Imaging cytosolic translocation of Mycobacteria with two-photon fluorescence resonance energy transfer microscopy

Yassel Acosta; Qi Zhang; Arifur Rahaman; Hugues Ouellet; Chuan Xiao; Jianjun Sun; Chunqiang Li

doi:10.1364/BOE.5.003990

Imaging cytosolic translocation of Mycobacteria with two-photon fluorescence resonance energy transfer microscopy

Biomed Opt Express. 2014 Oct 20;5(11):3990-4001. doi: 10.1364/BOE.5.003990. eCollection 2014 Nov 1.

Authors

Yassel Acosta¹, Qi Zhang², Arifur Rahaman³, Hugues Ouellet⁴, Chuan Xiao⁵, Jianjun Sun⁶, Chunqiang Li³

Affiliations

¹ Department of Physics, The University of Texas at El Paso, 500 W. University Ave., El Paso, Texas 79968, USA ; These authors contribute equally.
² Department of Biological Sciences, The University of Texas at El Paso, 500 W. University Ave., El Paso, Texas 79968, USA ; These authors contribute equally.
³ Department of Physics, The University of Texas at El Paso, 500 W. University Ave., El Paso, Texas 79968, USA.
⁴ Department of Biological Sciences, The University of Texas at El Paso, 500 W. University Ave., El Paso, Texas 79968, USA.
⁵ Department of Chemistry, The University of Texas at El Paso, 500 W. University Ave., El Paso, Texas 79968, USA.
⁶ Department of Biological Sciences, The University of Texas at El Paso, 500 W. University Ave., El Paso, Texas 79968, USA ; jsun@utep.edu.

Abstract

Transition from latency to active tuberculosis requires Mycobacterium tuberculosis (Mtb) to penetrate the phagosomal membrane and translocate to the cytosol of the host macrophage. Quantitative two-photon fluorescence resonance energy transfer (FRET) microscopy is developed to measure cytosolic translocation using Mycobacterium marinum (Mm) as a model organism for Mtb. Macrophages were infected with Mm or non-pathogenic Mycobacterium smegmatis (Ms) as a control, then loaded with a FRET substrate. Once translocation occurs, mycobacterium-bearing β-lactamase cleaves the substrate, resulting in decrease of FRET signal. Quantification of this FRET signal change revealed that Mm, but not Ms, is capable of translocating to the cytosol.

Keywords: (170.1530) Cell analysis; (170.2520) Fluorescence microscopy.