Published: Vol 11, Iss 8, Apr 20, 2021 DOI: 10.21769/BioProtoc.3991 Views: 3029
Reviewed by: 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-AMBackground
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.
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Acknowledgments
The study is supported by the 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 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.
Competing interest
The authors have no competing interests to report.
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Category
Microbiology > Microbe-host interactions > Bacterium
Cell Biology > Cell-based analysis > Bacterial infection
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