampered the progress of this study since the motive an essential role in the catalytic mechanism of the complex. was to identify the modifications in the levels of proteins to P. polychaetum extract represses AtpA expression which in correlate varied protein abundances with the drug action turn results in altered levels of free energy transduction. mechanism. This information would be valuable for the Energy limitation caused by the repression of these two development of new therapeuticals. enzymes may be coupled to the disappeance of MalE. During energy crisis, carbon import via periplasmic binding proteins is Analysis of 2-DE protein profiles essential for E. coli cells (Wang and Crowley 2005). Easton et al. (2006) suggested that use of low energy-requiring trans- The expression of the seven proteins identified decreased ports for carbon uptake may in turn result in the rapid markedly or they were not expressed at all and the expression consumption of such transporters (Ayudhya et al. 2009). of two proteins was induced under the influence of the plant Hence, energy limitation in the presence of the extract could extract. These proteins were involved in transport and eventually cause consumption of MalE. binding, membrane repair and maintenance, proteins synthe- Papaver polycaetum alkaloid extract affected the protein sis, energy metabolism, regulation and replication. biosynthesis machinery by repressing the cytoplasmic elon- Among the transport and binding proteins, a component gation factor Ts, EFTS. Elongation factors interact with ribosomes and catalyze formation of the acyl bond between of the oligopeptide permease periplasmic oligopeptide- binding protein OPPA was down-regulated. Besides its the incoming amino acid residue and the peptide chain to function as a carrier for peptides up to five amino acids extend the nascent polypeptide chain during the elongation long, there is evidence that OPPA may act as a carrier for stage of bacterial translation (Alberts et al. 2002; Jayasekera et aminoglycoside antibiotics (Acosta et al. 2000). Complete al. 2004). Because of its essential functions, EFTs has been disappearance of OPPA under P. polychaetum alkaloid regarded as a possible drug target (Jayasekera et al. 2004). extract stress may indicate that OPPA could act as a carrier The stress of the plant extract induced the expression of for berberine as its structure has a resemblance to amino the outer membrane lipoprotein, lipocalin (Blc). Normally, acids and aminoglycosides. In contrast to OPPA, the expression of the blc gene starts at the beginning of expression of the uncharacterized amino-acid ABC trans- stationary phase and serves as a starvation response porter ATP-binding protein YECC was induced. This function in E. coli. Structural analyses of the purified Blc protein is located in the cell inner membrane and belongs protein suggest a possible role in membrane repair or to the ABC transporter superfamily. It is probably part of a maintenance requiring lipid storage or transport (Valerie et binding protein-dependent transport system yecCS for an al. 2004) and in phospholipid binding (Bishop 2000). Since amino acid, responsible for energy coupling to the transport berberine reduces the expression of fimbrial subunits in E. system (Blattner et al. 1997). The repression of the coli, as reported by Sun et al. (1988), the induction of Blc periplasmic oligopeptide permease may be compensated may be a predictable consequence based on its membrane by induction of this system for amino acid uptake. repairing role. Some experiments suggest that bacterial Another protein belonging to the transport and binding lipocalins may play a significant role in resistance to family, maltose-binding protein MalE, was down-regulated. antibiotics (Bishop 2000). Therefore, induction of Blc can This is located in the periplasm and is involved in the high- also be correlated with the strong resistance to the extract. affinity maltose membrane transport system malEFGK Exposure to the plant extract repressed the expression of (Duplay et al. 1984). two other proteins, prophage dlp12 integrase (INTD), 716 Ann Microbiol (2010) 60:709–717 Amsterdam D (1996) Susceptibility testing of antimicrobials in liquid involved in replication, and transcription repressor frmR media. In: Loman V (ed) Antibiotics in laboratory medicine. (FRMR), involved in regulation of DNA synthesis. The Williams and Wilkins, Baltimore, pp 52–111 integrase INTD is essential for integration of the phage into the Bandow JE, Brötz H, Leichert LIO, Labischinski H, Hecker M (2003) host genome by site -specific recombination. In conjunction Proteomic approach to understanding antibiotic action. Antimicrob Agents Chemother 47:948–955 with excisionase, integrase is also a necessary element for Bishop RE (2000) The bacterial lipocalins. Biochim Biophys Acta excision of the prophage from the host genome (Lindsey et al. 1482:73–83 1989). Transcriptional repressors are proteins that bind to rd Blattner FR, Plunkett G 3 , Bloch CA, Perna NT, Burland V, Riley specific sites on DNA to cause looping and prevent