Abstract
Toxin-antitoxin (TA) systems are implicated in the downregulation of bacterial cell growth associated with stress survival and latent tuberculosis infection, yet the activities and intracellular targets of these TA toxins are largely uncharacterized. Here, we use a specialized RNA-seq approach to identify targets of a Mycobacterium tuberculosis VapC TA toxin, VapC-mt4 (also known as VapC4), which have eluded detection using conventional approaches. Distinct from the one other characterized VapC toxin in M. tuberculosis that cuts 23S rRNA at the sarcin-ricin loop, VapC-mt4 selectively targets three of the 45 M. tuberculosis tRNAs (tRNA(Ala2), tRNA(Ser26) and tRNA(Ser24)) for cleavage at, or adjacent to, their anticodons, resulting in the generation of tRNA halves. While tRNA cleavage is sometimes enlisted as a bacterial host defense mechanism, VapC-mt4 instead alters specific tRNAs to inhibit translation and modulate growth. This stress-linked activity of VapC-mt4 mirrors basic features of eukaryotic tRNases that also generate tRNA halves and inhibit translation in response to stress.
Highlights
Toxin–antitoxin (TA) systems are implicated in the downregulation of bacterial cell growth associated with stress survival and latent tuberculosis infection, yet the activities and intracellular targets of these TA toxins are largely uncharacterized
Our data suggested that tRNA(s) may be the primary targets for M. tuberculosis VapC-mt[4], we were unable to establish this connection using a battery of conventional biochemical, genetic and molecular biological approaches[19]
Bacterial transcripts possess a 50-triphosphate, 50-monophosphate or 50-hydroxyl (noncoding RNA intermediates and products cleaved by certain endoribonucleolytic toxins such as MazF or RNases such as RNase A and T1)
Summary
Toxin–antitoxin (TA) systems are implicated in the downregulation of bacterial cell growth associated with stress survival and latent tuberculosis infection, yet the activities and intracellular targets of these TA toxins are largely uncharacterized. Distinct from all other VapC toxins, and TA toxins in general, VapC-mt[4] arrests growth by translation inhibition resulting from selectively targeting three of the 45 tRNAs present in M. tuberculosis for cleavage at a single site in their anticodon loop This highly selective tRNA substrate discrimination is contingent on recognition of the consensus sequence in an appropriate structural context. VapC-mt4-tRNA-simulated docking experiments place the toxin active site in proximity to the cleavage site in the tRNA anticodon loop Overall, these studies bring to light a common theme between the role of VapC-mt[4] in this pathogen and stress responses in eukaryotic cells that engage cleaved tRNAs in unconventional roles
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