Toxin-antitoxin deletions in Legionella pneumophila reveal unusual cell death and contact-dependent survival responses during genotoxic stress [Lp_growth_phase_RNAseq]

Bacteria must contend with near constant environmental stresses, and to tolerate these cellular assaults, have evolved numerous stress response pathways and programs. One such strategy is the adoption of a dormant state, whereby cells restrict their growth and await a reversion to favourable conditions. A notable example of this is the formation of persister cells, which are a quiescent subpopulation that display increased tolerance to antibiotic killing. The genetics of persistence remain poorly understood, however bacterial genes called toxin-antitoxin (TA) systems have long been implicated in the persistence phenotype. TA systems are abundant genetic elements in bacteria that can function as growth toggling molecular switches, yet their role in persister formation is highly controversial. To address this, we constructed a pan-TA deletion strain (∆7TA) of the bacterial pathogen Legionella pneumophila to examine how a strain devoid of these genes tolerates cellular stress. We identified a single putative TA system (Lpg1604-05) that is activated specifically in response to genotoxic stress, however this system leads to cell death during stress rather than promoting survival. In the absence of Lpg1604-05, cells do not die during stress exposure and instead adopt a viable but non-culturable state. Strikingly, while persister survival during DNA stress is dramatically improved when Lpg1604-05 is deleted, this enhanced survival can be conferred to wild-type cells in a contact-dependent manner during co-culture with the deletion strain. This is dependent, however, on a sufficient proportion of the mixed population being deletion mutants. Despite having homology only to other TA systems, Lpg1604-05 displays non-canonical activity and we hypothesize that it has undergone exaptation, leading to integration into the cell’s stress response pathway and conservation within L. pneumophila’s core genome. Overall, our work demonstrates the stress-specific activation of a putative TA system and its involvement in facilitating cell death, rather than survival, during stressful conditions. These findings reveal both a new physiological function for TA systems in bacteria, as well as a heretofore undescribed phenomenon of contact-dependent survival within persister cells. This expands the functional scope of an omnipresent class of bacterial genes and suggests that domestication of mobilome genes may be an overlooked source of de novo genetic circuitry. These samples consist of wild-type (WT) Legionella pneumophila (LP) and a strain with a single TA system (lpg1604-05; DEL) deleted from its genome. Both strains were grown to two different growth phases to compare how their transcriptomes changed during stress exposure. Early exponential growth (OD1) and late exponential growth (OD3) were the time points when RNA was extracted for bulk sequencing. There are two biological replicates for each strain + growth phase.

细菌必须应对近乎持续不断的环境胁迫，为了耐受这些细胞内攻击，它们演化出了众多胁迫应答通路与程序。其中一种策略是进入休眠状态，此时细胞会限制自身生长，等待恢复至适宜环境。这类休眠状态的一个典型案例是持留菌（persister cells）的形成：持留菌是一类静息亚群，对抗生素杀伤的耐受性显著提升。
尽管持留性的遗传机制仍远未被阐明，但被称为毒素-抗毒素（toxin-antitoxin, TA）系统的细菌基因长期以来被认为与持留表型相关。TA系统是细菌中丰度颇高的遗传元件，可作为生长调控的分子开关，但其在持留菌形成中的作用却极具争议。
为解决这一问题，我们构建了病原菌嗜肺军团菌（Legionella pneumophila）的泛TA系统缺失菌株（∆7TA），以探究缺失这类基因的菌株如何耐受细胞胁迫。我们鉴定出一个单一的推定TA系统（Lpg1604-05），其仅在遗传毒性胁迫下被激活；然而该系统在胁迫过程中反而会导致细胞死亡，而非促进存活。
在缺失Lpg1604-05的情况下，细胞在胁迫暴露时不会死亡，反而会进入活的但不可培养（viable but non-culturable, VBNC）状态。值得注意的是，尽管缺失Lpg1604-05可显著提升DNA胁迫下的持留菌存活率，但这种增强的存活能力可通过与缺失菌株共培养的方式，以接触依赖的方式传递给野生型菌株。不过，这一现象依赖于混合种群中缺失突变体所占比例足够高。
尽管Lpg1604-05仅与其他TA系统具有同源性，但其展现出非典型的活性；我们推测该系统经历了功能外显适应（exaptation），整合进入细胞的胁迫应答通路，并在嗜肺军团菌的核心基因组中得以保守保留。
总体而言，我们的研究证明了推定TA系统的胁迫特异性激活，以及其在胁迫条件下促进细胞死亡而非存活的作用。这些发现不仅揭示了细菌TA系统的一种全新生理功能，还阐明了持留菌中此前未被报道的接触依赖存活现象。
本研究拓宽了这类普遍存在的细菌基因家族的功能范围，并提示可移动遗传元件基因（mobilome genes）的驯化可能是从头遗传回路形成的一个被忽视的来源。
本研究所用样本包括野生型（wild-type, WT）嗜肺军团菌（Legionella pneumophila, LP），以及基因组中删除了单一TA系统（lpg1604-05；DEL）的菌株。将两种菌株分别培养至两个不同的生长阶段，以比较它们在胁迫暴露过程中的转录组变化。我们选取了指数生长早期（OD₁）与指数生长晚期（OD₃）两个时间点提取RNA，用于批量转录组测序（bulk sequencing）。每个菌株+生长阶段组合均设置两个生物学重复（biological replicates）。