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

Javier Aguilera; Jianjun Sun

doi:10.21769/BioProtoc.3991

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

Bio Protoc. 2021 Apr 20;11(8):e3991. doi: 10.21769/BioProtoc.3991.

Authors

Javier Aguilera¹, Jianjun Sun¹

Affiliation

¹ Department of Biological Sciences and Border Biomedical Research Center, 500 West University Avenue, University of Texas at El Paso, El Paso TX, USA.

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: EsxA; CCF4-AM; EsxB; FRET; Mycobacterium marinum.

Abstract

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