用CCF4-AM FRET法检测RAW264.7巨噬细胞中海洋分枝杆菌的细胞质移位

Javier Aguilera; Jianjun Sun

doi:10.21769/BioProtoc.3991

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Measuring Cytosolic Translocation of Mycobacterium marinum in RAW264.7 Macrophages with a CCF4-AM FRET Assay

用CCF4-AM FRET法检测RAW264.7巨噬细胞中海洋分枝杆菌的细胞质移位

JA Javier Aguilera

JS Jianjun Sun email

发布: 2021年04月20日第11卷第8期 DOI: 10.21769/BioProtoc.3991 浏览次数: 3045

评审: Jason NeidlemanMei Po ChanAnonymous reviewer(s)

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Abstract

The CCF4-AM Förster resonance energy transfer (FRET) assay is a sensitive approach to measure bacterial cytosolic translocation in live cells. The FRET pair hydroxycoumarin (donor) and fluorescein (acceptor) are linked by a CCF4-AM β-lactam ring, the substrate of β-lactamase. The exogenously added, neutral charged-FRET reagent can diffuse across the membrane and stay in the cytosol only once it is charged in the cytosol. When bacteria translocate from subcellular organelles (e.g., phagosomes) to the cytosol, the bacteria-associated β-lactamase cleaves the β-lactam ring, resulting in loss of FRET signal. Here we describe the fluorometer-based approach optimized for direct measurement of cytosolic translocation as a result of the EsxAB complex of Mycobacterium marinum in RAW264.7 cells.

Keywords: CCF4-AM (CCF4-AM)

Mycobacterium marinum (海洋分支杆菌)

EsxA (EsxA)

EsxB (EsxB)

FRET (荧光共振能量转移)

Background

The development of a β-lactamase reporter assay gave rise to the subsequent Förster resonance energy transfer (FRET) assay (Zlokarnik et al., 1998; Cavrois et al., 2002). Two fluorescence dyes, hydroxycoumarin (donor) and fluorescein (acceptor), are linked via a CCF4-AM (CCF2-AM in earlier reports) β-lactam ring. When excited at 405 nm, hydroxycoumarin emits at 450 nm (blue), which subsequently excites fluorescein to emit at 527 nm (green). Cleavage of the β-lactam ring by bacteria-associated β-lactamase results in loss of FRET and direct emission of hydroxycoumarin (blue) (Jones and Padilla-Parra, 2016). The CCF4-AM FRET reporter assay is versatile and has been been used for a variety of different applications, such as tracking molecular fusions (Jones and Padilla-Parra, 2016; Shaikh, et al., 2019), tracking delivery of biological cargos to the cytosol (Stone et al., 2018), and monitoring translocation of bacteria within cells (Charpentier and Oswald, 2004). Since Mycobacterium marinum also possesses β-lactamase (Flores et al., 2005), Simeone and colleagues exploited the FRET assay to track cytosolic translocation of Mycobacterium tuberculosis in THP-1 cells (Simeone et al., 2012). Recently, we applied this method for investigation of the role of EsxAB in cytosolic translocation of Mycobacterium marinum (Acosta et al., 2014; Zhang et al., 2016; Aguilera et al., 2020). RAW264.7 cells were infected with M. marinum and the results were obtained under a fluorometer, providing a quick, quantitative method for analysis of results. Furthermore, as Mycobacterium marinum is a BSL-2 pathogen and contains similarities in its ESX-1 locus (Tobin and Ramakrishnan, 2008), it provides a safe, direct way to investigate the EsxAB complex. Here we describe our procedure for direct monitoring of cytosolic translocation of Mycobacterium marinum in RAW264.7 cells via the CCF4-AM FRET assay using a fluorometer.

Materials and Reagents

Inoculation loop (BD, catalog number: 220216)
Cell Scraper (Fisherbrand, catalog number: 08-100-241)
T-75 Flask (Thermo Scientific, catalog number: 12-566-440)
50 ml Centrifuge tube (Corning, catalog number: 352098)
6-well plate (Corning, catalog number: 3516)
10 ml syringe (Fisherbrand, catalog number: 14-955-459)
22 gauge needle (BD, catalog number: 305900)
27 gauge needle (BD, catalog number: 305109)
5 µm filter (Millex, catalog number: SLSV025LS)
Pasteur pipette (Fisherbrand, catalog number: 13-678-6b)
Aluminum Foil (Fisherbrand, catalog number: 01-213-102)
Thin Wall Cuvette (Fireflysci, catalog number: 30FL)
435 nm long-pass filter (Andover Corporation, catalog number: 435FG03-50S)
7H9 media (Difco, catalog number: 271310)
OADC Enrichment (BD, catalog number: 211886)
Tween-80 (Sigma-Aldrich, catalog number: P1754)
Kanamycin Monosulfate (Gold Bio, catalog number: K-120-5)
DMEM media (Gibco, catalog number: 10566-016)
Fetal Bovine Serum (Gibco, catalog number: A4766801)
Penicillin and Streptomycin (Gibco, catalog number: 15070063)
Phosphate buffered saline (Sigma-Aldrich, catalog number: P3813)
CCF4-AM FRET Loading Solutions (Life Technologies, catalog number: K1085)
Note: This kit contains Solution A, Solution B, and Solution C as described in the procedure.
DMSO (Sigma-Aldrich, catalog number: D8418)
4% Paraformaldehyde
NaCl (Fisherbrand, catalog number: 18-606-420)
MgCl (Fisherbrand, catalog number: 18-604-047)
CaCl₂ (Fisherbrand, catalog number: AA4428030)
KCl (Spectrum Chemical, catalog number: 18-605-507)
Glucose (Sigma-Aldrich, catalog number: G7021)
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid (HEPES)) (Fisher Bioreagents, catalog number: BP310)
Tris (Fisher Bioreagents, catalog number: BP152-500)
EM Media (see Recipes)

Equipment

Biosafety Cabinet (Labconco)
Vortex (VWR, catalog number: 58816-121)
Hemocytometer (Hausser Scientific, catalog number: S17040)
Inverted Light Microscope (VWR, catalog number: Vistavision)
Cell Imaging System (Life Technologies, catalog number: 4471136)
Spectrophotometer (Amersham Biosciences, model: Ultrospec 10)
Table Top Centrifuge (Eppendorf, catalog number: 5810R)
CO₂ Incubator (VWR, catalog number: 3078)
Fluorometer (ISS, catalog number: K2)
Incubator Shaker (VWR, catalog number: 1585)
Hot Plate/Stirrer (ThermoFisher Scientific, catalog number: SP131325)
pH meter (Mettler Toledo, catalog number: 30266626)

Procedure

English

中文翻译

文章信息

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如何引用

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

Aguilera, J. and Sun, J. (2021). Measuring Cytosolic Translocation of Mycobacterium marinum in RAW264.7 Macrophages with a CCF4-AM FRET Assay. Bio-protocol 11(8): e3991. DOI: 10.21769/BioProtoc.3991.
Aguilera, J., Karki, C. B., Li, L., Reyes, S. V., Estevao, I., Grajeda, B. I., Qi Zhang, 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.