M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, transcription of nearby genes. Looping alters the topology Shao Y (1997) The complete genome sequence of Escherichia of the DNA and may thereby prevent formation of the closed coli K-12. Science 277:1453–1474 or open RNA polymerase complex, activator binding or Bradford MM (1976) A rapid and sensitive method for the elongation (Alberts et al. 2002). The down-regulation of this quantitation of microgram of protein utilizing the principle of protein-dye binding. Anal Biochem 12:248–254 protein may imply that the sites for the transcription repressor Burbaum J, Tobal GM (2002) Proteomics in drug discovery. Curr were already occupied by berberine, due to its affinity for Opin Chem Biol 6:427–433 DNA, and transcription was blocked in the absence of frmR. Chen JJ, Duh CY, Chen IS (1999) New tetrahydroprotoberberine N- oxide alkaloids & cytotoxic constituents of corydalis tashiroi. Planta Med 65:643–646 Chen WH, Chan CL, Cai Z, Luo GL, Jiang Z-H (2004) Study on Conclusion noncovalent complexes of cytotoxic protoberberine alkaloids with double-stranded DNA by using electrospray ionization mass The proteome profiling technique provided an effective spectrometry. Bioorg Med Chem Lett 14:4955–4959 Cho YS, Schiller NL, Kahng HY, Oh KH (2007) Cellular responses approach to identify global changes in protein profiles and proteomic analysis of Escherichia coli exposed to green tea under the influence of P. polychaetum alkaloid extract. To polyphenols. Curr Microbiol 55:501–506 our knowledge, this is the first report on proteomic analysis Choi DS, Kim SJ, Jung MY (2001) Inhibitory activity of berberine on of bacteria exposed to an alkaloid extract as an antimicro- DNA strand cleavage induced by hydrogen peroxide and cytochrome c. Biosci Biotechnol Biochem 65:452–455 bial agent derived from P. polychaetum. In summary, the Cos P, Vlietinck AJ, Berghe DV, Maes L (2006) Anti-infective plant extract changed the levels of proteins which play vital potential of natural products: how to develop a stronger in vitro roles for maintenance such as protein synthesis, antimicro- ‘proof-of- concept’. J Ethnopharmacol 106:290–302 bial resistance, amino acid uptakes, and ATP synthesis. The Duplay P, Bedouelle H, Fowler A, Zabin I, Saurin W, Hofnung M (1984) Sequences of the malE gene and of its product, the altered proteins identified by this approach can further be maltose-binding protein of Escherichia coli K12. J Biol Chem characterized as potential drug targets. The experimental 259:10606–10613 findings of this study shed light on the mechanism of P. Easton JA, Thompson P, Crowder MW (2006) Time dependent polychaetum extract and berberine from a molecular perspec- translational response of E. coli to excess Zn(II). J Biomol Tech 17:303–307 tive. Further studies should lead to a better understanding of Futai M, Kanazawa H (1983) Structure and function of proton- the antimicrobial mode of action of the plant extract, translocating adenosine triphosphatase (F0F1): biochemical and specifically berberine, and will contribute to the development molecular biological approaches. Microbiol Rev 47:285–312 of novel plant-based therapeutic drugs. A still open question Hanson BA (2005) Understanding medicinal plants: their chemistry and therapeutic action. Haworth, Binghamton is how beberine enters the bacterial cells to kill them. In this Herrmann R, Ruppert T (2006) Proteome of Mycoplasma pneumoniae. respect, a significant finding could be obtained by analyzing Methods Biochem Anal 49:39–56 the interaction between berberine and oligo-peptide permease. Hooven L, Baird W (2008) Proteomic analysis of MCF-7 cells treated with benzo[a]pyrene, dibenzo[a]pyrene, coal tar extract, and diesel exhaust extract. J Toxicol 249:1–10 Acknowledgements This work was supported by Marmara University, Ingledew WJ, Poole RK (1984) The respiratory chains of Escherichia Research Fund Projects FEN-C-YLP-060308-0041 and FEN-D-040609- coli. Microbiol Rev 48:222–271 0178 and TUBITAK-MAG Project 108 M597. Isarankura-Na-Ayudhya P, Isarankura-Na-Ayudhya C, Treeranapaniboon L, Kasikun K, Thipheaw K, Prachayasittikul V (2009) Proteomics profiling of Escherichia coli in response to heavy metals stress. 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Proteomic response of Escherichia coli to the alkaloid extract of Papaver polychaetum

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Springer Journals
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Copyright © 2010 by Springer-Verlag and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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10.1007/s13213-010-0118-0
