Mtb-Timer: a fluorescent reporter to visualize Mycobacterium tuberculosis replication and antibiotic responses

Area of Science:

• Microbiology
• Molecular Biology
• Immunology

Background:

• Tuberculosis (TB) treatment is complicated by phenotypic heterogeneity in Mycobacterium tuberculosis (Mtb) populations.
• Existing tools for monitoring single Mtb replication and antibiotic responses are limited.
• Understanding bacterial growth dynamics is crucial for developing effective TB therapies.

Purpose of the Study:

• To develop and validate Mtb-Timer, a novel fluorescent reporter system for visualizing Mtb replication and antibiotic responses.
• To enable single-cell level monitoring of Mtb growth dynamics both in vitro and in cellulo.
• To quantify distinct bacterial subpopulations and monitor real-time changes during antibiotic treatment.

Main Methods:

• Development of an Mtb H37Rv strain expressing the Timer fluorescent protein (TimerMtb).
• Monitoring of Mtb replication using single-cell imaging and flow cytometry.
• Assessment of antibiotic responses (isoniazid, rifampicin, ethambutol, pyrazinamide) using Mtb-Timer in vitro and in primary human monocyte-derived macrophages (in cellulo).

Main Results:

• Mtb-Timer successfully monitored Mtb replication in vitro at the single-cell level.
• Exposure to first-line anti-TB antibiotics resulted in a dose-dependent green-to-red photoconversion.
• Intracellular Mtb populations in macrophages exhibited significant phenotypic heterogeneity, with Mtb-Timer revealing distinct subpopulations and antibiotic effects.

Conclusions:

• Mtb-Timer is a robust tool for monitoring Mycobacterium tuberculosis replication and antibiotic responses.
• The system is effective for both in vitro and in cellulo studies using imaging and flow cytometry.
• Mtb-Timer provides valuable insights into bacterial heterogeneity and drug efficacy in tuberculosis research.