参见作者原研究论文

本实验方案简略版
Apr 2020
Advertisement

本文章节


 

A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins
一种以测定成孔蛋白膜通透性的基于荧光淬灭的脂质体渗漏测定方法   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have great adaptability in size, lipid composition, and loading cargo. Incorporating the fluorescent dye/quencher pair, 8-Aminonaphthalene-1,3,6-Trisulfonic Acid (ANTS) and p-Xylene-Bis-Pyridinium Bromide (DPX), in liposomes is an effective approach for measuring membrane leakage. When ANTS and DPX are encapsulated in a liposome, the fluorescence of ANTS is quenched by DPX. However, disruption of liposome integrity and subsequent leakage result in measurable fluorescence emitted by ANTS. Here, we report our protocol for optimal liposome preparation for measuring liposome leakage by fluorescence dequenching.

Keywords: Fluorescence dequenching (荧光淬灭), Liposome leakage (脂质体渗漏), ANTS (ANTS), DPX (DPX), EsxA (EsxA), EsxB (EsxB), Mycobacterium tuberculosis (结核杆菌)

Background

Pore-forming toxins (PFTs) are a family of virulence factors produced by microbial pathogens for host invasion (Alouf et al., 2005). The major action of PFTs is forming pores on lipid membranes, resulting in membrane lysis and/or translocation of other effector proteins into host cells (Bischofberger et al., 2009). Liposomes have long been used as a biological membrane model for protein-membrane interactions due to their great adaptability in size, lipid composition, and loading cargo (Chatterjee and Agarwal, 1988). A common way of using liposomes to measure pore formation or membrane leakage is to encapsulate ions or fluorescent dyes inside liposomes. Upon liposome rupture, the released ions or fluorescent dyes emit measurable signals. We first used a K+ release assay to measure pore formation on liposomal membranes by the anthrax toxin (Sun et al., 2007 and 2008; Sun and Collier, 2010). Later, the fluorescence dye and quencher pair ANTS/DPX caught our attention (Nieva et al., 1989; Ruiz-Argüello et al., 1996). Compared with the K+ release assay, which requires a bulky K+ probe and frequent changes of costly probe membranes, the ANTS/DPX dequenching assay featured higher sensitivity, more accurate and consistent measurements, and lower cost. Therefore, over the past ten years, we have transitioned to the ANTS/DPX dequenching assay. With this assay, we have successfully measured the membrane permeabilizing activity (MPA) of Mycobacterium tuberculosis (Mtb) virulence factors. The two effector proteins, 6-kDa early secreted antigenic target (ESAT-6) or EsxA and 10-kDa culture filtrate protein (CFP-10) or EsxB have been tested with various experimental designs and settings (De Leon et al., 2012; Ma et al., 2015; Peng et al., 2016; Zhang et al., 2016; Aguilera et al., 2020; Ray et al., 2019). EsxAB is a heterodimer implicated in penetrating the phagosomal membrane of macrophages, allowing for translocation of Mtb into the cytosol (De Leon et al., 2012; Ma et al., 2015; Zhang et al., 2016; Aguilera et al., 2020). Recently, we have identified a new lipid composition for liposomes that greatly enhances the MPA of EsxA (Ray et al., 2019; Vazquez-Reyes et al., 2020). Here, we describe in detail the fluorescence dequenching based liposome leakage assay with our new lipid composition.


Materials and Reagents

  1. Screw cap glass vials with PTFE screwcap (Spectrum Chemical, catalog number: 985-92215)

  2. Cuvette spinbar (VWR, catalog number: 58949-030)

  3. Quartz UV cuvette (Fireflysci, catalog number: 1FLUV10)

  4. 515 nm filter (Andover Corporation, catalog number: 515FG05-50S)

  5. 1 ml syringe (BD, catalog number: 309659)

  6. 9’’ Pasteur pipet (Fisher scientific, catalog number 13-678-6b)

  7. Saint-Gobain Tygon tubing (Saint-Gobain, catalog number: ACF00017F)

  8. Glass tube rack (Fisher Scientific, catalog number: K7492100100)

  9. HiTrap® desalting columns (Millipore-Sigma, catalog number: GE17-1408-01)

  10. Gel loading pipet tips (Fisherbrand, catalog number: 02-707-139)

  11. 10 mm filter supports (Avanti Polar Lipids, catalog number: 610014)

  12. 0.2 µm membrane (Whatman, catalog number: 800281)

  13. Aluminum foil (Fisherbrand, catalog number: 01-213-100)

  14. 1 ml sample loop (Thermo Scientific, catalog number: 03-052-382)

  15. Chloroform (Fisher Chemical, catalog number: c298)

  16. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, catalog number: 850375)

  17. 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (nickel salt) (18:1 DGS-NTA(Ni) (Avanti Polar Lipids, catalog number: 790404)

  18. 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) (Avanti Polar Lipids, catalog number: 840457)

  19. 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, catalog number: 850457)

  20. Ethanol (Fisher Chemical, catalog number: a4094)

  21. HPLC grade water (Fisher Chemical, catalog number: W5-4)

  22. Tris-base (Fisher Chemical, catalog number: BP152)

  23. Sodium chloride (Fisher Chemical, catalog number: BP358)

  24. Sodium hydroxide (Fisher Chemical, catalog number: S318)

  25. Hydrochloric acid (Fluka, catalog number: 7647)

  26. Dry ice

  27. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) (Fisher Bioreagents, catalog number: BP310)

