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

Javier Aguilera; Salvador  Vazquez-Reyes; Jianjun Sun

doi:10.21769/BioProtoc.4025

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

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

JA Javier Aguilera

SV Salvador Vazquez-Reyes

JS Jianjun Sun email

发布: 2021年05月20日第11卷第10期 DOI: 10.21769/BioProtoc.4025 浏览次数: 5994

评审: Guangzhi ZhangHimanshu SharmaShailesh Kumar

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Biological Chemistry, featuring study using the protocol.

Apr 2020

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

磷酸肌醇包衣颗粒结合试验

Manuel Gálvez-Santisteban [...] Fernando Martin-Belmonte

2014年02月05日 12205 阅读

表面等离子共振 (SPR)法分析蛋白脂质的相互作用

Olga Lucia Baron and David Pauron

2014年09月20日 13522 阅读

革兰阴性菌中内外膜通透性的同期测定

Bo Ma [...] Zheng Hou

2020年03月05日 5899 阅读

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)

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

Screw cap glass vials with PTFE screwcap (Spectrum Chemical, catalog number: 985-92215)
Cuvette spinbar (VWR, catalog number: 58949-030)
Quartz UV cuvette (Fireflysci, catalog number: 1FLUV10)
515 nm filter (Andover Corporation, catalog number: 515FG05-50S)
1 ml syringe (BD, catalog number: 309659)
9’’ Pasteur pipet (Fisher scientific, catalog number 13-678-6b)
Saint-Gobain Tygon tubing (Saint-Gobain, catalog number: ACF00017F)
Glass tube rack (Fisher Scientific, catalog number: K7492100100)
HiTrap^® desalting columns (Millipore-Sigma, catalog number: GE17-1408-01)
Gel loading pipet tips (Fisherbrand, catalog number: 02-707-139)
10 mm filter supports (Avanti Polar Lipids, catalog number: 610014)
0.2 µm membrane (Whatman, catalog number: 800281)
Aluminum foil (Fisherbrand, catalog number: 01-213-100)
1 ml sample loop (Thermo Scientific, catalog number: 03-052-382)
Chloroform (Fisher Chemical, catalog number: c298)
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, catalog number: 850375)
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)
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) (Avanti Polar Lipids, catalog number: 840457)
1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, catalog number: 850457)
Ethanol (Fisher Chemical, catalog number: a4094)
HPLC grade water (Fisher Chemical, catalog number: W5-4)
Tris-base (Fisher Chemical, catalog number: BP152)
Sodium chloride (Fisher Chemical, catalog number: BP358)
Sodium hydroxide (Fisher Chemical, catalog number: S318)
Hydrochloric acid (Fluka, catalog number: 7647)
Dry ice
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) (Fisher Bioreagents, catalog number: BP310)
8-Aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (Setareh Biotech, catalog number: 6951)
p-Xylene-bis-pyridinium bromide (DPX) (Setareh Biotech, catalog number: 6271)
Triton X-100 (Sigma-Aldrich, catalog number: X100)
Sodium acetate (Sigma-Aldrich, catalog number: S2889)
Methanol (Fisher, catalog number: A453-500)
Gel filtration buffer (see Recipes)
pH 4 buffer (see Recipes)

Equipment

Glass tubes (VWR, catalog number: 89000-502)
50 ml beaker (Pyrex, catalog number: 02-540G)
Lab mounting stand (Humboldt MFG Company, catalog number: H-21217)
Nitrogen gas tank
Gas tank regulator (VWR, catalog number: 55850-277)
Large top desiccator (Corning Life Sciences, catalog number: CLS3120250)
Hot plate/stirrer (Thermo Fisher Scientific, catalog number: SP131325)
Extruder set with heating block (Avanti Polar Lipids, catalog number: 610000)
pH meter (Mettler Toledo, catalog number: 30266626)
Vortex (VWR, catalog number: 58816-121)
Scale (Mettler Toledo, catalog number: ML54)
AKTA FPLC system (General Electric, catalog number: UPC-900 and P920)
Fluorometer (ISS, catalog number: K2)
Water bath (VWR, catalog number: 13271-086)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

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.
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.