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

ABSTRACT: 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.

ORGANISM(S): Legionella pneumophila

PROVIDER: GSE278701 | GEO | 2025/07/31

REPOSITORIES: GEO

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Project description: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.

Project description:The ability to eliminate bacterial persister cells is still a medical challenge that has yet to be overcome. These cells represent a unique subpopulation within bacterial communities and are characterized by a reduced susceptibility to antibiotics with growth retardation. In this study, we investigated the molecular basis of persister formation in Salmonella Typhimurium 14028s under aminoglycoside stress.We analyzed the crystal structure of the STM14_5441–STM14_5442 complex, which belongs to the type II toxin-antitoxin system, and identified key ribosome binding residues in STM14_5441. Changes in the antibiotic susceptibility of Salmonella caused by the loss of the ribosome binding property of STM14_5441 were assessed. We conducted intracellular ATP assays under aminoglycoside stress and RNA-seq analysis following STM14_5441 induction.Our studies demonstrated the critical role of STM14_5441 in the formation of persister cells in Salmonella, particularly those under aminoglycoside stress. We observed that a loss of ribosome binding in STM14_5441 resulted in increased antibiotic susceptibility. Additionally, intracellular ATP assays revealed increased ATP levels in bacterial persister cells, and RNA-seq analysis identified several genes that play a role in the formation of these persister cells.The present data suggest that persister forms under aminoglycoside stress through the following mechanisms: i) inhibition of membrane hyperpolarization by impeding F1Fo ATP synthase activity and ii) enhanced poststress recovery by ATP storage and increased protein synthesis capacity. Based on this suggestion, we reannotated the STM14_5441-STM14_5442 TA pair as the ResTA (RNA cleavage-induced energy storage toxin-antitoxin) system. Furthermore, new insights into the function of TA systems may lay the groundwork for developing novel strategies to target bacterial persister cells, thereby preventing the accelerated emergence of antibiotic resistance in bacterial populations.

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

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