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Identification and Quantification of Secondary Metabolites by LC-MS from Plant-associated Pseudomonas aurantiaca and Pseudomonas chlororaphis
采用LC-MS法鉴定和定量植物相关桔黄假单胞菌和绿针铜绿假单胞菌中次级代谢产物   

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Journal of Microbiology and Biotechnology
Mar 2017

 

Abstract

Increased antibiotic resistance of plants and human pathogens and continuous use of chemical fertilizers has pushed microbiologists to explore new microbial sources as potential antagonists. In this study, eight strains of Pseudomonas aurantiaca and Pseudomonas chlororaphis, have been isolated from different plant sources and screened for their antagonistic and plant growth promoting potential (Shahid et al., 2017). All strains were compared with reference strain PB-St2 and their secondary metabolites were isolated by the use of solvent partitioning and subjected to LC/ESI/MS for confirmation of compounds. The ESI-mass spectra obtained were used to characterize the surfactants ionization behavior and [M + H]+ and [M + Na]+ ions were monitored for phenazines, derivatives of lahorenoic acid and cyclic lipopeptide (WLIP). LC-MS and HPLC methods were developed to see the elution of dominant metabolites in a single run to avoid the labor and separate methods of detection for all compounds. The method was found suitable and distinctively separated the compounds at different retention times in gradient flow. This method can be helpful to explore the metabolome of Pseudomonas sp. overall and in identification and quantification of strain specific metabolites.

Keywords: Phenazines (吩嗪), White-line inducing principle (WLIP) (白线诱导原理(WLIP)), Lahorenoic acid (Lahorenoic acid), Pseudomonas aurantiaca (桔黄假单胞菌), Pseudomonas chlororaphis (绿针铜绿假单胞菌), LC-MS (LC-MS)

Background

Authors in this research group are working on bioformulations and have isolated a large number of bacteria, including pseudomonads with potential plant growth promoting abilities. Pseudomonads are known for their biocontrol ability through antibacterial and antifungal secondary metabolites. These secondary metabolites successfully counter several phytopathogens of economically important crops and Pseudomonas based biofertilizers developed so far, have been marketed as commercial products (Mandryk et al., 2007; Rovera et al., 2014). Pseudomonas aurantiaca and P. chlororaphis strains are well known biocontrol agents due to the production of phenazines and cyclic lipopeptides and their broad spectrum antagonistic activities. Although many compounds have been reported by Pseudomonas aurantiaca and P. chlororaphis to date, the list of unidentified compounds is long (Chin et al., 2001; Hu et al., 2014).

Major metabolites produced by our strains were, white line inducing principle (WLIP), 2-hydroxy phenazine (2-OH-PHZ), phenazine-1 carboxylic acid (PCA) and Lahorenoic acid A. Previously reported methods for the detection of these compounds usually account only for HPLC, and comparison of results with standards. Moreover, detailed information about their MS/MS spectra and LC-MS methods is not available. Many researchers described the short HPLC runs for the detection of phenazines that did not account for the metabolites that elute at later stages (El-sayed et al., 2008; Upadhyay and Srivastava, 2011).

Lahorenoic acid A was exclusively reported by our group (Mehnaz et al., 2013) and since then has not been reported by any other group. WLIP was also reported first time from Pseudomonas aurantiaca PB-St2 in the same paper, though it has been reported earlier from other species of Pseudomonas (Meng et al., 2014). Keeping these findings in mind, a method was established that accounts for all the basic details in detection of these compounds from Pseudomonas aurantiaca in particular and other pseudomonads in general. LC-MS method was established with long run time and gradient flow, to make the better separation of these compounds. For confirmation of results, MS/MS fragmentation patterns of all compounds were also observed. The same LC-MS method was used for HPLC detection of these compounds, however, a different column was used. Many research groups working on metabolomics do not have access to standards of every compound. It was difficult to get the standards by our group as well and therefore, purified fractions of these compounds manually collected through HPLC, were used as reference standards for relative quantification of detected compounds. This method is significant as it provides the details about four essential and main secondary metabolites of Pseudomonas sp. in a single LC-MS and HPLC run.

