Elucidating population-wide mycobacterial replication dynamics at the single-cell level.

Area of Science:

• Microbiology
• Infectious Diseases
• Cell Biology

Background:

• Mycobacterium tuberculosis infections present diverse clinical outcomes, often persisting in a latent, asymptomatic state.
• Persistent Mycobacterium tuberculosis infections are thought to originate from viable, non-replicating (VBNR) cells, which are antibiotic-tolerant and require extended treatment.
• Understanding these persistent mycobacteria is challenging due to their difficulty in isolation.

Purpose of the Study:

• To develop and apply a novel replication reporter system for Mycobacterium tuberculosis to investigate within-host bacterial population heterogeneity.
• To characterize intracellular Mycobacterium tuberculosis populations in macrophages and identify distinct bacterial subpopulations.

Main Methods:

• Development of a dual fluorescence reporter system utilizing constitutive and inducible reporters for tracking and measuring bacterial replication.
• Application of fluorescence single-cell analysis to study Mycobacterium tuberculosis within murine macrophages.
• Assessment of bacterial populations using d-cycloserine treatment to evaluate drug tolerance.

Main Results:

• Identification of a distinct subpopulation of non-growing Mycobacterium tuberculosis within murine macrophages.
• Observation of both VBNR and actively replicating Mycobacterium tuberculosis within the same macrophage after 48 hours of infection.
• Evidence suggesting macrophage uptake enriches for non- or slowly replicating bacteria, likely enriched for drug-tolerant persisters.

Conclusions:

• The novel dual fluorescence reporter system is successfully applied in vitro and in macrophage infection models.
• The system provides valuable insights into the heterogeneity of Mycobacterium tuberculosis populations, particularly the presence of non-replicating persister cells.
• These findings contribute to understanding the persistence mechanisms of Mycobacterium tuberculosis and may inform strategies for prolonged treatment.