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Single-cell evidence for plasmid addiction mediated by toxin-antitoxin systems.

ABSTRACT: Toxin-antitoxin (TA) systems are small selfish genetic modules that increase vertical stability of their replicons. They have long been thought to stabilize plasmids by killing cells that fail to inherit a plasmid copy through a phenomenon called post-segregational killing (PSK) or addiction. While this model has been widely accepted, no direct observation of PSK was reported in the literature. Here, we devised a system that enables visualization of plasmid loss and PSK at the single-cell level using meganuclease-driven plasmid curing. Using the ccd system, we show that cells deprived of a ccd-encoding plasmid show hallmarks of DNA damage, i.e. filamentation and induction of the SOS response. Activation of ccd triggered cell death in most plasmid-free segregants, although some intoxicated cells were able to resume growth, showing that PSK-induced damage can be repaired in a SOS-dependent manner. Damage induced by ccd activates resident lambdoid prophages, which potentiate the killing effect of ccd. The loss of a model plasmid containing TA systems encoding toxins presenting various molecular mechanisms induced different morphological changes, growth arrest and loss of viability. Our experimental setup enables further studies of TA-induced phenotypes and suggests that PSK is a general mechanism for plasmid stabilization by TA systems.

SUBMITTER: Fraikin N

PROVIDER: S-EPMC10899753 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Single-cell evidence for plasmid addiction mediated by toxin-antitoxin systems.

Fraikin Nathan N Van Melderen Laurence L

Nucleic acids research 20240201 4

Toxin-antitoxin (TA) systems are small selfish genetic modules that increase vertical stability of their replicons. They have long been thought to stabilize plasmids by killing cells that fail to inherit a plasmid copy through a phenomenon called post-segregational killing (PSK) or addiction. While this model has been widely accepted, no direct observation of PSK was reported in the literature. Here, we devised a system that enables visualization of plasmid loss and PSK at the single-cell level ...[more]

PMID: 38224456

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Project description:Rickettsia spp. can cause mild to severe human disease. These intracellular bacteria are associated with arthropods, nematodes and trematodes, and usually, are efficiently transmitted transovarially to the progeny of the invertebrate host. We recently demonstrated foreign gene acquisition by lateral gene transfer in Rickettsia genomes. The unexpected presence of laterally transferred toxin-antitoxin (TA) genetic elements (including vapBC) in several Rickettsia genomes has not been connected with the pathogenic process or the host-bacteria relationship. We suspect that vapBC are selfish genetic elements that addict eukaryotic hosts to Rickettsia. We identified a statistical link between the transovarial transmission of Rickettsia in invertebrate hosts and the presence of TA operons, specifically vapBC, in the Rickettsia genome. These TA are neighboring to type IV secretion genes. Tunel assays and whole-genome expression of infected cells showed that antibiotic eradication of TA-containing Rickettsia from the host in cell culture initiates a proapoptotic program. Rickettsia VapC toxins inhibit the growth of transformed Escherichia coli and Saccharomyces cerevisiae. Rickettsia toxin presents in vitro RNase activity. Annexin-V staining and time-lapse video showed that intracytoplasmic injections of VapC toxins in cells cause apoptosis. These data demonstrate that host cells may develop a dependence on Rickettsia spp. expressing the vapBC operon. This would constitute a new evolutionary M-bM-^@M-^\mafia strategyM-bM-^@M-^] of intracellular bacteria based on host addiction. Fresh cells from the human microvascular endothelial cell line (HMEC-1) [26] were infected with R. felis California-2 strain in the presence and absence of antibiotics, at a rate of 5 bacteria per eukaryotic cell. Then, we added or not antibiotics (chloramphenicol 50 M-BM-5g/ml or doxycycline to 40 M-BM-5g/ml) in both experimental (R.felis-infected) and control, mock-infected cells for 6 hours. The cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen). DNA contamination was removed using the Turbo DNA-free Kit (Ambion). RNA were labeled using the Quick Amp Labeling Kit One-color (Agilent) and hybridized onto a Whole Human Genome Microarray, 4x44K (Agilent) as recommended by the manufacturer. Arrays were scanned with DNA Microarray Scanner (Agilent), and data were extracted using Feature Extractor (Agilent).

Dataset Information

Single-cell evidence for plasmid addiction mediated by toxin-antitoxin systems.

Publications

Single-cell evidence for plasmid addiction mediated by toxin-antitoxin systems.

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