Materials and Reagents

  1. Pipette tips (AxygenTM 1,000 μl Universal Pipette Tips) (Corning, Axygen®, catalog number: T-1000-B )
  2. Sterile syringe filters of 0.2 µm (Corning, catalog number: 431225 )
  3. Pasteur pipettes (e.g., FisherbrandTM disposable borosilicate glass Pasteur pipettes, Fisher Scientific, catalog number: 13-678-20A )
  4. pH indicator strips (pH 0-14 Universal Indicator) (Merck, catalog number: 1095350001 )
  5. Nine bacterial strains including eight isolates of Pseudomonas chlororaphis subsp. aurantiaca (GS-1, GS-3, GS-4, GS-6, GS-7, ARS-38, PBSt-2, FS-2) and one Pseudomonas chlororaphis subsp. chlororaphis (RP-4) isolate
  6. Hydrochloric acid (HCl), 36.5-38% (Sigma-Aldrich, catalog number: 320331 )
  7. Ethyl acetate, EMSURE® ACS, ISO, Reag. Ph Eur. (Merck, catalog number: 1096232500 )
  8. Sodium sulfate, anhydrous, ≥ 99% (Sigma-Aldrich, catalog number: 1614807 )
  9. Methanol gradient grade for liquid chromatography, LiChrosolv® Reag. Ph Eur (Merck, catalog number: 1060072500 )
  10. Chloroform, EMSURE® ACS, ISO, Reag. Ph Eur. (Merck, catalog number: 1024452500 )
  11. Water, LC-MS grade, LiChrosolv® (Merck, catalog number: 1153332500 )
  12. Dimethyl sulfoxide (DMSO), ≥ 99% (Sigma-Aldrich, catalog number: 472301 )
  13. Formic acid, 98-100%, EMSURE® ACS, ISO, Reag. Ph Eur. (Merck, catalog number: 1002640510 )
  14. Acetonitrile gradient grade for liquid chromatography, LiChrosolv® Reag. Ph Eur (Merck, catalog number: 1000302500 )
  15. Protease-peptone, microbiology grade (Sigma-Aldrich, catalog number: 82450 )
  16. Glycerol, molecular biology grade, ≥ 99.5% (Sigma-Aldrich, catalog number: G9012 )
  17. Potassium phosphate dibasic (K2HPO4), ACS reagent, ≥ 98% (Sigma-Aldrich, catalog number: P3786 )
  18. Magnesium sulfate heptahydrate (MgSO4·7H2O), ACS reagent, ≥ 98% (Sigma-Aldrich, catalog number: 230391 )
  19. Agar (for solid medium only), powder for Microbiology (Sigma-Aldrich, catalog number: 05040 )

Equipment

  1. Microbiological static incubator (e.g., Memmert, model: IF55 standard incubator )
  2. Microbiological incubator shaker (e.g., IKA, model: KS 4000 i control )
  3. Pipettes (e.g., Sartorius, catalog numbers: 728020 , 728050 , 728060 and 728070 )
  4. Large-volume centrifuge (e.g., HERMLE Labortechnik, model: Centrifuge ZK 496 )
  5. Erlenmeyer flasks (e.g., PYREXTM, 1,000 ml, 500 ml, Corning, PYREX®, catalog number: 4980-1L )
  6. Separatory funnels with stopcock and ring stand (e.g., 1,000 ml, 500 ml, VWR, catalog number: 89426-640 )
  7. Autoclave
  8. Glass beakers (e.g., PYREXTM, 1,000 ml, 500 ml, 100 ml, 50 ml, DWK Life Sciences, KIMBLE, catalog number: 14000-2000 )
  9. Glass stirrer
  10. Rotary evaporator (e.g., Buchi Rotavapor® R-100 (BÜCHI Labortechnik, model: Rotavapor® R-100 ) with Re-circulating Chiller F-100/F-105 (BÜCHI Labortechnik, model: Recirculating Chiller F-100/F-105 ), Vacuum Pump V-100 (BÜCHI Labortechnik, model: Vacuum Pump V-100 ), water bath and Interface I-100 (BÜCHI Labortechnik, model: Interface I-100 ))
  11. Fume hood (e.g., Labconco, model: 4’ Protector XStream Laboratory Hood, catalog number: 110410000 )
  12. LC-MS (e.g., Thermo Finnegan LC-MS, ESI Ion Trap Mass Spectrometer, Thermo Electron, model: LCQ Advantage Max )
  13. TLC plates (Silica Gel 60G F254 20 x 20 cm, e.g., Merck, catalog number: 100390 )
  14. C18 column for MS (e.g., Thermo Hypersil Gold C18 column, Length 250 mm, 4.6 mm ID, 5 µm particle size, Thermo Fisher Scientific, catalog number: 25005-254630 , Lot No. 7648, S/N 0871381M)
  15. HPLC system (e.g., WATERS, model: e2995 Separations Module , 2998 Photodiode Array (PDA) Detector)
  16. HPLC column (e.g., Nucleosil C18 column, 4.6 x 250 mm, 5 µM; Macherey-Nagel, Germany)

Software

  1. Xcalibur 2.0 software (XcaliburTM Software, control and process data from LC-MS instruments. It is WindowsTM based software that provides method setup, data acquisition, data processing and reporting. Data files are retrieved in Qual Browser and are processed for interpretation and analysis)

Procedure

  1. Culture conditions
    1. Total nine bacterial strains including eight isolates of Pseudomonas chlororaphis subsp. aurantiaca (GS-1, GS-3, GS-4, GS6, GS-7, ARS-38, PBSt-2, FS-2) and one isolate Pseudomonas chlororaphis subsp. chlororaphis (RP-4) were included in this research. These bacterial strains are 16S rRNA identified and screened for their PGPR activities (Shahid et al., 2017). All bacterial strains were streaked on King’s B agar plates (see Recipes) and plates were incubated at 28 ± 2 °C for 48 h.
    2. A single colony of each bacterial strain; GS-1, GS-3, GS-4, GS6, GS-7, ARS-38, PBSt-2, FS-2 and, RP-4 was separately used to inoculate 10 ml of King’s B broth. Cultures were incubated in a shaking incubator for 24 h at 150 rpm and 28 ± 2 °C. Next day, each bacterial culture was individually inoculated (2%) in 500 ml King’s B broth and flasks were incubated at 28 ± 2 °C, 150 rpm for 96 h.