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Ann Microbiol (2010) 60:709–717 DOI 10.1007/s13213-010-0118-0 ORIGINAL ARTICLE Proteomic response of Escherichia coli to the alkaloid extract of Papaver polychaetum Çağakan Ozbalci & Çağlayan Unsal & Dilek Kazan & Berna Sariyar-Akbulut Received: 2 March 2010 /Accepted: 30 July 2010 /Published online: 31 August 2010 Springer-Verlag and the University of Milan 2010 Abstract The cellular response of Escherichia coli exposed synthase, pyridine nucleotide transhydrogenase STHA) and to alkaloids extracted from a biennial endemic plant, regulation. These results provide clues for understanding the Papaver polychaetum, was explored using proteome analy- mechanism of the alkaloid extract-induced stress and cytotox- sis. Following determination of the minimum inhibitory icity on E. coli. The altered proteins can serve as potential concentration of the berberine-containing plant extract as targets for development of innovative therapeutic agents. 1,250 μg/mL, E. coli cells were grown in the presence of . . . 750 μg/mL extract. The response of the bacteria to the Keywords Proteomics Antimicrobial Escherichia coli extract, with berberine found as the major alkaloid, was Papaver polychaetum Berberine analyzed on two-dimensional gels. The differentially expressed proteins in the presence of 750 μg/mL extract were identified by matrix-assisted laser desorption/ionization-time Introduction of flight mass spectrometry. These proteins included those that play vital roles for maintenance such as protein synthesis Widespread bacterial drug resistance to current cheap and (elongation factor-Ts), transport (oligopeptide-binding protein effective first-choice drugs raises the number of untreat- A, uncharacterized amino-acid ABC transporter ATP binding able bacterial infections and adds urgency to the search protein YECC), energy metabolism (alpha-subunit of ATP for new infection-fighting strategies (World Health Orga- nization 2002; Vlieghe et al. 2009). In addition to careful use of existing antimicrobials and better hygiene condi- tions, development of novel therapeuticals is the key to Electronic supplementary material The online version of this article fighting the remarkable adaptability of microorganisms. (doi:10.1007/s13213-010-0118-0) contains supplementary material, which is available to authorized users. The challenge in the search for new antimicrobial classes : : lies in the timely knowledge of the molecular mechanism of Ç. Ozbalci D. Kazan B. Sariyar-Akbulut (*) Department of Bioengineering, Faculty of Engineering, action of the drug and the related bacterial response Marmara University, (Bandow et al. 2003). Göztepe Campus, Kadiköy, Use of plants for medicinal purposes has been practised 34722 Istanbul, Turkey for many centuries (Summer 2000; Hanson 2005; van Wyk e-mail: berna.akbulut@marmara.edu.tr and Wink 2005). Due to their unmatched availability of Ç. Unsal chemical diversity, plant extracts, either as pure compounds Department of Pharmacognosy, Faculty of Pharmacy, or as standardized extracts, provide unlimited opportunities Istanbul University, for new drugs to counter multi-resistant microorganisms 34116 Beyazıt, Istanbul, Turkey (Cos et al. 2006). Papaver polychaetum, belonging to the D. Kazan plant kingdom Papaveraceae, is a biennial endemic species TUBITAK-MAM, (southern Turkey) possessing antimicrobial activity (Ünsal Research Institute for Genetic Engineering and Biotechnology, et al. 2007, 2009). The alkaloid found in P. polychaetum is PK: 21, berberine (Sariyar 2002) that is commonly used for various 41470 Gebze-Kocaeli, Turkey 710 Ann Microbiol (2010) 60:709–717 medicinal purposes and is effective against a broad range of was partitioned between CHCl -H O (1:1) to afford a CHCl - 3 2 3 bacteria, protozoa, fungi and viruses. Evidence from soluble fraction (Fr. A, 1,383 mg) and dried over anhydrous piecewise descriptions of individual interactions indicated Na SO (Chen et al. 1999). TLC examination of Fr. A in 2 4 that the effect of berberine is on nucleic acids since it different solvent systems revealed the presence of only one intercalates into DNA molecules (Burbaum and Tobal alkaloid. Next, 60 mg of Fr. A were separated on preparative 2002). However, if evaluated by the global analysis of TLC yielding 28 mg of alkaloid. The structure of this alkaloid changes in the protein expression profiles, the cellular was identified as berberine by comparing its physical and response to either pure berberine or berberine in an extract spectral data and TLC R values with an authentic sample. will be expected to be of different complexities. Proteins constitute the vast majority of drug targets against Stress treatment with the alkaloid extract and survival test which pharmaceutical drug design processes are initiated. In this respect, proteomics is gaining widespread use in drug Minimum inhibitory concentration (MIC) determination discovery and drug development programs allowing the dynamic study of interrelationships between proteins in Minimum inhibitory concentration (MIC) was defined as the microbial systems following drug treatment. This, in turn, lowest concentration of P. polychaetum alkaloid extract which contributes important insight for understanding the mechanistic completely inhibited E. coli growth at 37°C after incubation basis for drug action (Yoshida et al. 2001). There are a couple for 18–24 h (Amsterdam 1996). The broth dilution method of reports concerning the antimicrobial effects of berberine, (microdilution) was