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.
Highlights
It is estimated that one-third of the world’s population is latently infected with Mycobacterium tuberculosis (Mtb), and 5-10% of these latently infected individuals develop into active tuberculosis, which results in over 2 million deaths each year [1]
Macrophages were infected with Mm or non-pathogenic Mycobacterium smegmatis (Ms) as a control, loaded with a fluorescence resonance energy transfer (FRET) substrate
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
Summary
It is estimated that one-third of the world’s population is latently infected with Mycobacterium tuberculosis (Mtb), and 5-10% of these latently infected individuals develop into active tuberculosis, which results in over 2 million deaths each year [1]. After bacteria disrupt the phagosomal membrane, bacterial β–lactamase cleaves CCF4 at the cephalosporin core, which breaks the donor and the acceptor apart and results in loss of FRET signal. By monitoring this FRET signal change, researchers have studied the spatial and temporal progress in pathogeninduced vacuole rupture. The blue signal is deflected by the second dichroic beam splitter, and is transmitted through a 417-477 nm band-pass filter (FF02-447/60, Semrock, Rochester, NY), and is detected by a photomultiplier tube (PMT) (R10699, Hamamatsu, Bridgewater, NJ). Details of this microscope design can be found in reference [14]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