  2. Extraction procedure
    1. After 96 h of incubation, harvest cultures and centrifuge at 3,376 x g for 40 min at 4 °C. Collect supernatants in separate flasks.
    2. All supernatants are acidified to pH 2 with 1 N HCl and pH is monitored with pH-indicator strips.
    3. Acidified supernatants are extracted twice with equal volume of ethyl acetate. For this, in each flask with 500 ml of acidified supernatant, add 500 ml of ethyl acetate. Put flasks in a shaking incubator for 10-15 min and then transfer materials into separatory funnels. The materials will separate into two phases, organic phase and water phase. The upper phase is organic phase while the lower phase has culture supernatant. Collect them into separate beakers. Re-extract the collected culture supernatant with ethyl acetate and combine the re-extracted organic layer into the previous collected organic phase. 
    4. Add 20-30 g of anhydrous sodium sulphate to the beaker with organic phase and stir with a glass stirrer. This step is essential to remove any moisture from the organic layers. Then, transfer this content to another clean and dry beaker (avoiding any salt particles) and finally in the rotary evaporator flask.
    5. Turn on all basic units of rotary evaporator including central interface, glass assemblies, water bath, chiller, and vacuum. Set water bath temperature to 40-45 °C, and adjust rotary rotations accordingly to prevent any bumping of liquid in glass assemblies. When the liquid phase is completely dried, separate the round bottom flask with dried residue from rotary evaporator glass assembly.
    6. Re-dissolve residues of extracts in methanol and chloroform (2 ml methanol:2 ml chloroform), completely dry in a fume hood and store at 4 °C.

  3. Identification of bacterial compounds using LC-MS
    1. For characterization of bacterial secondary metabolites, dissolve the extracts in 1.5 ml of methanol and 500 µl of chloroform.
      Note: Please be sure that extracts are completely dissolved and no un-dissolved residue left in the vials. If any portion of extracts remains undissolved, collect the methanol and chloroform soluble part in separate vials and dissolve the remaining undissolved part in DMSO (Dimethyl sulfoxide).
    2. Take 500 µl of these extracts and individually filter with sterile syringe filters of 0.2 µm (Fisher Scientific).
    3. Subject the extracts of all strains to Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC/ESI/MS), for identification of secondary metabolites.
    4. Set up the instrument and perform LC-ESI MS/MS runs using a Thermo Finnegan HPLC system, coupled to an LCQ Advantage Max ESI-Ion Trap Mass Spectrometer (Thermo Electron, USA).
    5. Chromatographic separations are achieved using Thermo Hypersil Gold C18 column (4.6 x 250 mm, 5 µm particle size). Set the temperature of the column compartment at 25 °C, and load 20 µl of injection volume on the column.
    6. A gradient used to separate the metabolites consists of two solvent systems. Solvent A is 0.1% formic acid in water and solvent B is 0.1% formic acid in acetonitrile. Total LC-MS/MS runtime is 55 min, and set the flow rate at 0.7 ml/min.

  4. Gradient conditions of LC-MS/MS
    1. Set gradient conditions as follows (Table 1):

      Table 1. Gradient run used for LC-MS and HPLC analyses


    2. ESI positive mode is used for the runs with data dependent protocol with a mass range of 150-1,500 a.m.u. and two scan events are employed for this data. The first scan event is a full scan of 150-1,500 and the second scan is dependent on the most abundant ions in the first scan triggering their MS2 acquisition.
    3. Data are acquired at the normalized collision energy of 30%. The heated capillary is maintained at 350 °C, and sheath and auxiliary/sweep gases are at 60 and 25 arbitrary units, respectively.
    4. Set the source voltage to 4.5 kV with 10 V capillary voltage. The ESI-mass spectra obtained are used to characterize the surfactant ionization behavior; [M + H]+ and [M + Na]+ ions are monitored for phenazines, i.e., 2-hydroxy-phenazine(2-Oh-Phz) and phenazine-1-carboxylic acid (PCA); Lahorenoic acid A, and cyclic lipopeptide (WLIP). In addition, the ESI-MS/MS fragmentation behavior of identified peaks is investigated to confirm the structure of these secondary metabolites. Figures (Figures 1-3 and Supplemental Figures S1-S10) describe the structures, extracted ion current (XIC) chromatograms, mass spectrums and MS/MS fragmentation behavior of detected metabolites. Chemical formulas, exact masses and observed m/z values for detected metabolites have been given in Table 2.