used to determine MIC. Serial two-fold but we are unaware of any published studies on proteomic dilutions of the plant extract and berberine dissolved in analysis of bacteria exposed to berberine or berberine DMSO were prepared in sterile 96-well U-bottomed, micro- containing plant extracts. titer plates; 50 μL of the cell culture (10 /mL) was also In this study, Escherichia coli served as a model to deposited in each well. MIC was evaluated with reference to investigate the physiological changes caused by P. polychaetum control cells, mixed with DMSO or fresh growth media. alkaloid extract. E. coli is the most widely studied bacterium in the world, and has an extreme importance as a model organism Cell culture conditions in many research fields due to its rapid growth rate and simple nutritional requirements (Ingledew and Poole 1984). In Escherichia coli cells (50 μL), grown to 0.7 at OD 600 nm additiontothese,incontrasttomanyother organisms, most were used to inoculate 50 mL of fresh media in 250-mL of its gene products have been functionally assigned making it erlenmeyer flasks. Cells were incubated at 37°C and 180 rpm. ideal for proteomic studies (Nandi et al. 2004;Herrmannand Final concentration of the extract or berberine in growth Ruppert 2006). The method used in this work will facilitate the media was 750 μg/mL. In addition to the control group, identification of target proteins involved in key biological culture with DMSO supplement was maintained to evaluate processes in E. coli that may serve as potential drug targets. the differences caused by the solvent. To keep growth volume constant, water was added to the control culture. Cell growth was monitored spectrophotometrically at OD 600 nm. Materials and methods Viable cell count was determined by plating samples of liquid cultures. Bacterial strain and growth conditions Sample preparation: protein extraction Escherichia coli ATCC 29425 was routinely cultured and maintained in Luria-Bertani (LB) broth (per liter: 10 g Following 18 h of incubation, cells were collected by tryptone, 5 g yeast extract, 10 g NaCl) or on LB agar plates centrifugation at 4°C and 6,000 rpm for 20 min. Super- (Cho et al. 2007) at 37°C for 18 h. natants were discarded and the cells were washed twice with 50 mM Tris buffer (pH 7.8). ProteoPrep® Sample Plant material, extraction and analysis of the alkaloids Extraction Kit (SIGMA PROT-TOT) was used for the extraction of cellular proteins using the protocol provided Papaver polychaetum was collected from İçel-Arslanköy by the manufacturer. Based on cell yield, 150–250 mg of (southern part of Turkey) in August 2005 (altitude 2,100 m). cell pellets were resuspended in 1.5 ml of extraction buffer Voucher specimens were identified by N. Sadıkoğlu and are and incubated for 15 min at room temperature. Liquid deposited in the Herbarium of the Faculty of Pharmacy, nitrogen was used for multiple cycles of freeze–thaw for cell Istanbul University (ISTE 83225). Aerial parts of P. poly- lysis without oxidation. Suspensions containing whole cell chaetum (300 g) were extracted with MeOH and the extract proteins were centrifuged at 15,000 g for 10 min at 15°C. was concentrated under reduced pressure. The MeOH extract Supernatants containing the cellular proteins were decanted Ann Microbiol (2010) 60:709–717 711 into clean tubes and tributylphospine was added to a final modifications of the protocol described by Hooven and concentration of 5 mM as a reducing agent. The mixture Baird (2008). Peptides formed by tryptic cleavage reactions was incubated 1 h at room temperature. Proteins were were mixed with 1 μl saturated alpha-cyano-4 hydroxycin- alkylated with 15 mM iodoacetamide for 1.5 h at room namic acid matrix solution (0.5 mg/mL alpha-cyano-4 temperature. Protein samples were centrifuged at 20,000 g hydroxycinnamic acid diluted with 0.1% trifluoroacetic for 5 min at room temperature to prevent insoluble material acid in 49.5% ethanol and 49.5% acetonitrile), and 1 μLof contamination and the supernatants were aliquoted and the resulting peptide–matrix mixture was spotted onto the stored at −80°C for further applications. MALDI target plate. Alcohol dehydrogenase was used as Protein concentration was estimated by the Bradford the calibration standard and Glu-fibrinopeptide was used as protein assay (Bradford 1976). the standard. Mass spectrometric analysis of the peptides was performed on a MALDI-time-of-flight (TOF) micro LR Two-dimensional gel electrophoresis (Waters, Manchester, UK), equipped with a pulsed nitrogen laser (k=337 nm). The instrument operated in positive ion Non-equilibrium pH gradient electrophoresis (NEpHGE) reflectron mode with the source voltage set to 15,000 V. The technique (Klose and Kobalz 1985) has been used for the pulse voltage was optimized at 3,250 V, the detector and two-dimensional separation of bacterial proteins. Samples reflectron voltages were set to 1,850 and 500 V, respectively. were separated in the first dimension on capillary rods Measurements were performed in the mass range m/z 500– using polyacrylamide gels containing 