GSE160767
PerSorted dim and lit cells of MSM-mEos2
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 pre-exist in small numbers within Mycobacterium spp. cultures growing under optimal conditions, but which dramatically increased in proportion under stress conditions. The translationally dormant subpopulation exhibited multidrug tolerance and regrowth properties consistent with 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. Overall design: Dim and lit cells (n= 300,000) were PerSorted into 1 mL TriZol having ~ 1000 bone marrow derived macrophage cells. RNA was extracted with phenol chloroform method described previously (PMID:30833303), rRNA was depleted with Illumina rRNA depletion kit, and mycobacterium RNA was enriched with Path-seq methodology described previously (PMID:30833303). A total of 100,240 probes were designed to cover M. smegmatis mc2155 (Agilent probe library ELID number S3206492).