Characterization and elimination of stochastically generated persister subpopulation in mycobacteria (original) (raw)

New Results

Summary

Mycobacterium tuberculosis (MTB) is able to persist in the host for long periods of time, even during antibiotic treatment. Eliminating persister cells, which are implicated as the primary reason for treatment failure, is essential for shortening TB treatment regimen. Here, we report a novel methodology, Per-Sort, to identify and sort miniscule numbers of translationally dormant mycobacterial cells within an isogenic mycobacterium population. Using Per-Sort we have discovered that translationally dormant cells pre-exist (under optimal growth conditions) as a fraction of a percent of isogenic mycobacterial cultures, suggesting they are generated stochastically as a bet hedging strategy. We show that this pre-existing translationally dormant subpopulation of cells are tolerant to antibiotics, small in size, low in oxidative metabolism, and expand in number upon nutrient starvation. Finally, through transcriptional profiling at single cell resolution, we’ve determined that the pre-existing persisters are a heterogeneous mix of vapC30, mazF, and relA/spoT overexpressing cells that are eliminated and sensitized to antibiotic killing through induction of respiration.

Highlights

We have developed a novel method, Per-Sort, to isolate a small proportion of translationally dormant cells that pre-exist in Mycobacterium spp. cultures growing under optimal conditions but dramatically increase in proportion under stressful conditions.
The pre-existing translationally dormant cells have lower oxygen consumption, significantly longer lag phase to initiate growth in nutrient rich medium, and high tolerance to >10x MIC concentrations of isoniazid (INH) and rifampicin (RIF), indicating they are a subpopulation of persister cells.
Single-cell expression profiling demonstrated that the persisters are a heterogenous mixture of toxin (VapC30 and MazF) and alarmone response (RelA/SpoT) expressing cells.
A shared outcome of high toxin and alarmone response is reduced cellular oxidative metabolism, which we demonstrate is reversed upon addition of L-cysteine to reduce the proportion of pre-existing persister cells and increase killing by INH and RIF.

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