  28. 8-Aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (Setareh Biotech, catalog number: 6951)

  29. p-Xylene-bis-pyridinium bromide (DPX) (Setareh Biotech, catalog number: 6271)

  30. Triton X-100 (Sigma-Aldrich, catalog number: X100)

  31. Sodium acetate (Sigma-Aldrich, catalog number: S2889)

  32. Methanol (Fisher, catalog number: A453-500)

  33. Gel filtration buffer (see Recipes)

  34. pH 4 buffer (see Recipes)

Equipment

  1. Glass tubes (VWR, catalog number: 89000-502)

  2. 50 ml beaker (Pyrex, catalog number: 02-540G)

  3. Lab mounting stand (Humboldt MFG Company, catalog number: H-21217)

  4. Nitrogen gas tank

  5. Gas tank regulator (VWR, catalog number: 55850-277)

  6. Large top desiccator (Corning Life Sciences, catalog number: CLS3120250)

  7. Hot plate/stirrer (Thermo Fisher Scientific, catalog number: SP131325)

  8. Extruder set with heating block (Avanti Polar Lipids, catalog number: 610000)

  9. pH meter (Mettler Toledo, catalog number: 30266626)

  10. Vortex (VWR, catalog number: 58816-121)

  11. Scale (Mettler Toledo, catalog number: ML54)

  12. AKTA FPLC system (General Electric, catalog number: UPC-900 and P920)

  13. Fluorometer (ISS, catalog number: K2)

  14. Water bath (VWR, catalog number: 13271-086)

Procedure

  1. Drying phospholipids

    1. If working with lipid powders, create a lipid stock solution.

    2. Measure 50-150 mg of lipids into a glass vial with a screw cap.

    3. Add 5-15 ml of 3:2 chloroform:methanol as needed to maintain concentration at 10 mg/ml for ease of usage.

      Note: Cover the vial using PTFE lined caps; otherwise, the chloroform may dissolve plastics and contaminate the sample.

    4. Measure the appropriate amount of lipids (10 mg will be enough for approximately 30 reactions) into a glass tube.

      Note: Liposomes can be made of single lipid forms [e.g., 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG), and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)] or their combinations. For example, the Ni2+-chelating lipid, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (nickel salt) or (DGS-NTA(Ni)), can attach His-tagged proteins to the membranes (Sun et al., 2007 and 2008; Jacquez et al., 2014). The new liposome developed for measuring the MPA of EsxAB is composed of POPC to POPG at a 4:1 molar ratio.

    5. Use a clamp stand to hold the vial in place.

    6. Using a second clamp, position a Pasteur pipette approximately 2-5 mm away from the liquid in the tube.

    7. Connect the tubing to the end of the Pasteur pipette and open the valve to allow gentle airflow.

    8. Evaporate the chloroform from the vial until there is no liquid visible.

    9. Remove the vial from the stand and place it inside a vacuum chamber at room temperature for at least 3 h or overnight.

    10. Store the dry lipids at -20 °C. Lipids may be stored under these conditions for up to a month.


  2. Preparing column and FPLC for desalting

    1. A 5-ml desalting column is needed for removing the excess ANTS/DPX and for buffer exchange. If necessary, two desalting columns can be connected in tandem to improve desalting results.

    2. Pass at least 5 column volumes (CV) or, in this case, 50 ml of water through the column to remove the ethanol from the column.

    3. Pass at least 5 CV (50 ml) of gel filtration buffer through the column.

    4. Prepare the machine for desalting by passing 50 ml of water through the machine at 5 ml/min.

    5. Pass 50 ml of gel filtration buffer through the machine at 5 ml/min.

    6. Connect the column to the machine and pass an additional 100 ml of gel filtration buffer.

    7. Clean the injection valve by injecting 1 ml of gel filtration buffer.

    8. Repeat Step B7 at least two more times.


  3. Preparing the lipid-ANTS/DPX solution

    1. Prepare a freezing solution in a beaker by adding dry ice to > 95% ethanol. The freezing solution is used to flash freeze the lipids containing ANTS/DPX. Alternatively, liquid nitrogen can be used to flash freeze. It is ideal to keep the freezing solution below -50 °C.

    2. Fill another beaker with lukewarm water for thawing the lipids.

    3. Re-suspend lipids in 5 mM HEPES (pH 7.4), 1 ml for every 10 mg of lipids.

    4. Vortex until the solution is well suspended.

    5. Measure the solution volume and add 50 mM of ANTS and DPX into the tube.

    6. Mix by pipetting up and down and cover the tube with aluminum foil.

    7. Place the tube into the freezing solution and allow it to freeze.

    8. Move the tube into warm water to thaw the lipids.

    9. Repeat Steps C7-C8 for five more freeze/thaw cycles.

      Note: Six freeze/thaw cycles are usually sufficient to solubilize the lipids. If problems occur during the extrusion, such as difficulties in passing the liquid through the membranes, perform more freeze/thaw cycles.