9 M urea, 3.5% 3,000 with a suppression mass gate set to m/z 500 to prevent acrylamide, 0.3% piperazine diacrylamide and 4% ampho- detector saturation from matrix cluster peaks and an extraction lyte mixture (pH 2–11). Next, 300 and 60 μg protein- delay of 600 ns. All spectra were processed and analyzed containing samples were loaded onto the anodic side of the using the MassLynx 4.0 software (Waters, Milford, MA, tubular gels for colloidal coomasie brilliant blue (CBB)- USA). MASCOT search engine (Matrix Science, London, G250 and silver staining, respectively. Optimized running UK, http:www.matrixscience.com) was employed to assign voltages were 100 V, 60 min; 200 V, 60 min; 400 V, monoisotopic peptide masses. Peptide mass tolerance was set 990 min; 600 V, 60 min; and 1,000 V, 30 min. At the end of at 1 Da. Maximum number of missed cleavages was set to 1 each run, gels were incubated 10 min at room temperature with trypsin as the protease. Taxonomy to be searched was in 1% DTT solution. The second dimensional separation selected as E. coli to increase the specificity of the results. was performed using 12% acrylamide gels (Laemmli 1970) at 120 mA for 15 min and 150 mA for 135 min. Protein Statistical analysis spots were visualized either with silver or colloidal CBB G- Experiments were performed at least five times. All data 250 staining prior to mass spectrometric analysis. Stained gels were scanned and the ProgenesisSameSpots software were plotted as the mean ± standard deviations, and (free trial version) was used to detect and match spots. statistically significant differences were determined using the t test (MS-office Excel). The gels have been compared In-gel tryptic digestion of proteins in CBB-G250 stained using the tools in Progenensis software. Following scan- gels ning, the images have been automatically aligned and samespots have been detected with background subtraction, The entire gel slab was rinsed with HPLC grade water and normalization and matching. The lists of spots statistically the spots of interest were excised with a clean scalpel. For ordered by p value from the one-way ANOVA analysis destaining, these spots were transferred to clean tubes and were then viewed. By going through the spot rank table, top incubated for 30 min in 100 μL 1:1 (v/v) 100 mM ranked spots have been accepted for further study. ammonium bicarbonate/acetonitrile mixture with occasional vortexing. Gel pieces were shrunk by dehydration in acetonitrile. Dried gel pieces were then swollen with Results sufficient trypsin containing buffer (6 ng/μL trypsin in 50 mM ammonnium carbonate) for 30 min on ice. Gels Survival of E. coli under the alkaloid extract stress were further incubated at 37°C overnight to have sufficient peptide recovery (Shevchenko et al. 2007). Initial efforts involved the minimization of the amount of toxic solvent DMSO. Consequently, concentration of the Mass spectroscopy and databank searching extract and berberine in the stock solution was fixed to 9.0 mg/mL. Matrix-assisted laser desorption/ionization mass spectrom- MIC of P. polychaetum alkaloid extract was found as etry (MALDI-MS) analysis was performed with slight 1,250 μg/mL for E. coli. For the same cells, the MIC value 712 Ann Microbiol (2010) 60:709–717 1,E+12 obtained with pure berberine was identical. This was not unexpected since the only alkaloid found in P. polychaetum was berberine. Taking this value as the upper limit, 1,E+09 concentration of P. polychaetum extract was adjusted to 750 μg/mL to investigate the alterations the extract 1,E+06 enforces on E. coli. The results were evaluated with reference to control cells, also following the changes caused by DMSO. 1,E+03 Figure 1 revealed that DMSO significantly retarded cell growth and reduced growth rate. Toxic effects of the 1,E+00 extract and berberine became apparent only after 2 h of 11 14 16 growth. Growth time (hr) Lag phase was clearly longer for the drug-treated cells. Nevertheless, all E. coli cultures grown in the presence of Fig. 2 Number of viable cells; control (vertical shading), DMSO DMSO (w/o drug) entered stationary phase at around the supplemented (dotted shading), and alkaloid extract (diagonal shading) supplemented 10th hour of growth. Growth period in the presence of the drugs was shorter. The OD 600 nm was as low as 1.0 for the drug-treated cells whereas it was 2.2 for the cells grown under the effect of DMSO only. underlying mechanisms of cell death upon exposure to P. polychaetum alkaloid extract. Differences were considered Further analysis was performed to correlate optical density measurements to cell viability. Number of colony as significant when they were due to drug treatment but not forming units in cultures after 11, 14 and 16 h of growth DMSO. Approximately 600 protein spots were detected on have been plotted in Fig. 2. the 2-DE gel with isoelectric focusing from pH 4.5 through During the course of growth between 11 and 16 h, the 8.0. Representative 2-D analytical gel images are shown in Fig. 3. viable cell number remained relatively constant versus the control and the DMSO