Srinivas Vivek, Arrieta-Ortiz Mario L, Kaur Amardeep, Peterson Eliza J R, Baliga Nitin S
Institute for Systems Biology, Seattle, Washington, USA.
Institute for Systems Biology, Seattle, Washington, USA
mSystems. 2020 Dec 1;5(6):e01127-20. doi: 10.1128/mSystems.01127-20.
(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 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 , , and / 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. (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 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.
结核分枝杆菌（MTB）通过产生表型多样性来在感染过程中遇到的恶劣条件下持续存在并存活。MTB通过进入不同的生理状态来避免免疫效应器和抗菌杀伤。存活的细胞，即持留菌，是结核病（TB）及时且无复发治疗的主要障碍。我们首次展示了PerSort，一种在无抗生素或其他压力的情况下分离和表征持留菌的方法。我们证明了PerSort在分离翻译静止细胞方面的价值，这些细胞在最佳条件下生长的菌种培养物中少量预先存在，但在应激条件下比例显著增加。翻译静止亚群表现出与持留菌细胞一致的多药耐受性和再生长特性。此外，PerSort实现了单细胞转录谱分析，提供了证据表明翻译静止持留菌是通过多种机制产生的，包括……、……和……过表达。最后，我们证明尽管持留菌细胞产生的机制多种多样，但它们汇聚到一种相似的低氧代谢状态，通过激活呼吸作用可以逆转这种状态，从而迅速消除在宿主相关应激条件下形成的持留菌。我们得出结论，PerSort为研究MTB持留菌提供了一种新工具，能够制定有针对性的策略来改善和缩短结核病的治疗。（MTB）通过产生表型异质性的耐药物亚群在抗生素治疗下持续存在并存活。存活的细胞，即持留菌，是结核病无复发治疗的主要障碍，结核病每年已导致超过180万人死亡，并且随着多重耐药菌株的出现变得更加致命。这项研究描述了PerSort，一种细胞分选方法，用于在不进行抗生素治疗的情况下分离和表征在菌种培养物中少量预先存在的翻译静止持留菌。对这个亚群的表征发现了分枝杆菌持留菌出现的多种机制，并揭示了它们休眠和多药耐受生理状态的生理基础。该分析发现使用L-半胱氨酸激活氧呼吸生理可以消除预先存在的持留菌亚群，增强在宿主相关应激下对分枝杆菌的快速抗生素杀伤。PerSort作为研究MTB持留菌的一种新工具，能够制定有针对性的策略来改善和缩短结核病的治疗。
