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A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Published on: January 17, 2014

A Fluorogenic Trehalose Probe for Tracking Phagocytosed Mycobacterium tuberculosis.

Tingting Dai¹, Jinghang Xie², Qihua Zhu^2,3,4

¹Department of Chemistry, Stanford University, Stanford, California 94305, United States.

Journal of the American Chemical Society

|August 20, 2020

Summary

Researchers developed a new fluorescent probe, CDG-Tre, to track live Mycobacterium tuberculosis (Mtb) bacteria within host cells. This tool aids in understanding tuberculosis pathogenesis and host-pathogen interactions.

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Area of Science:

Microbiology
Cell Biology
Infectious Diseases

Background:

Tuberculosis (TB) is a global epidemic caused by Mycobacterium tuberculosis (Mtb).
Tracking viable Mtb replication within macrophages is crucial for understanding host-pathogen interactions.
Existing tools lack specificity or ease of use for live Mtb tracking.

Purpose of the Study:

To develop a novel fluorogenic probe for real-time monitoring of live Mtb replication within host cells.
To enable visualization of Mtb bacilli during host-pathogen interactions.
To provide a tool for studying Mtb physiology and pathogenesis.

Main Methods:

Development of a cephalosphorinase-dependent green trehalose (CDG-Tre) fluorogenic probe.
Utilizing BlaC, a unique Mtb β-lactamase, for probe activation.
Incorporation of the fluorescent product into the Mtb cell wall via the trehalose metabolic pathway.
Assessing probe performance in labeling live Bacille Calmette-Guérin (BCG) within macrophages.

Main Results:

CDG-Tre enables fluorescence labeling of single live BCG cells within macrophages at concentrations as low as 2 μM.
The probe exhibits high selectivity for mycobacteria, distinguishing them from other Corynebacterineae species.
The fluorescent signal is generated upon BlaC activation and integrated into the bacterial cell wall.

Conclusions:

CDG-Tre is a versatile tool for tracking Mtb during both pre- and post-phagocytosis.
This probe facilitates the elucidation of fundamental physiological and pathological processes related to the Mtb mycomembrane.
The findings offer new avenues for studying Mtb infections and developing anti-TB strategies.