  4. Producing liposomes via extrusion

    1. Assemble the extruder according to the manufacturer’s specifications.

    2. Draw up 1 ml of gel filtration buffer using one of the syringes and pass the buffer through the extruder until the liquid has been transferred to the other syringe.

      Note: You should feel a little bit of resistance. If there is no resistance, it is possible the membrane has moved during assembly or has torn.

    3. Remove the buffer from the syringe.

    4. Draw up to 1 ml of liposomes into one of the syringes.

    5. Pass the liposomes through the membrane until the liquid has been transferred from one syringe to the other.

    6. Repeat Step D5 for a total of 20 times.

    7. Inspect the fluid after extrusion; it should have changed from an opaque to a transparent, yellow-tinged liquid (Figure 1).



      Figure 1. Comparison of the lipid solution before and after extrusion. A. The lipid solution before extrusion has an opaque color. B. The lipid solution becomes more transparent after extrusion.


    8. Dispense the lipids into a clean glass tube.


  5. Removal of excess fluorescent dyes via desalting chromatography

    1. After pre-equilibration of the column and machine and cleaning the injection loop, inject the liposomes into the injection loop.

    2. Run the desalting program at a 0.5 ml/min flow rate, using gel filtration buffer, and collect 0.5 ml per fraction.

    3. Allow 50 ml of buffer to pass through the column.

    4. The liposomes will be eluted out of the column after approximately 2 ml have passed through.

    5. Collect fractions containing the liposomes, usually in six tubes for a total 3 ml of liposomes (Figure 2).



      Figure 2. Most lipids will elute after flowing approximately 2 ml of buffer through the column. A typical yield of 3 ml is expected using this procedure. The blue line represents mAu, and the brown line represents conductance.


    6. Combine all liposome samples, cover with aluminum foil, and store at 4 °C.

    7. Liposomes at this stage can be stored for approximately 1-3 weeks.

    8. Liposomes prepared with this procedure are usually consistent in concentration. However, technical replicates with the same batch of lipids are needed for every biological replicate to obtain consistent results.

    9. Clean the column by passing at least 5 CV + 30 ml (the volume of the machine) of water through the machine.

    10. Pass at least five CV in addition to 30 ml of 20% ethanol through the machine for column storage.


  6. Liposome leakage assay

    1. The quality of the liposomes should be tested before analyzing the protein of interest by adding 2% (v/v) Triton X-100, which completely lyses the liposomes, quickly revealing a strong fluorescence signal.

    2. Set fluorometer excitation to 350 nm and record emissions at 520 nm and continue to measure at 1-second intervals for 5-10 min.

    3. Cross polarizers are applied at the excitation and emission paths to reduce background. If necessary, apply a 515 nm long-pass filter at the emission path to reduce background scatter.

    4. If available, connect a water bath to the water inlet and outlet of the sample chamber to maintain a constant temperature. This is especially important with proteins that interact at the physiological temperature, as is the case with EsxAB.

    5. To a UV cuvette, add 1.15 ml of gel filtration buffer, 150 µl of 1 M NaAc buffer (pH 4), and 100 µl of liposomes.

    6. Place the sample cuvette inside the sample chamber and close the lid.

    7. After approximately 30 s, use a gel loading pipette tip to inject 100 µl of 2% Triton X-100 into the cuvette through the hole on the cover lid. The ANTS fluorescence should increase at least 5-fold (Figure 3). This will serve as a positive control.

      1. The fluorescence intensity will drop for a second or two due to the dilution of the mix inside the cuvette, but it will begin to rise shortly after.

      2. A negative control consists of the sample prepared with liposomes and the gel filtration buffer.

    8. Continue experimenting by testing cytolytic proteins; prepare the cuvette as in Step F5 (Figure 3).



      Figure 3. Raw plots of an ANTS/DPX assay. The background fluorescence intensity is approximately 100,000 units. The fluorescence intensity increased 5-fold after adding 2% Triton X-100. With EsxA, a protein known to have MPA, we observed a gradual increase in fluorescence. EsxB, a protein without MPA, did not increase fluorescence intensity. The negative control consisting of the only gel filtration buffer did not increase fluorescence intensity.


    9. Place the UV cuvette in the sample chamber and close the lid.

    10. After about 30 s, use a gel loading pipette tip to add 100 µl of protein into the cuvette.

      1. Protein concentrations can vary, but a total of 100 µg is usually used in our experiments.

      2. Depending on the nature of the experiment, the protein can be loaded into the cuvette instead of the pH 4 buffer. Then, after 30 s, adding the pH 4 buffer will cause EsxAB to lyse the membrane.

Data analysis

  1. The fluorescence intensity is normalized at the time point the protein was added by subtracting the lowest intensity value from all other values, removing the background fluorescence.

  2. The lowest intensity value is now deemed the measurement at T = 0, and all values from T = 0 until the end of the experiment are averaged for all replicates.

    The dilution of the lipids by adding the sample results in a small drop in fluorescence. Measuring from the moment the protein is added allows determination of the increase in fluorescence as the liposomes are lysed.

  3. The average intensity is graphed against the time to obtain a curve (Figure 4A).

    The plateau followed by a one-phase association can also be used to obtain an association constant.

