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

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

doi:10.1364/boe.5.003990

Cited by 26 publications

(20 citation statements)

References 24 publications

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“…Since the incident light in two-photon RET is lower in energy compared to RET, photo-destruction of living tissue can be circumvented. Therefore, biological applications of this process have arisen, including photodynamic therapy [153][154][155][156][157][158][159][160] and bioimaging [155,[160][161][162][163].…”

Section: Recent Ret Research Nanomaterials For Energy Transfermentioning

confidence: 99%

Resonance Energy Transfer: From Fundamental Theory to Recent Applications

2019

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Resonance energy transfer (RET), the transport of electronic energy from one atom or molecule to another, has significant importance to a number of diverse areas of science. Since the pioneering experiments on RET by Cario and Franck in 1922, the theoretical understanding of the process has been continually refined. This review presents a historical account of the post-Förster outlook on RET, based on quantum electrodynamics, up to the present-day viewpoint. It is through this quantum framework that the short-range, R −6 distance dependence of Förster theory was unified with the long-range, radiative transfer governed by the inverse-square law. Crucial to the theoretical knowledge of RET is the electric dipole-electric dipole coupling tensor; we outline its mathematical derivation with a view to explaining some key physical concepts of RET. The higher order interactions that involve magnetic dipoles and electric quadrupoles are also discussed. To conclude, a survey is provided on the latest research, which includes transfer between nanomaterials, enhancement due to surface plasmons, possibilities outside the usual ultraviolet or visible range and RET within a cavity.

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Section: Recent Ret Research Nanomaterials For Energy Transfermentioning

confidence: 99%

Resonance Energy Transfer: From Fundamental Theory to Recent Applications

2019

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“…Mycobacterial cytosolic translocation was measured by CCF-4 FRET assay as previously described (17,22,50). Briefly, RAW264.7 cells were plated in triplicate in a 6-well plate at a density of 2.5 x 10 6 cells/well.…”

Section: Ccf-4 Fret Assaymentioning

confidence: 99%

Nα-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence

Aguilera

Karki

et al. 2020

Journal of Biological Chemistry

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The Mycobacterium tuberculosis virulence factor EsxA and its chaperone EsxB are secreted as a heterodimer (EsxA:B) and are crucial for mycobacterial escape from phagosomes and cytosolic translocation. Current findings support the idea that for EsxA to interact with host membranes, EsxA must dissociate from EsxB at low pH. However, the molecular mechanism by which the EsxA:B heterodimer separates is not clear. In the present study, using liposome-leakage and cytotoxicity assays, LC-MS/MS–based proteomics, and CCF-4 FRET analysis, we obtained evidence that the Nα-acetylation of the Thr-2 residue on EsxA, a post-translational modification that is present in mycobacteria but absent in Escherichia coli, is required for the EsxA:B separation. Substitutions at Thr-2 that precluded Nα-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the host membrane, resulting in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamics simulations revealed that at low pH, the Nα-acetylated Thr-2 makes direct and frequent “bind-and-release” contacts with EsxB, which generates a force that pulls EsxB away from EsxA. In summary, our findings provide evidence that the Nα-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence.

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“…With pH-indicator dyes the total fluorescence emission is not as quantitative because the total increase in fluorescence might be a result of the gradual phagosomal acidification or the increase in the number of phagosomes formed161819202122. Moreover, bacterial species might escape the phagosome owing to their ability to withstand the acidic microenvironment, as shown with Förster Resonance Energy Transfer (FRET) experiments23. Thus phagocytic-based research is increasingly dependent on multiple fluorescence labels, full spectral measurements, complex data analysis, efficient sample-screening, fluorescence ratio-metric methods and combinations of spectrofluorometry, fluorescence microscopy and flow cytometry9131415172425.…”

mentioning

confidence: 99%

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

Houston

2017

Sci Rep

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Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

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

Cited by 26 publications

References 24 publications

Resonance Energy Transfer: From Fundamental Theory to Recent Applications

Resonance Energy Transfer: From Fundamental Theory to Recent Applications

Nα-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

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