      Figure 1. Structures of the compounds isolated from Pseudomonas aurantiaca and Pseudomonas chlororaphis. A. 2-hydroxyphenazine, B. phenazine-1-carboxylic acid, C. white-line-inducing principle, D. lahorenoic acid A (Mehanz et al., 2013).

      Table 2. Chemical formulas, exact masses and observed m/z values for detected metabolites



      Figure 2. Extracted ion current (XIC) chromatograms for 2-hydroxyphenazine (2-OH-Phz), m/z 197, [M + H]+ of four strains; ARS-38, RP-4, FS-2 and PB-St2


      Figure 3. Mass spectrum of 2-Hydroxyphenazine, m/z [M + H] + 197.07

  5. Thin layer chromatography (TLC) of identified secondary metabolites
    Identified four secondary metabolites can also be analyzed by thin layer chromatography (TLC), using PB-St2 as a reference strain. PB-St2 is used as reference strain as all of its secondary metabolites have previously been published (Mehnaz et al., 2009 and 2013).
    1. For thin layer chromatography, load methanol fractions of all bacterial extracts (10 µl) on TLC plates (Silica Gel 60G F254 20 x 20 cm). Mobile phase contains chloroform: acetone: acetic acid (78.4:20:1.6, v/v/v) and samples are spot inoculated on TLC plate for separation (Figure 4).
    2. Air dry TLC plates (approximately for 5-10 min) and analyze for showing the separation of phenazines, i.e., phenazine-1-carboxylic acid (PCA) and 2-hydroxy phenazine (2-OH-Phz).


      Figure 4. Thin layer chromatogram of phenazine-1-carboxylic acid (PCA) and 2-hydroxy phenazine (2-OH-PHZ), present in cell-free supernatants of bacterial extracts of RP-4, ARS-38, GS-4 and FS-2. PB-St2 fractions were used as reference standard for this TLC analysis.

  6. Quantification of secondary metabolites
    1. Manually collect the pure fractions of these compounds using HPLC from PB-St2 Pseudomonas aurantiaca isolate (or any bacterium that needs to be analyzed for its secondary metabolites) and analyze on LC-MS for their purity (Figure 5). The method for LC-MS is the same as used in HPLC analysis. The total run time is 55 min, with 20 µl injection volume and with same acetonitrile and water gradient run, as described in Table 1.
    2. Analyze samples on Waters HPLC System (e2995, separations module) with 299 h photodiode-array (PDA) detector using a Nucleosil C18 column (4.6 x 250 mm, 5 µM; Macherey-Nagel, Germany). Collected fractions of these compounds are used as reference standard to quantify the production of these compounds from all bacteria used in this study.


      Figure 5. HPLC analysis of enriched P. aurantiaca PB-St2 fractions of detected metabolites

Data analysis

All LC-MS/MS data were acquired and data files were processed using Xcalibur 2.0 software. Data files were initially open using Xcalibur FTMS raw data files. These files displayed total ion chromatogram (TIC) on the top and m/z spectra of total scan at the bottom. Using software’s basic features, data were acquired for reported four compounds and their ESI-MS/MS fragmentation patterns were also observed to confirm the structure of these secondary metabolites. ESI-MS/MS fragmentation was also confirmed with Wishart Research Group’s (CFM-ID), Competitive Fragmentation Modeling for Metabolite Identification (cfmid.wishartlab.com).

Notes

  1. Avoid any plastic equipment during the preparation of samples for HPLC or LC-MS and also at the time of extraction. This may add some plasticizers in your prepared samples that often result in false peaks in LC-MS and HPLC run.
  2. Amount of the culture medium can also be reduced accordingly. This method used the fractions extracted from 500 ml of King’s B broth. It can be reduced to 100 ml or 50 ml according to the requirement. Amount of organic solvents used will also get reduced with it.
  3. For extraction with ethyl acetate, the supernatant is acidified to pH 2, while using dichloromethane (DCM) for extraction of secondary metabolites, the supernatant is not acidified/pH not changed.

Recipes

  1. King’s B agar and broth
    Protease peptone 20 g/L
    Glycerol 20 g/L
    Anhydrous K2HPO4 1.5 g/L
    MgSO4·7H2O, 6.09 ml of 1 M solution
    Agar (for solid medium only), 15 g/L
    Deionized H2O, 1,000 ml
    Completely dissolve protease peptone, glycerol and anhydrous K2HPO4 in 500 ml of deionized H2O and adjust the pH to 7.2. Make up the final volume to 994 ml and autoclave for 20 min. Separately, prepare 1 M solution of MgSO4·7H2O and autoclave it. Add 6.00 ml of this solution to the autoclaved medium for making up the final volume to 1,000 ml

Acknowledgments

The original article has been published in Journal of Microbiology and Biotechnology (Shahid et al., 2017). Authors gratefully acknowledge the support of Alexander Von Humboldt Foundation, Bonn, Germany, for equipment grant and Higher Education Commission (HEC; Project No. 20-3134), Pakistan, for supporting this research work. Authors declare that the research was conducted in the absence of any financial or commercial relationships that could be constructed as a potential conflict of interest.