  4. The average of the highest fluorescence intensity values for each sample are graphed in a bar graph (Figure 4B).



    Figure 4. Plot of sample liposome experiment after normalization. A. The data were normalized to remove background fluorescence and to account for the drop in fluorescence caused by the dilution of the liposomes by the sample. B. The peak fluorescence was graphed with error bars representing the standard deviation. Statistical significance is represented here as well.


  5. The values are tested for normality using the Shapiro-Wilk test.

  6. If the data fails to differ from normal, it is tested for significance with a One-way ANOVA; otherwise, a Tukey-Kramer test is performed.

Recipes

  1. Gel filtration buffer

    20 mM Tris, pH 7.4

    10 mM NaCl

  2. pH 4 Buffer

    1 M NaAC, pH 4.01

Acknowledgments

This study was supported by grants from NIGMS (SC1GM095475 to J. Sun), National Center for Research Resources (5G12RR008124), and National Institute on Minority Health and Health Disparities (G12MD007592). Javier Aguilera was supported by a National Institutes of Health Grant (R25GM069621 to R. Aguilera) via the RISE program for graduate students. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

    This protocol was adapted from Nieva et al.,1989 (DOI: 10.1021/bi00444a032) and optimized for the EsxA/EsxB complex.

Competing interests

Authors have no competing interests to declare.

References

  1. Aguilera, J., Karki, C. B., Li, L., Vazquez Reyes, S., Estevao, I., Grajeda, B. I., Zhang, Q., Arico, C. D., Ouellet, H. and Sun, J. (2020). Nα-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence. J Biol Chem 295(17): 5785-5794.
  2. Alouf, J. E., Ladant, D., and Popoff, M. R. (2005). The comprehensive sourcebook of bacterial protein toxins. 3rd Edition. Academic Press. ISBN: 9780080456980.
  3. Bischofberger, M., Gonzalez, M. R. and van der Goot, F. G. (2009). Membrane injury by pore-forming proteins. Curr Opin Cell Biol 21(4): 589-95.
  4. Chatterjee, S. N. and Agarwal, S. (1988). Liposomes as membrane model for study of lipid peroxidation. Free Radic Biol Med 4(1): 51-72.
  5. De Leon, J., Jiang, G., Ma, Y., Rubin, E., Fortune, S. and Sun, J. (2012). Mycobacterium tuberculosis ESAT-6 exhibits a unique membrane-interacting activity that is not found in its ortholog from non-pathogenic Mycobacterium smegmatis. J Biol Chem 287(53): 44184-91.
  6. Jacquez, P., Lei, N., Weigt, D., Xiao, C. and Sun, J. (2014). Expression and purification of the functional ectodomain of human anthrax toxin receptor 2 in Escherichia coli Origami B cells with assistance of bacterial Trigger Factor.Protein Expr Purif 95: 149-155.
  7. Ma, Y., Keil, V. and Sun, J. (2015). Characterization of Mycobacterium tuberculosis EsxA membrane insertion: roles of N- and C-terminal flexible arms and central helix-turn-helix motif. J Biol Chem 290(11): 7314-22.
  8. Nieva, J. L., Goni, F. M. and Alonso, A. (1989). Liposome fusion catalytically induced by phospholipase C. Biochemistry 28(18): 7364-7.
  9. Peng, X., Jiang, G., Liu, W., Zhang, Q., Qian, W. and Sun, J. (2016). Characterization of differential pore-forming activities of ESAT-6 proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis. FEBS Lett 590(4): 509-19.
  10. Ray, S., Vazquez Reyes, S., Xiao, C. and Sun, J. (2019). Effects of membrane lipid composition on Mycobacterium tuberculosis EsxA membrane insertion: A dual play of fluidity and charge. Tuberculosis (Edinb) 118: 101854.
  11. Ruiz-Argüello, M. B., Basanez, G., Goni, F. M. and Alonso, A. (1996). Different effects of enzyme-generated ceramides and diacylglycerols in phospholipid membrane fusion and leakage. J Biol Chem 271(43): 26616-21.
  12. Sun, J. and Collier, R. J. (2010). Disulfide bonds in the ectodomain of anthrax toxin receptor 2 are required for the receptor-bound protective-antigen pore to function. PLoS One 5(5): e10553.
  13. Sun, J., Lang, A. E., Aktories, K. and Collier, R. J. (2008). Phenylalanine-427 of anthrax protective antigen functions in both pore formation and protein translocation. Proc Natl Acad Sci U S A 105(11): 4346-51.
  14. Sun, J., Vernier, G., Wigelsworth, D. J. and Collier, R. J. (2007). Insertion of anthrax protective antigen into liposomal membranes: effects of a receptor. J Biol Chem 282(2): 1059-65.
  15. Vazquez-Reyes, S., Ray, S., Aguilera, J., Sun, J. 2020) Development of a new liposome model that enhances membrane permeabilizing activity of Mycobacterial tuberculosis EsxAB heterodimer.
  16. Zhang, Q., Wang, D., Jiang, G., Liu, W., Deng, Q., Li, X., Qian, W., Ouellet, H. and Sun, J. (2016). EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5.Sci Rep 6: 32618.

