PerSort facilitates characterization and elimination of persister subpopulation in mycobacteria

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 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.SummaryWe have developed a novel method, PerSort, to isolate translationally dormant cells that pre-exist in small numbers within Mycobacterium spp. cultures growing under naïve conditions (i.e., absence of antibiotic treatment), but dramatically increase in proportion under stress conditions. The translationally dormant cells have high tolerance to isoniazid and rifampicin, and can regenerate the parental population structure in standard media, albeit after a significantly longer lag phase, indicating they are persister cells. Single-cell expression profiling demonstrated that the translationally dormant persister subpopulation is a mixture of vapC30, mazF, and relA/spoT overexpressing cells, indicating there are multiple pathways to become a persister cell. Regardless of the mechanism by which they are generated, the persister cells have reduced oxidative metabolism, which is reversed upon addition of L-cysteine to effect complete clearance by INH and RIF under host-related stress.

Moxifloxacin-mediated killing of Mycobacterium tuberculosis involves respiratory downshift, reductive stress, and ROS accumulation

“…NAC also potentiated moxifloxacin lethality (5-to 7-fold) with nutrient-starved, dormant M. tuberculosis (Fig 6D). These data agree with recent results in which activation of oxidative metabolism and respiration, through addition of L-cysteine to nutrient-starved cultures, reduced the fraction of persister subpopulations and enhanced the in vitro lethal activity of isoniazid and rifampicin (Srinivas et al, 2020). As expected, a similar treatment with NAC failed to enhance moxifloxacin lethality under hypoxic conditions (Fig 6E), consistent with NAC functioning by inducing oxygen consumption and elevating ROS levels.…”

Protozoan persister-like cells and drug treatment failure

“…Once those conditions pass, this population can return to a proliferative state and recolonizes the environment. Although the dormant state enables bacteria to survive stress, it is not necessarily triggered by stress, instead dormancy occurs spontaneously [4][5][6][7] and recent preliminary data point to new approaches to identify and isolate low numbers of spontaneously arising Mycobacterium smegmatis persisters 9 ; that is, a subpopulation of cells is prepared for the sudden onset of stress.…”

Transcriptome signature of cell viability predicts drug response and drug interaction for Tuberculosis

“…Additionally, the ability to enrich and amplify RNA should allow DRonA to be used with samples where bacterial cell numbers are below the limit of detection of bacteriological assays. The high sensitivity and the autonomy from culturing, makes DRonA especially suitable to evaluate the efficacy of treatment regimens on drug tolerant Mtb subpopulations, such as persister cells 27 .…”