References

  1. Chin, A. W. T. F., van den Broek, D., de Voer, G., van der Drift, K. M., Tuinman, S., Thomas-Oates, J. E., Lugtenberg, B. J. and Bloemberg, G. V. (2001). Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted into the growth medium. Mol Plant Microbe Interact 14(8): 969-979.
  2. El-Sayed, W., El-Megeed, M., El-Razik, A. B., Soliman, K. H. and Ibrahim, S. A. (2008). Isolation and identification of phenazine-1-carboxylic acid from different Pseudomonas isolates and its biological activity against Alternaria solani. Res J Agric Biol Sci. 4 (6): 892-901.
  3. Hu, W., Gao, Q., Hamada, M. S., Dawood, D. H., Zheng, J., Chen, Y. and Ma, Z. (2014). Potential of Pseudomonas chlororaphis subsp. aurantiaca strain Pcho10 as a biocontrol agent against Fusarium graminearum. Phytopathology 104(12): 1289-1297.
  4. Mandryk, M. N., Kolomiets, E. I. and Dey, E. S. (2007). Characterization of antimicrobial compounds produced by Pseudomonas aurantiaca S-1. Pol J Microbiol 56(4): 245-250.
  5. Mehnaz, S., Baig, D. N., Jamil, F., Weselowski, B. and Lazarovits, G. (2009). Characterization of a phenazine and hexanoyl homoserine lactone producing Pseudomonas aurantiaca strain PB-St2, isolated from sugarcane stem. J Microbiol Biotechnol 19(12): 1688-1694.
  6. Mehnaz, S., Saleem, R. S., Yameen, B., Pianet, I., Schnakenburg, G., Pietraszkiewicz, H., Valeriote, F., Josten, M., Sahl, H. G., Franzblau, S. G. and Gross, H. (2013). Lahorenoic acids A-C, ortho-dialkyl-substituted aromatic acids from the biocontrol strain Pseudomonas aurantiaca PB-St2. J Nat Prod 76(2): 135-141.
  7. Meng, J., Fan, Y., Su, M., Chen, C., Ren, T., Wang, J. and Zhao, Q. (2014). WLIP derived from Lasiosphaera fenzlii Reich exhibits anti-tumor activity and induces cell cycle arrest through PPAR-γ associated pathways. Int Immunopharmacol 19(1): 37-44.
  8. Rovera, M., Pastor, N., Niederhauser, M. and Rosas, S. B. (2014). Evaluation of Pseudomonas chlororaphis subsp. aurantiaca SR1 for growth promotion of soybean and for control of Macrophomina phaseolina. Biocont Sci Tech 24: 1012-1025.
  9. Shahid, I., Rizwan, M., Baig, D. N., Saleem, R. S., Malik, K. A. and Mehnaz, S. (2017). Secondary metabolites production and plant growth promotion by Pseudomonas chlororaphis and P. aurantiaca strains isolated from cactus, cotton, and Para grass. J Microbiol Biotechnol 27(3): 480-491.
  10. Upadhyay, A. and Srivastava, S. (2011). Phenazine-1-carboxylic acid is a more important contributor to biocontrol Fusarium oxysporum than pyrrolnitrin in Pseudomonas fluorescens strain Psd. Microbiol Res 166: 323-335.

简介

哺乳动物正呼吸道病毒(呼肠孤病毒)利用成孔肽穿透宿主细胞膜。 在病毒进入过程中,这一步对于提供含核心颗粒的基因组至关重要。 该协议描述了用于测量呼肠孤病毒诱导的孔形成的体外测定。

【背景】呼肠孤病毒是无包膜的双链RNA病毒,其由两个同心蛋白质壳组成:内衣壳(核心)和外衣壳(Dryden等人,1993; Zhang等人, / ,2005; Dermody et al ,2013)。在附着之后,病毒颗粒被内吞(Borsa et al。,1979; Ehrlich et al。,2004; Maginnis et al。,2006; Maginnis和宿主组织蛋白酶蛋白酶降解σ3外壳蛋白(Chang和Zweerink,1971; Silverstein等人,1972; Borsa等人,et al。 1981; Sturzenbecker等人,1987; Dermody等人,1993; Baer和Dermody,1997; Ebert等人, 2002年)。这个过程产生一个亚稳中间体,称为感染性亚病毒颗粒(ISVP),其中细胞穿透蛋白μ1被暴露(Dryden等人,1993)。呼肠孤病毒ISVPs进行第二次构象改变以将含有基因组的核心沉积到宿主细胞的细胞质中。被改变的粒子被称为ISVP *(Chandran et al。,2002)。 ISVP-to-ISVP *转换在释放μ1-衍生的成孔肽方面达到高潮(Nibert等人,1991; Zhang等人,2005; Chandran 2002年; Odegard等人,2004; Nibert等人,2005; Agosto等人。 ,2006; Ivanovic等人,2008)。释放的肽在内体膜内形成孔隙,其被认为介导核心递送(Agosto et al。,2006; Ivanovic et al。,2008; Zhang等人al 。,2009)。