简介

[摘要]在多种生物中都发现了成孔毒素(PFT s)。其功能是对基本的生存或致病许多种。PFT经常与脂质膜相互作用。大单层囊泡(LUV),也被称为脂质体,已被广泛用作可靠膜模型小号用于测试的PFT活动。脂质体在大小,脂质组成, 和装载货物。结合了荧光染料/淬灭剂对,8-氨基萘-1,3,6-三磺酸(ANTS)和对二甲苯双-吡啶溴化物(DPX),在脂质体中是一种有效的方法用于测量膜渗漏。当ANTS和DPX封装在脂质体中时,ANTS的荧光会被DPX淬灭。然而,脂质体完整性的破坏和随后的泄漏导致ANTS发出可测量的荧光。在这里,我们报告我们的最佳协议脂质体的测量电准备ING荧光脱猝灭脂质体渗漏。


[背景]成孔毒素(的PFT)是由主机入侵微生物病原体的毒力产生因子家族(Alouf等人。,2005)。的PFT的主要作用是形式ING孔上脂膜,造成膜的裂解和/或易位其他效应蛋白进入宿主细胞(Bischofberger等人。,2009)。脂质体小号公顷VE长期用作生物膜模型用于蛋白质-膜相互作用小号由于它们在大小,脂质组成极大的适应性,和装载货物(Chatterjee的和瓦尔,1988)。使用脂质体来测量孔的形成或膜渗漏的一种常见方式是封装离子或fluorescen吨脂质体内染料秒。ü PON的脂质体破裂,所述释放d离子或fluorescen吨染料EMIT可测量信号。我们首先使用一个ķ +释放测定以测量在脂质体膜孔形成小号通过的炭疽毒素(太阳等人,2007。和2008; Sun和科利尔,2010)。以后,荧光染料和淬灭剂对ANTS / DPX Ç一个ught (我们的注意力Nieva等人。,1989; Ruiz-阿圭略等人。,1996)。相比与所述ķ +释放测定,这需要一个庞大的ķ +探针和昂贵探针膜的频繁变化,所述ANTS / DPX去淬灭测定功能更高的灵敏度,更准确和一致的测量小号,和更低的成本。因此,在过去的十几年,我们已经转移到了蚂蚁/ DPX脱猝灭法。与此测定中,我们已经成功地测量到的膜透活性(MPA)的结核分枝杆菌(Mtb的)的毒力因子。这两种效应蛋白,6-kDa早期分泌抗原靶标(ESAT-6)或EsxA和10-kDa培养滤液蛋白(CFP-10)或EsxB已通过各种实验设计和设置进行了测试(De Leon等,2012)。 ; Ma等人,2015; Peng等人,2016; Zhang等人,2016; Aguilera等人,2020; Ray等人,20 19 )。EsxAB是一种异二聚体,与巨噬细胞的吞噬膜穿透有关,允许Mtb易位到细胞质中(De Leon等,2012; Ma等,2015; Zhang等,2016; Aguilera等。(2020年)。最近,我们已经确定了新的脂质成分的脂质体小号,大大提高了MPA的EsxA (雷等人,2019;巴斯克斯-雷耶斯等,2020)。在这里,我们用我们的新脂质组合物详细描述了基于荧光猝灭的脂质体泄漏测定法。

关键字:荧光淬灭, 脂质体渗漏, ANTS, DPX, EsxA, EsxB, 结核杆菌



材料和试剂


带PTFE螺帽的螺口玻璃小瓶(Spectrum Chemical,目录号:985-92215)
比色杯旋转杆(VWR,货号:58949-030)
石英UV比色皿(Fireflysci ,目录号:1FLUV10)
515 nm滤光片(Andover Corporation,目录号:515FG05-50S)
1 ml注射器(BD,货号:309659)
9英寸巴斯德吸管(Fisher Scientific,产品目录号13-678-6b)
Saint-Gobain Tygon管(Saint-Gobain,目录号:ACF00017F)
玻璃管架(Fisher Scientific,目录号:K7492100100)
的HiTrap ®脱盐柱(Millipore公司-Sigma,目录号:GE17-1408-01)
凝胶上样移液器吸头(Fisherbrand ,目录号02-707-139 )
10毫米滤镜支架(Avanti Polar Lipids,货号:610014)
0.2 µm膜(Whatman,目录号:800281)
铝箔(Fisherbrand ,目录号01-213-100)
1 ml样品定量环(Thermo Scientific,目录号:03-052-382)
氯仿(Fisher C hemical,目录号:c298)
1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)(Avanti极性脂质,目录号:850375)
1,2-二油酰基-sn-甘油-3-[(N-(5-氨基-1-羧基戊基)亚氨基二乙酸)琥珀酰](镍盐)(18:1 DGS-NTA(Ni)(Avanti极性脂质,目录编号:790404)
1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(钠盐)(POPG)(Avanti极性脂质,目录号:840457)
1-棕榈酰基-2-油酰基-甘油-3-磷酸胆碱(POPC)(Avanti极性脂质,目录号:850457)
乙醇(Fisher Chemical,目录号:a4094)
HPLC级水(Fisher Chemical,目录号:W5-4)
Tris-base(Fisher Chemical,目录号:BP152)
氯化钠(Fisher Chemical,目录号:BP358)
氢氧化钠(Fisher Chemical,目录号:S318)
盐酸(Fluka ,目录号:7647)
干冰
4-(2-羟乙基)哌嗪-1-乙磺酸,N-(2-羟乙基)哌嗪-N'-(2-乙磺酸)(HEPES)(Fisher B代理试剂,目录号:BP310)
8-氨基萘-1,3,6-三磺酸二钠盐(Setareh Biotech,目录号:6951)
对二甲苯二溴化吡啶鎓(DPX)(Setareh Biotech,目录号:6271)
Triton X - 100 (Sigma-Aldrich,目录号:X100)
醋酸钠(Sigma-Aldrich,目录号:S2889)
甲醇(Fisher,目录号:A453-500)
凝胶过滤缓冲液(请参阅食谱)
pH 4缓冲液(请参见配方)