supplemented cultures. In contrast, The images from P. polychaetum alkaloid extract and berberine-exposed cells were almost identical, showing that the presence of the extract caused a sharp reduction in cell viability after 14 h of growth. the effect of the antimicrobial alkaloid extract and berberine were very similar and that the trace amounts of organic Effect of plant alkaloid extract on global protein expression acids, phenolic compounds or pigments remained in the extract had no significant effect. Silver-stained gel images profiles of E. coli were analyzed to find a total of 28 differentially expressed Comparative proteomic analysis has been used to identify proteins due to drug treatment. target-related proteins which will lead to the elucidation of Spots that exhibited significant increase or decrease in abundance due to drug treatment, but not DMSO, have been indicated in magnified views of control and extract treated cultures on Fig. 4. Identification of differentially expressed proteins Peptide mass fingerprinting using MALDI-TOF was per- formed to identify the proteins that demonstrated altered expression in 2-DE. Peptide mass fingerprints (PMFs) were 0,1 obtained for the selected protein spots and all PMFs were Control searched with MASCOT software in Swissprot database for identification. The result has high confidence if the protein DMSO 0,01 was ranked at the best hit with a significant score and high plant ext. sequence coverage. berberine Finally of the 28 differentially expressed protein spots, 0,001 0 4 8 12 16 20 10 of them showed >2-fold increase and, out of these 10 Time (hrs) spots, for 9 of them significant matches were obtained from the protein database. Results of the selected proteins are Fig. 1 Growth profiles of E. coli in the presence of DMSO, berberine and, alkaloid extract with reference to control culture presented in Table 1. OD600nm Colony forming units Ann Microbiol (2010) 60:709–717 713 Fig. 3 Proteome maps of cultures in the presence of DMSO (b), 750 μg/mL plant extract (c), and 750 μg/mL berberine (d) with reference to control culture (a). The images are representative of five replicate gels Table 1 shows MALDI-TOF MS analysis of the proteins. that Papaver species also process significant antimicrobial The accession number, theoretical and predicted molecular activities (Ünsal et al. 2007, 2009). The antimicrobial weight and pI, sequence coverage and score of each protein activity of the alkaloid berberine from P. polychaetum has spot are given. been attributed to its specificity for the minor groove of AT- rich duplexes in DNA sequences (Saran et al. 1995; Choi et al. 2001; Sriwilaijareon et al. 2002; Mazzini et al. 2003; Discussion Chen et al. 2004; Qin et al. 2006). In addition to this information, Sun et al. (1988) reported that berberine For decades, plants of the genus Papaver have known to chloride blocked adhesion by reducing the synthesis of accumulate a rich spectrum of different alkaloids (Preininger fimbrial subunits and the expression of assembled fimbriae 1986; Sariyar 2002; Ziegler et al. 2006). These plants have (1988). This could be associated with the antimicrobial been regarded as important sources of narcotic alkaloids, property of berberine since adhesion to the tissue surface is such as morphine, codeine and thebaine (Ziegler et al. the first step for bacteria to establish infection. Unfortu- 2006; Salehi et al. 2007). More recent studies have shown nately, they observed that E. coli growth was reduced by 714 Ann Microbiol (2010) 60:709–717 Fig. 4 Magnified views of the A8 A8 a b proteome map of the plant A7 A7 extract treated culture (a,c) with A10 A9 A10 A9 reference to culture control (b,d) A15 A15 A19 A13 A19 A13 A16 A16 A18 A17 A18 A17 A12 A12 A14 A14 c d A1 A2 A1 A2 A23 A3 A3 A23 A22 A22 A25 A25 A26 A26 A27 A27 A21 A21 A11 A28 A29 A11 A28 A29 A4 A5 A6 A4 A5 A6 A31 A31 only 10% in the presence of 300 μg/mL berberine chloride, pumps. However, it could be as effective as other although 90% of its ability to adhere was lost (Wang et al. antimicrobial agents against E. coli when administered 2008). Stermitz et al. (2000) reported that berberine is together with an MDR inhibitor (Stermitz et al. 2000). pumped out by bacterial multi-drug resistance (MDR) Since information for this antimicrobial alkaloid was Table 1 Identification of differentially expressed protein spots in the presence of P. polychaetum alkaloid extract Spot no./protein description Accession Sequence Protein Theoretical molecular mass Experimental molecular mass number coverage (%) score (kDa)/pI (kDa)/pI Transport and binding Amino acid/peptide 1 OPPA_ECOLI ↓ P23843 22 47 60.9/6.05 66/6.3 10 YECC_ECOLI ↑ P37774 35 43 27.7/8.89 25/6.6 Sugar 6 MALE_ECOLI ↓ P02928 22 66 43.4/5.53 40/5.6 Membrane repair and maintenance 9 BLC_ECOLI ↑ P39281 24 31 19.8/8.81 22/6.6 Protein synthesis 4 EFTS_ECO24 ↓ P02997 28 54 30.5/5.22 34/5.3 Energy metabolism 3 ATPA_ECOLI ↓ P00822 30 103 55.2/5.80 55/5.3 2 STHA_ECO24 ↓ P27306 21 41 51.6/6.09 66/6.4 Regulation DNA synthesis 8 FRMR_ECO24 ↓ A7ZIA5 29 37 10.3/5.84 20/6.6 Replication 5 INTD_ECOLI ↓ P24218 16 30 45.1/9.76 36/5.6 Ann Microbiol (2010) 60:709–717 715 limited, an exhaustive study with the alkaloid extract of P. Upon exposure to P. polychaetum alkaloid extract, polychaetum was conducted for the comparative analysis of proteins from energy metabolism, soluble pyridine nucleo- cells exhibiting diverse phenotypes under drug stress. tide transhydrogenase STHA and alpha-subunit of ATP synthase ATPA, were down-regulated. STHA is proposed Survival of E. coli exposed to P. polychaetum alkaloid to be localized in the cytoplasmic space and involved in the extract conversion of NADPH to NADH. The down-regulation of STHA may be an indication of the repression in the Cell growth in the presence of the extract indicated that the respiratory activities of the cells under stress. The alpha extract or berberine can be considered to be an antimicro- subunit of ATP synthase is a peripheral membrane protein bial agent against E. coli only when available at higher found integrated to the membrane. The ATP synthase complex concentrations than the agents generally regarded as uses the proton gradient across the membrane to drive ATP antimicrobials. MIC is usually reported to be in the order synthesis from ADP and inorganic phosphate. Under fermen- of <50 μg/mL range for effective antimicrobials. tative conditions, it energizes the inner membrane by Due to the operation of MDR pumps, this value is as catalyzing the extrusion of protons at the expense of ATP high as 1,250 μg/mL for the extract. The high MIC value hydrolysis (Futai and Kanazawa 1983). The alpha-subunit has has not hampered the progress of this study since the motive an essential role in the catalytic mechanism of the complex. was to identify the modifications in the levels of proteins to P. polychaetum extract represses AtpA expression which in correlate varied protein abundances with the drug action turn results in altered levels of free energy transduction. mechanism. This information would be valuable for the Energy limitation caused by the repression of these two development of new therapeuticals. enzymes may be coupled to the disappeance of MalE. During energy crisis, carbon import via periplasmic binding proteins is Analysis of 2-DE protein profiles essential for E. coli cells (Wang and Crowley 2005). Easton et al. (2006) suggested that use of low energy-requiring trans- The expression of the seven proteins identified decreased ports for carbon uptake may in turn result in the rapid markedly or they were not expressed at all and the expression consumption of such transporters (Ayudhya et al. 2009). of two proteins was induced under the influence of the plant Hence, energy limitation in the presence of the extract could extract. These proteins were involved in transport and eventually cause consumption of MalE. binding, membrane repair and maintenance, proteins synthe- Papaver polycaetum alkaloid extract affected the protein sis, energy metabolism, regulation and replication. biosynthesis machinery by repressing the cytoplasmic elon- Among the transport and binding proteins, a component gation factor Ts, EFTS. Elongation factors interact with ribosomes and catalyze formation of the acyl bond between of the oligopeptide permease periplasmic oligopeptide- binding protein OPPA was down-regulated. Besides its the incoming amino acid residue and the peptide chain to function as a carrier for peptides up to five amino acids extend the nascent polypeptide chain during the elongation long, there is evidence that OPPA may act as a carrier for stage of bacterial translation (Alberts et al. 2002; Jayasekera et aminoglycoside antibiotics (Acosta et al. 2000). Complete al. 2004). Because of its essential functions, EFTs has been disappearance of OPPA under P. polychaetum alkaloid regarded as a possible drug target (Jayasekera et al. 2004). extract stress may indicate that OPPA could act as a carrier The stress of the plant extract induced the expression of for berberine as its structure has a resemblance to amino the outer membrane lipoprotein, lipocalin (Blc). Normally, acids and aminoglycosides. In contrast to OPPA, the expression of the blc gene starts at the beginning of expression of the uncharacterized amino-acid ABC trans- stationary phase and serves as a starvation response porter ATP-binding protein YECC was induced. This function in E. coli. Structural analyses of the purified Blc protein is located in the cell inner membrane and belongs protein suggest a possible role in membrane repair or to the ABC transporter superfamily. It is probably part of a maintenance requiring lipid storage or transport (Valerie et binding protein-dependent transport system yecCS for an al. 2004) and in phospholipid binding (Bishop 2000). Since amino acid, responsible for energy coupling to the transport berberine reduces the expression of fimbrial subunits in E. system (Blattner et al. 1997). The repression of the coli, as reported by Sun et al. (1988), the induction of Blc periplasmic oligopeptide permease may be compensated may be a predictable consequence based on its membrane by induction of