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Published on: February 23, 2021
PerSort Facilitates Characterization and Elimination of Persister Subpopulation in Mycobacteria
Vivek Srinivas1, Mario L Arrieta-Ortiz1, Amardeep Kaur1
1Institute for Systems Biology, Seattle, Washington, USA.
Abstract:
Mycobacterium tuberculosis (MTB) generates phenotypic diversity to persist and survive the harsh conditions encountered during infection. MTB avoids immune effectors and antibacterial killing by entering into distinct physiological states. The surviving cells, persisters, are a major barrier to the timely and relapse-free treatment of tuberculosis (TB). We present for the first time, PerSort, a method to isolate and characterize persisters in the absence of antibiotic or other pressure. We demonstrate the value of PerSort to isolate translationally dormant cells that preexisted in small numbers within Mycobacterium species cultures growing under optimal conditions but that dramatically increased in proportion under stress conditions. The translationally dormant subpopulation exhibited multidrug tolerance and regrowth properties consistent with those of persister cells. Furthermore, PerSort enabled single-cell transcriptional profiling that provided evidence that the translationally dormant persisters were generated through a variety of mechanisms, including vapC30, mazF, and relA/spoT overexpression. Finally, we demonstrate that notwithstanding the varied mechanisms by which the persister cells were generated, they converge on a similar low-oxygen metabolic state that was reversed through activation of respiration to rapidly eliminate persisters fostered under host-relevant stress conditions. We conclude that PerSort provides a new tool to study MTB persisters, enabling targeted strategies to improve and shorten the treatment of TB.IMPORTANCE Mycobacterium tuberculosis (MTB) persists and survives antibiotic treatments by generating phenotypically heterogeneous drug-tolerant subpopulations. The surviving cells, persisters, are a major barrier to the relapse-free treatment of tuberculosis (TB), which is already killing >1.8 million people every year and becoming deadlier with the emergence of multidrug-resistant strains. This study describes PerSort, a cell sorting method to isolate and characterize, without antibiotic treatment, translationally dormant persisters that preexist in small numbers within Mycobacterium cultures. Characterization of this subpopulation has discovered multiple mechanisms by which mycobacterial persisters emerge and unveiled the physiological basis for their dormant and multidrug-tolerant physiological state. This analysis has discovered that activating oxygen respiratory physiology using l-cysteine eliminates preexisting persister subpopulations, potentiating rapid antibiotic killing of mycobacteria under host-relevant stress. PerSort serves as a new tool to study MTB persisters for enabling targeted strategies to improve and shorten the treatment of TB.
Insights
Mycobacterium tuberculosis persisters, key to tuberculosis treatment failure, can now be isolated without antibiotics using the novel PerSort method. Activating respiration rapidly eliminates these dormant cells, offering new strategies to shorten TB treatment.
Area of Science:
- Microbiology
- Infectious Diseases
- Molecular Biology
Background:
- Mycobacterium tuberculosis (MTB) exhibits phenotypic diversity, forming persister cells that survive harsh conditions and antibiotic treatment.
- Persister cells are a significant obstacle to effective and relapse-free tuberculosis (TB) treatment, contributing to treatment failure and the rise of multidrug-resistant strains.
- Existing methods for persister isolation often require antibiotic pressure, limiting understanding of their natural emergence and characteristics.
Purpose of the Study:
- To develop and validate PerSort, a novel method for isolating and characterizing Mycobacterium tuberculosis persisters without antibiotic or other external pressure.
- To investigate the mechanisms underlying the generation of translationally dormant persister cells.
- To identify physiological targets for the rapid elimination of persister cells.
Main Methods:
- Development of PerSort, a cell sorting technique to isolate persister populations from Mycobacterium cultures.
- Characterization of isolated persisters, including assessment of translational dormancy, multidrug tolerance, and regrowth potential.
- Single-cell transcriptional profiling to elucidate the molecular mechanisms of persister formation.
- Investigation of metabolic states and potential therapeutic interventions for persister elimination.
Main Results:
- PerSort successfully isolated translationally dormant persister cells from Mycobacterium cultures under both optimal and stress conditions.
- These persisters exhibited multidrug tolerance and regrowth properties consistent with clinical observations.
- Single-cell analysis revealed diverse mechanisms of persister generation, including overexpression of genes like vapC30, mazF, and relA/spoT.
- Persisters converged on a low-oxygen metabolic state, which could be reversed by activating respiration (e.g., using L-cysteine) to enable rapid elimination.
Conclusions:
- PerSort is a valuable new tool for studying Mycobacterium tuberculosis persisters, enabling their isolation and characterization without antibiotic pressure.
- Understanding the diverse mechanisms and convergent metabolic state of persisters provides critical insights into their survival strategies.
- Targeting the respiratory metabolism of persister cells offers a promising strategy to enhance the efficacy and shorten the duration of tuberculosis treatment.