设备


玻璃管(VWR,货号:89000-502 )
50毫升烧杯(派热克斯(Pyrex),货号:02-540G)
实验室安装架(Humboldt MFG Company,目录号:H-21217)
氮气储气罐
气罐调节器(VWR,目录号:55850-277)
大型顶部干燥器(Corning Life Sciences,目录号:CLS3120250)
热板/搅拌器(Thermo Fisher Scientific,目录号:SP131325)
带有加热块的挤出机套件(Avanti Polar Lipids,目录号:610000)
pH计(梅特勒-托利多(Mettler Toledo),目录号:30266626)
涡流(VWR,货号:58816-121)
秤(梅特勒-托利多,目录号:ML54)
AKTA FPLC系统(通用电气,目录号:UPC-900和P920)
荧光计(ISS,目录号:K2)
水浴(VWR,目录号:13271-086)


程序


干燥磷脂
如果使用脂质粉,请创建脂质储备液。
用螺帽将50-150 mg的脂质测量到玻璃小瓶中。
根据需要添加5-15毫升3:2氯仿:甲醇,以将浓度保持在10毫克/毫升,以便于使用。
注意:用衬有PTFE的盖子盖住样品瓶;否则,氯仿可能会溶解塑料并污染样品。


在玻璃管中测量适当量的脂质(10 mg足以进行约30个反应)。
注意:脂质体可以由单一的脂质形式小号[例如,1,2- Dioleoyl- SN -glycero -3-磷酸胆碱(DOPC),1-棕榈酰-2-油酰-sn-甘油-3-磷酸(1 “-rac甘油)(钠盐)(POPG) ,和1-棕榈酰-2-油酰-甘油-3-磷酸胆碱(POPC)]或它们的组合。例如,Ni 2+螯合脂质,1,2-二油酰基-sn-甘油-3-[(N-(5-氨基-1-羧基戊基)亚氨基二乙酸)琥珀酰](镍盐)或(DGS-NTA (Ni)),可以将带有His标签的蛋白质附着到膜上(Sun等,2007和2008; Jacquez等,2014)。为测量EsxAB的MPA而开发的新型脂质体由POPC与POPG组成,摩尔比为4:1。


使用钳座将小瓶固定到位。
使用第二个夹具,将Pasteur移液器放置在距离试管中液体约2-5 mm的位置。
连接的管道巴斯德移液管端,打开阀门,让柔和的气流中。
从小瓶中蒸发出氯仿,直到看不到液体为止。
从支架上取下样品瓶,并将其放在室温下的真空室内至少3小时或过夜。
将干脂质储存在-20 ℃。在这些条件下,脂质最多可以保存一个月。


准备色谱柱和FPLC进行脱盐
1.需要5毫升的脱盐柱以去除多余的ANTS / DPX和交换缓冲液。如有必要,可以串联连接两个脱盐柱,以提高脱盐效果。     

2.通行证至少5倍柱体积(CV)或,在这种情况下,通过该柱50ml的水,以除去所述乙醇从该柱。     

3.使至少5 CV (50 ml )的凝胶过滤缓冲液通过色谱柱。     

4.使机器以5毫升/分钟的速度通入50毫升水,以准备进行脱盐处理。     

5.以5 ml / min的速度使50 ml的凝胶过滤缓冲液通过机器。     

6.将色谱柱连接到机器上,并通过100 ml的凝胶过滤缓冲液。     

7.注入1 ml的凝胶过滤缓冲液清洁注入阀。     

8.重复步骤B 7至少两次。     



Prepar荷兰国际集团的脂质蚂蚁/ DPX解决方案
通过向> 95%的乙醇中添加干冰,在烧杯中制备冷冻溶液。冷冻溶液用于使包含ANTS / DPX的脂质快速冷冻。可替代地,液体可以使用氮气,以闪烁冻结。理想的是将冷冻溶液保持在-50 °C以下。
用温水装满另一个烧杯,以解冻脂质。
将脂质重悬于5 mM HEPES (pH 7.4 )中,每10 mg脂质中应含1 ml脂质。
涡旋直至溶液完全悬浮。
测量该溶液的体积,并添加ANTS和DPX 50mM的到管中。
上下吹打混合,并用铝箔覆盖管。
将试管放入冷冻溶液中,使其冷冻。
将试管移入温水中以解冻脂质。
重复步骤C7-C8进行五个以上的冻结/解冻循环。
注:六˚F reeze /解冻循环是通常足以溶解脂肪。如果在挤出过程中出现问题,例如使液体无法通过膜的困难,请执行更多的冷冻/解冻循环。