定义呼肠弧病毒进入的许多构象变化可以在体外重现:(i)通过用糜蛋白酶消化纯化的病毒体产生ISVP(Joklik,1972; Borsa等人)。 ,(2)ISVP *的形成可以利用热(Middleton et al。,2002),大单价阳离子(Borsa et al。,1973b) ,来自μ1的肽(Agosto et al。,2008),红细胞(Chandran等人,2002; Sarkar和Danthi,2010)或脂质(Snyder和Danthi,2015年和2016年)。因此,使用生物化学和基于细胞的方法研究与呼肠孤病毒进入相关的问题。在这个协议中,我们描述了一个体外试验,概括了ISVP到ISVP *的转化和随后的孔形成。

关键字:吩嗪, 白线诱导原理(WLIP), Lahorenoic acid, 桔黄假单胞菌, 绿针铜绿假单胞菌, LC-MS

材料和试剂

  1. 移液器吸头
    1. 0.1-10μl容量(USA Scientific,目录号:1111-3700)
    2. 1-200μl容量(VWR,目录号:89079-474)
    3. 容量100-1,250μl(VWR,目录号:53508-924)
  2. PCR 8孔管条(VWR,目录号:20170-004)
  3. 50毫升离心管(VWR,目录号:89039-660)
  4. 1.7 ml微量离心管(MIDSCI,目录号:AVSS1700)
  5. 真空驱动和一次性瓶顶0.22微米过滤器(默克,目录号:SCGPT05RE)
  6. 平底,96孔板(Greiner Bio One International,目录号:655180)
  7. 纯化的呼肠孤病毒储液(参见Berard和Coombs,2009; Kobayashi等人,2010,用于繁殖和纯化过程)
  8. 碎冰
  9. 标准SDS-PAGE材料和试剂(例如,10%SDS-聚丙烯酰胺微型凝胶)
  10. 考马斯亮蓝染色和脱色溶液(Bio-Rad Laboratories,目录号:1610435)
  11. 柠檬酸小牛血(科罗拉多血清公司,目录号:31023)
  12. 漂白剂(Biz4USA,Janitorial Supplies,目录号:CLO30966CT)
  13. 2-氨基-2-(羟甲基)-1,3-丙二醇(Tris)(MP Biomedicals,目录号:02103133)
  14. 氯化钠(NaCl)(Merck,目录号:SX0420-3)
  15. 0.1N盐酸(Sigma-Aldrich,目录号:2104)
  16. 0.1N氢氧化钠(Sigma-Aldrich,目录号:2105)
  17. N-α-甲苯磺酰-L-赖氨酸氯甲基酮(TLCK)处理的胰凝乳蛋白酶(Worthington Biochemical,目录号:LS001432)
  18. 苯基甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  19. 异丙醇(Avantor Performance Materials,Macron,目录号:3032-02)
  20. Dulbecco磷酸盐缓冲盐水(Thermo Fisher Scientific,Gibco TM,产品目录号:21600044)
  21. 氯化镁六水合物(MgCl 2•6H 2 O)(Sigma-Aldrich,目录号:M9272)
  22. 超纯脱氧核糖核酸酶/无RNA酶的蒸馏水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  23. Triton X-100(TX-100)(Sigma-Aldrich,目录号:X100)
  24. 50%的漂白剂(见食谱)
  25. 病毒存储缓冲区(VB)(请参阅食谱)
  26. 2毫克/毫升TLCK处理胰凝乳蛋白酶(见食谱)
  27. 100毫米苯甲基磺酰氟(PMSF)(见食谱)
  28. 补充有2mM MgCl 2(PBS Mg)的磷酸盐缓冲盐水(参见食谱)
  29. 10%Triton X-100(TX-100)(见食谱)