this system for amino acid uptake. repairing role. Some experiments suggest that bacterial Another protein belonging to the transport and binding lipocalins may play a significant role in resistance to family, maltose-binding protein MalE, was down-regulated. antibiotics (Bishop 2000). Therefore, induction of Blc can This is located in the periplasm and is involved in the high- also be correlated with the strong resistance to the extract. affinity maltose membrane transport system malEFGK Exposure to the plant extract repressed the expression of (Duplay et al. 1984). two other proteins, prophage dlp12 integrase (INTD), 716 Ann Microbiol (2010) 60:709–717 Amsterdam D (1996) Susceptibility testing of antimicrobials in liquid involved in replication, and transcription repressor frmR media. In: Loman V (ed) Antibiotics in laboratory medicine. (FRMR), involved in regulation of DNA synthesis. The Williams and Wilkins, Baltimore, pp 52–111 integrase INTD is essential for integration of the phage into the Bandow JE, Brötz H, Leichert LIO, Labischinski H, Hecker M (2003) host genome by site -specific recombination. In conjunction Proteomic approach to understanding antibiotic action. Antimicrob Agents Chemother 47:948–955 with excisionase, integrase is also a necessary element for Bishop RE (2000) The bacterial lipocalins. Biochim Biophys Acta excision of the prophage from the host genome (Lindsey et al. 1482:73–83 1989). Transcriptional repressors are proteins that bind to rd Blattner FR, Plunkett G 3 , Bloch CA, Perna NT, Burland V, Riley specific sites on DNA to cause looping and prevent M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, transcription of nearby genes. Looping alters the topology Shao Y (1997) The complete genome sequence of Escherichia of the DNA and may thereby prevent formation of the closed coli K-12. Science 277:1453–1474 or open RNA polymerase complex, activator binding or Bradford MM (1976) A rapid and sensitive method for the elongation (Alberts et al. 2002). The down-regulation of this quantitation of microgram of protein utilizing the principle of protein-dye binding. Anal Biochem 12:248–254 protein may imply that the sites for the transcription repressor Burbaum J, Tobal GM (2002) Proteomics in drug discovery. Curr were already occupied by berberine, due to its affinity for Opin Chem Biol 6:427–433 DNA, and transcription was blocked in the absence of frmR. Chen JJ, Duh CY, Chen IS (1999) New tetrahydroprotoberberine N- oxide alkaloids & cytotoxic constituents of corydalis tashiroi. Planta Med 65:643–646 Chen WH, Chan CL, Cai Z, Luo GL, Jiang Z-H (2004) Study on Conclusion noncovalent complexes of cytotoxic protoberberine alkaloids with double-stranded DNA by using electrospray ionization mass The proteome profiling technique provided an effective spectrometry. Bioorg Med Chem Lett 14:4955–4959 Cho YS, Schiller NL, Kahng HY, Oh KH (2007) Cellular responses approach to identify global changes in protein profiles and proteomic analysis of Escherichia coli exposed to green tea under the influence of P. polychaetum alkaloid extract. To polyphenols. 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J Biol Chem characterized as potential drug targets. The experimental 259:10606–10613 findings of this study shed light on the mechanism of P. Easton JA, Thompson P, Crowder MW (2006) Time dependent polychaetum extract and berberine from a molecular perspec- translational response of E. coli to excess Zn(II). J Biomol Tech 17:303–307 tive. Further studies should lead to a better understanding of Futai M, Kanazawa H (1983) Structure and function of proton- the antimicrobial mode of action of the plant extract, translocating adenosine triphosphatase (F0F1): biochemical and specifically berberine, and will contribute to the development molecular biological approaches. Microbiol Rev 47:285–312 of novel plant-based therapeutic drugs. A still open question Hanson BA (2005) Understanding medicinal plants: their chemistry and therapeutic action. Haworth, Binghamton is how beberine enters the bacterial cells to kill them. In this Herrmann R, Ruppert T (2006) Proteome of Mycoplasma pneumoniae. respect, a significant finding could be obtained by analyzing Methods Biochem Anal 49:39–56 the interaction between berberine and oligo-peptide permease. Hooven L, Baird W (2008) Proteomic analysis of MCF-7 cells treated with benzo[a]pyrene, dibenzo[a]pyrene, coal tar extract, and diesel exhaust extract. J Toxicol 249:1–10 Acknowledgements This work was supported by Marmara University, Ingledew WJ, Poole RK (1984) The respiratory chains of Escherichia Research Fund Projects FEN-C-YLP-060308-0041 and FEN-D-040609- coli. Microbiol Rev 48:222–271 0178 and TUBITAK-MAG Project 108 M597. Isarankura-Na-Ayudhya P, Isarankura-Na-Ayudhya C, Treeranapaniboon L, Kasikun K, Thipheaw K, Prachayasittikul V (2009) Proteomics profiling of Escherichia coli in response to heavy metals stress. 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Annals of Microbiology – Springer Journals

Published: Aug 31, 2010

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