通过挤出生产脂质体
组装的根据挤出机的制造商的规格。
制定1毫升的凝胶过滤缓冲液使用的注射器中的一个,并通过挤出机通过缓冲,直到液体被转移到其他注射器。
注意:您可能会感到一点阻力。我f无阻力,所以能够在膜已经在装配过程中移动或已经撕裂。


除去的从注射器缓冲器。
制定到1毫升脂质体到一个注射器的。
通过膜传递的脂质体,直到液体已经从一个针筒转移到另一个。
重复小号TEP D5 ,总共20次。
挤出后检查流体;它应该从改变一个不透明到透明的,黄色的色彩的液体(图1)。




图1的比较的前和挤出后的脂质溶液。一。挤出之前的脂质溶液具有不透明的颜色。乙。挤出后脂质溶液变得更透明。


将脂质分配到干净的玻璃管中。


通过脱盐色谱去除多余的荧光染料
在对柱子和机器进行预平衡并清洗进样环后,将脂质体注入进样环。
运行脱盐程序以0.5ml /分钟的流速,使用凝胶过滤缓冲液,并收集0.5每馏分毫升。
允许50 ml缓冲液通过色谱柱。
脂质体将柱洗脱出来后约2mL公顷VE通过。
通常在六个试管中收集含有脂质体的级分,以收集总计3 ml的脂质体(图2)。




图2.大多数流动大约2毫升缓冲液后的脂质将洗脱通过该柱。使用该程序,预期的典型产量为3 ml。蓝线表示mAu ,棕线表示电导。


合并所有脂质体样品,盖上铝箔,并在4°C下保存。
在这一阶段的脂质体可以存储约1-3周。
用这种方法制备的脂质体的浓度通常是一致的。但是,每个生物复制都需要使用具有相同批次脂质的技术复制来获得一致的结果。
通过使至少5CV的清洁柱+ 30毫升(该体积的机器)的水通过该机器。
通过至少五个CV除了30毫升20%乙醇的第ř ough机器为列存储。


脂质体泄漏测定
Ť脂质体的质量他应测试b安伏ANALY懋目的蛋白质中加入2%(V / V)Ť瑞通X - 100,其完全裂解脂质体,迅速揭示一个强荧光信号。
将荧光计的激发设置为350 nm,并记录520 nm的发射,并以1秒的间隔继续测量5-10分钟。
Ç罗斯两极分化RS在所施加的激发和发射路径,以减少背景。我˚F必要,应用515nm的长-pass滤波器在发射路径,以减少背景散射。
如果可用,水浴连接到水入口和出口的样品室的维持一个恒定的温度。这是与蛋白质,在相互作用是特别重要的生理温度,因为是与壳体EsxAB 。
向紫外线比色杯中,加入1.15 ml凝胶过滤缓冲液,150 µl 1 M NaAc缓冲液(pH 4)和100 µl脂质体。
将样品比色杯放入样品室中,然后合上盖子。
一个后pproximately 30秒,使用凝胶加样枪头注入100微升的2%Ť瑞通X - 100通过在盖盖在孔到试管。ANTS荧光应增加至少5倍(图3)。这将起到积极的控制作用。
荧光我ntensity将一个或两个第二次下降,由于试管内混合稀释,但它会开始后不久上涨。
阴性对照由小号样品的制备与脂质体和凝胶过滤缓冲液中。
通过测试溶细胞蛋白继续进行实验;制备的反应杯在小号TEP ˚F 5(图3)。




图3.原始情节小号的一个ANTS / DPX测定。背景荧光强度约为100,000个单位。加入2%T riton X -100后,荧光强度增加了5倍。随着EsxA ,已知的蛋白质具有MPA ,我们观察到逐渐增加的荧光。EsxB ,没有蛋白质MPA ,也不会增加荧光强度。自由以下组成的阴性对照的只凝胶过滤缓冲液并不能增加荧光强度。


将紫外线比色皿放入样品室中,然后盖上盖子。
经过约30秒,使用凝胶加样枪头添加100 μ蛋白的升中的比色皿TE 。
P rotein Ç oncentrations可以变化,但一共有100微克的是通常在我们的实验中使用。
根据实验的性质,可以将蛋白质而不是pH 4缓冲液加载到比色杯中。然后,在30秒钟后,添加pH 4缓冲液将导致EsxAB裂解膜。


数据一nalysis


通过从所有其他值中减去最低强度值,除去背景荧光,在添加蛋白质的时间点对荧光强度进行标准化。
最低强度值现在被视为测量在T = 0 ,并且在T的所有值= 0直到的端部的试验的平均,所有重复。
通过添加样品来稀释脂质会导致荧光的少量下降。中号从添加了蛋白质的瞬间easuring允许determin的通货膨胀的荧光的增加为脂质体裂解。


平均强度作图针对该时间获得的曲线(图4甲)。
该p lateau接着是一个-相关联也可以被用来获得的缔合常数。


每个样品的最高荧光强度值的平均值以条形图绘制(图4B)。




图4 。标准化后的样品脂质体实验图。一。数据进行归一化,以除去背景荧光和以考虑了在荧光下降引起的稀释的由脂质体的样品。乙。用表示标准偏差的误差线对荧光峰进行绘图。统计意义也显示在这里。


