Abstract
The Mycobacterium tuberculosis genome harbors an unusually large number of toxin-antitoxin (TA) modules. Curiously, over half of these are VapBC (virulence-associated protein) family members. Nonetheless, the cellular target, precise mode of action, and physiological role of the VapC toxins in this important pathogen remain unclear. To better understand the function of this toxin family, we studied the features and biochemical properties of a prototype M. tuberculosis VapBC TA system, vapBC-mt4 (Rv0596c-Rv0595c). VapC-mt4 expression resulted in growth arrest, a hallmark of all TA toxins, in Escherichia coli, Mycobacterium smegmatis, and M. tuberculosis. Its expression led to translation inhibition accompanied by a gradual decrease in the steady-state levels of several mRNAs. VapC-mt4 exhibited sequence-specific endoribonuclease activity on mRNA templates at ACGC and AC(A/U)GC sequences. However, the cleavage activity of VapC-mt4 was comparatively weak relative to the TA toxin MazF-mt1 (Rv2801c). Unlike other TA toxins, translation inhibition and growth arrest preceded mRNA cleavage, suggesting that the RNA binding property of VapC-mt4, not RNA cleavage, initiates toxicity. In support of this hypothesis, expression of VapC-mt4 led to an increase in the recovery of total RNA with time in contrast to TA toxins that inhibit translation via direct mRNA cleavage. Additionally, VapC-mt4 exhibited stable, sequence-specific RNA binding in an electrophoretic mobility shift assay. Finally, VapC-mt4 inhibited protein synthesis in a cell-free system without cleaving the corresponding mRNA. Therefore, the activity of VapC-mt4 is mechanistically distinct from other TA toxins because it appears to primarily inhibit translation through selective, stable binding to RNA.
Highlights
Mycobacterium tuberculosis harbors a highly expanded number of toxin-antitoxin (TA) systems
In Escherichia coli, activation of toxin-antitoxin systems is triggered by various stresses (8 –11) and induces a state of cell growth arrest [5, 6] with striking similarities to the slowly or non-replicating state thought to occur in M. tuberculosis during the latent infection [12]
We determined whether expression of any of the 23 VapC toxins originally annotated in the genome of M. tuberculosis [4] caused growth arrest in E. coli
Summary
Mycobacterium tuberculosis harbors a highly expanded number of toxin-antitoxin (TA) systems. Unlike other TA toxins, translation inhibition and growth arrest preceded mRNA cleavage, suggesting that the RNA binding property of VapC-mt, not RNA cleavage, initiates toxicity. In Escherichia coli, activation of toxin-antitoxin systems is triggered by various stresses (8 –11) and induces a state of cell growth arrest [5, 6] with striking similarities to the slowly or non-replicating state thought to occur in M. tuberculosis during the latent infection [12]. Recently the Shigella and Salmonella VapC toxins were shown to cleave tRNAfMet at a single site between the anticodon stem and loop [35] These contradictory results suggest that not all VapCs function by cleaving RNA and that the specific mechanism of action may vary among different members of this family of PIN domain proteins. VapC-mt toxicity appears to result from the stable, selective binding of RNA containing an ACGC or AC(A/U)GC consensus sequence
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