设备

  1. 个人防护设备(PPE)
    1. 实验室外套
    2. 手套
    3. 眼睛保护
  2. 生物安全2级(BSL-2)实验室设施
  3. BSL-2认证的组织培养罩
  4. 固体和液体废物容器
  5. 高压灭菌器
  6. 真空泵和吸气器
  7. 冰桶
  8. -20°C冷冻机
  9. 微量移液器
    1. 0.1-2.5μl容量(Eppendorf,目录号:3123000012)
    2. 2-20μl容量(Eppendorf,目录号:3123000039)
    3. 20-200μl容量(Eppendorf,目录号:3123000055)
    4. 100-1,000μl容量(Eppendorf,目录号:3123000063)
  10. 数字pH计(VWR,型号:SB70P)
  11. 数字实验室天平(梅特勒 - 托利多,型号:PB1502-S)
  12. NanoDrop分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:ND-1000)
  13. 热板搅拌器(VWR,目录号:12365-382)
  14. 磁力搅拌棒(VWR,目录号:58948-273)
  15. 微量离心机(Eppendorf,型号:5424)
  16. 热循环仪(Bio-Rad Laboratories,型号:S1000TM)
  17. 温控水浴(VWR,目录号:89501-466)
  18. 凝胶成像系统(LI-COR,型号:Odyssey®Classic)
  19. 酶标仪(Molecular Devices,型号:FilterMax F5 Multi-Mode)
  20. 250毫升玻璃烧杯(VWR,目录号:89000-204)
  21. 1000毫升玻璃烧杯(VWR,目录号:89000-212)
  22. 100毫升量筒(VWR,目录号:65000-006)
  23. 1000毫升量筒(VWR,目录号:65000-012)
  24. 100毫升储存瓶(VWR,目录号:89000-234)
  25. 1000毫升储存瓶(VWR,目录号:89000-240)
    注:此产品已停产。

软件

  1. Image Studio Lite(LI-COR)
  2. SoftMax Pro(Molecular Devices)

程序

  1. 产生感染性亚病毒颗粒(ISVPs)
    1. 如先前所述(Berard和Coombs,2009; Kobayashi等人,2010)繁殖和纯化呼肠孤病毒毒粒。使用NanoDrop分光光度计,通过测量纯化的病毒原液在260nm处的光密度(OD 260 = 1,OD 260 = 2.1×10 -1, > 12颗粒/ ml)(Smith等人,1969)。
    2. 在8孔管条的1根管中,将2×10 11病毒体稀释到90μl冰冷的VB中(见食谱)。
    3. 加10μl冰冷的2mg / ml TLCK处理的胰凝乳蛋白酶(见食谱)到稀释的病毒中。
      上下移液3-4次 注意:对于未消化的对照,用10μl冰冷的VB取代10μlTLCK处理的胰凝乳蛋白酶。

    4. 在32°C的热循环仪孵育反应20分钟 注意:在这些条件下,σ3被降解(Joklik,1972; Borsa等,1973a),而μ1被裂解(Nibert和Fields,1992; Chandran等,1999)。 >
    5. 消化后,通过加入1μl100mM PMSF淬灭糜蛋白酶活性(参见食谱)。
      上下移液3-4次

    6. 在冰上孵育反应20分钟
    7. 为确认产生了ISVP,在10%SDS-聚丙烯酰胺微型凝胶上每泳道运行2×10 10个颗粒。
      在200V恒定温度下运行凝胶40-45分钟
    8. 通过考马斯亮蓝染色显示蛋白质条带(见数据分析,图1)。
    9. 将ISVPs储存在冰上,2-3小时内进行溶血实验。

  2. 牛红细胞(RBCs)的制备

    1. 在冰上或4°C执行所有步骤
    2. 将1毫升柠檬酸牛血液转移到微量离心管中。
      注:柠檬酸牛血液应在抽签日期的2周内使用。

    3. 在500gxg离心5分钟沉淀RBCs 注:红细胞是溶血实验膜的来源。
    4. 吸出并丢弃上清液。
    5. 在1毫升冰冷PBS(参见食谱)中重悬红细胞。
      轻轻地上下移液
    6. 重复步骤B3-B5,直到造粒后上清液保持澄清。
    7. 以30%(体积/体积)浓度重新悬浮在冰冷PBS中的RBCs。
      轻轻地轻弹管子的一边 注意:使用微量离心管上的体积标记来评估RBC颗粒体积。
    8. 将红细胞储存在冰上,立即用于溶血实验

  3. ISVP诱导的溶血试验
    1. 在单独的离心管中,在冰上组装下列反应物:
      1. 33.3μlVB +3.7μl30%RBCs(0%溶血对照)
      2. 30.3μlVB +3.7μl30%RBCs +3μl10%TX-100(100%溶血对照,见食谱)
      3. 3.3μlVB +3.7μl30%RBCs +30μlISVPs
    2. 通过轻轻地轻弹管子的侧面来混合反应。
    3. 孵育反应1小时(T3D呼肠孤病毒)或2小时(T1L呼肠孤病毒)在37°C在水浴。
      注意:在这些条件下,诱导了ISVP对ISVP *的转换(Chandran等,2002; Sarkar和Danthi,2010),并释放了μ1-来源的成孔肽(Nibert等,1991; Chandran等,2002; Odegard等,2004; Nibert等,2005; Zhang等,2005; Agosto等,2006; Ivanovic等,2008)。 >
    4. 将反应物放置在冰上20分钟。

    5. 在500gxg离心5分钟沉淀完整的RBCs 注意:这个步骤应该在4°C。
    6. 将20μl上清液转移到96孔板的各个孔中。
    7. 用80μl的VB稀释每个转移的上清液。
      上下移液3-4次
    8. 为了量化释放的血红蛋白的量(即RBC裂解),使用酶标仪在405nm处测量稀释的上清液的吸光度(em)。在SoftMax Pro软件中记录 A 值。
    9. 计算溶血百分比(见数据分析)。