使用Shapiro-Wilk检验测试这些值的正态性。
如果数据与正常值不同,则使用单向方差分析对其进行显着性检验。否则,执行Tukey-Kramer测试。


菜谱


凝胶过滤缓冲液
20 mM Tris,pH 7.4


10毫米氯化钠


pH 4缓冲液
1 M NaAC ,pH 4.01


致谢


钍是研究是由赠款NIGMS(SC1GM095475到J.日),国家研究资源中心(5G12RR008124)的支持,以及对少数民族健康与健康差距(G12MD007592)研究所。哈维尔·阿奎莱拉(Javier Aguilera)由美国国立卫生研究院(R25GM069621授予R. Aguilera)通过RISE计划为研究生提供了支持。内容仅由作者承担,并不一定代表美国国立卫生研究院的正式观点。


该协议改编自Nieva等人。,1989(DOI:10.1021 / bi00444a032)并针对EsxA / EsxB复合体进行了优化。


竞争我nterests


作者没有竞争利益要声明。


参考


Aguilera,J.,Karki,CB,Li,L.,Vazquez Reyes,S.,Estevao ,I.,Grajeda ,BI,Zhang,Q.,Arico ,CD,Ouellet,H.和Sun,J.(2020) 。Ñ α的毒力因子的-Acetylation EsxA需要分枝杆菌胞质易位和毒力。生物化学杂志295(17):5785-5794。
Alouf ,JE,Ladant ,D。,和Popoff,MR(2005)。细菌蛋白毒素综合资料手册。第三版。学术出版社。国际标准书号(ISBN):9780080456980 。
Bischofberger,M.,MR Gonzalez和FG van der Goot (2009)。造孔蛋白对膜的伤害。Curr Opin Cell Biol 21(4):589-95。
Chatterjee,SN和Agarwal,S.(1988年)。脂质体作为膜模型用于脂质过氧化的研究。Free Radic Biol Med 4(1):51-72。
De Leon,J.,Jiang,G.,Ma,Y.,Rubin,E.,Fortune,S. and Sun,J.(2012年)。结核分枝杆菌ESAT-6表现出独特的膜相互作用活性,这是非致病性耻垢分枝杆菌在其直系同源物中未发现的。生物化学杂志287(53):44184-91。
Jacquez,P.,Lei,N.,Weigt ,D.,Xiao,C. and Sun,J.(2014年)。借助于细菌触发因子,人炭疽毒素受体2的功能胞外域在大肠杆菌Origami B细胞中的表达和纯化。Protein Expr Purif 95:149-155。
Ma,Y.,Keil,V.和Sun,J.(2015)。结核分枝杆菌EsxA膜插入的表征:N和C末端柔性臂和中央螺旋-转-螺旋基序的作用。生物化学杂志290(11):7314-22。
Nieva ,JL,Goni ,FM和Alonso,A。(1989)。磷脂酶C催化诱导的脂质体融合。生物化学28(18):7364-7。
彭X.,姜G.,刘W.,张Q.,钱W.和孙J.(2016)。表征结核分枝杆菌和耻垢分枝杆菌的ESAT-6蛋白的不同成孔活性。FEBS Lett 590(4):509-19。
Ray,S.,Vazquez Reyes,S.,Xiao,C.和Sun,J.(2019年)。膜脂质成分对结核分枝杆菌EsxA膜插入的影响:流动性和电荷的双重作用。结核病(Edinb )118:101854。
Ruiz- Argüello ,MB,Basanez ,G.,Goni ,FM和Alonso,A。(1996)。酶产生的神经酰胺和二酰基甘油在磷脂膜融合和渗漏中的不同作用。生物化学杂志271(43):26616-21。
Sun,J.和Collier,RJ(2010)。炭疽毒素受体2胞外域中的二硫键是受体结合的保护性抗原孔发挥功能所必需的。PLoS One 5(5):e10553。
Sun,J.,Lang,AE,Aktories ,K.和Collier,RJ(2008)。炭疽保护性抗原的苯丙氨酸427在孔形成和蛋白质转运中均起作用。PROC国家科科学院科学USA 105(11):4346-51。
Sun,J.,Vernier,G.,Wigelsworth ,DJ和Collier,RJ(2007)。炭疽保护性抗原插入脂质体膜:受体的作用。生物化学杂志282(2):1059至1065年。
Vazquez-Reyes,S.,Ray,S.,Aguilera,J.,Sun,J.2020 )开发了一种新的脂质体模型,该模型增强了结核分枝杆菌EsxAB异二聚体的膜透化活性。
Zhang,Q.,Wang,D.,Jiang,G.,Liu,W.,Deng,Q.,Li,X.,Qian,W.,Ouellet,H.和Sun,J.(2016)。EsxA膜透化活性在分枝杆菌胞质转运和毒力中起关键作用:谷氨酰胺5的单残基突变的影响。Sci Rep 6:32618。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Aguilera, J., Vazquez-Reyes, S. and Sun, J. (2021). A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins. Bio-protocol 11(10): e4025. DOI: 10.21769/BioProtoc.4025.
  2. Aguilera, J., Karki, C. B., Li, L., Vazquez Reyes, S., Estevao, I., Grajeda, B. I., Zhang, Q., Arico, C. D., Ouellet, H. and Sun, J. (2020). Nα-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence. J Biol Chem 295(17): 5785-5794.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。