数据分析

  1. 产生感染性亚病毒颗粒(ISVPs)
    1. 使用凝胶成像系统和Image Studio Lite软件记录和分析结果(图1)。
      1. 病毒体含有λ1,2,3,μ1C,σ2和σ3。
      2. ISVPs包含λ1,2,3,μ1C,δ和σ2。
      注意:δ的出现,μ1C的损失以及σ3的损失表明从病毒粒子到ISVP的转变。 λ1,2,3和σ2应保持不变。


      图1.呼肠孤病毒和ISVPs的SDS-PAGE凝胶

  2. ISVP诱导的溶血试验
    1. 所有的溶血实验都应该重复进行至少三次独立的重复。
    2. 使用以下公式计算溶血百分比:

      [( )]×100

      1. 缓冲
      2. TX-100 代表来源于VB,RBCs和TX-100的上清液。
      3. 样品
    3. 当比较不同呼肠孤病毒毒株的溶血能力时,使用学生t检验来计算p值。
    4. 使用图形软件绘制溶血百分比。
      注意:对于T3D呼肠孤病毒,在37°C孵育1小时后,通常会观察到40-60%的溶血。

笔记

  1. 如果可能的话,所有的程序都在BSL-2认证的组织培养罩中进行。
  2. 实验室人员应使用适当的PPE。
  3. 所有的固体废物都是在处理之前进行高压灭菌。
  4. 所有的液体废物在处理之前都用50%的漂白剂灭活。

食谱

  1. 50%漂白
    在储存瓶中,将50ml 100%漂白剂稀释到50ml超纯H 2 O中,
  2. 病毒储存缓冲液(VB)(10mM Tris,pH 7.4,15mM MgCl 2和150mM NaCl)
    1. 在玻璃烧杯中,将以下成分溶解在900ml超纯H2O2中:
      1.21克Tris
      3.05克MgCl2•6H2O 2 O. 8.77克NaCl
    2. 使用磁力搅拌棒在搅拌板上在室温下混合
    3. 用0.1N盐酸调节至pH7.4
    4. 在量筒中,用超纯水将终体积加至1000毫升
    5. 将溶液转移到储存瓶中
    6. 通过高压灭菌消毒
    7. 在室温下储存3。
  3. 2毫克/毫升α-甲苯磺酰-L-赖氨酸氯甲基酮(TLCK)处理的胰凝乳蛋白酶
    1. 在离心管中,将100mg TLCK处理的胰凝乳蛋白酶溶于50ml超纯H 2 O
    2. 在室温下轻轻颠倒管子直至溶液变澄清
    3. 将1ml等分试样转移至微量离心管中
    4. 在-20°C储存
  4. 100毫米苯甲基磺酰氟(PMSF)
    1. 在微量离心管中,将17.4mg PMSF溶解在1ml异丙醇中
    2. 在室温下轻轻颠倒管子直至溶液变澄清
    3. 在-20°C储存
  5. 补充有2mM MgCl 2(PBS Mg)的磷酸盐缓冲盐水
    1. 在玻璃烧杯中,将以下成分溶解在900ml超纯H2O2中:
      9.55克Dulbecco磷酸盐缓冲液
      0.41g MgCl 2•6H 2 O.
    2. 使用磁力搅拌棒在搅拌板上在室温下混合
    3. 调整到pH值7.4
    4. 在量筒中,用超纯水将终体积加至1000毫升
    5. 通过0.22μm瓶顶过滤器过滤消毒
    6. 在室温下储存
  6. 10%Triton X-100(TX-100)
    1. 在玻璃烧杯中,将以下成分溶解在80ml超纯H2O2中:
      0.12克Tris
      0.31g MgCl 2•6H 2 O 0.88g NaCl
      10ml的100%TX-100
    2. 使用磁力搅拌棒在搅拌板上在室温下混合
    3. 调整到pH值7.4
    4. 在量筒中,用超纯水将终体积加至100ml
    5. 将溶液转移到储存瓶中
    6. 通过高压灭菌消毒
    7. 在室温下储存

致谢

该协议是从以前发表的研究(Chandran等人,2002; Sarkar和Danthi,2010)改编的。在该出版物中报道的研究得到了国立卫生研究院的过敏和传染病研究所的支持,奖项号码分别为1R01AI110637(至P.D.)和F32AI126643(至A.J.S.),印第安纳大学布卢明顿分校。内容完全是作者的责任,不一定代表出资者的官方观点。作者宣称没有利益冲突。

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引用:Shahid, I., Rizwan, M. and Mehnaz, S. (2018). Identification and Quantification of Secondary Metabolites by LC-MS from Plant-associated Pseudomonas aurantiaca and Pseudomonas chlororaphis. Bio-protocol 8(2): e2702. DOI: 10.21769/BioProtoc.2702.
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