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Growth and translation inhibition through sequence-specific RNA binding by Mycobacterium tuberculosis VapC toxin.

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.

SUBMITTER: Sharp JD

PROVIDER: S-EPMC3339977 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Type II toxin-antitoxin (TA) systems play a critical role in the establishment and maintenance of bacterial dormancy. They are composed of a protein toxin and its cognate protein antitoxin. They function to regulate growth under conditions of stress, such as starvation or antibiotic treatment. As cellular proteases degrade the antitoxin, which normally binds and neutralizes the toxin, this frees the toxin to act on its cellular targets and arrest bacterial growth. TA systems are of particular concern in regard to pathogenic organisms, such as nontypeable Haemophilus influenzae (NTHi), as dormancy may lead to chronic infections and failure of antibiotic treatment. Many targets of VapC toxins have not been identified, to date, and this knowledge is crucial to understanding how toxins control the establishment and maintenance of bacterial dormancy. Accordingly, we characterized the target specificity of the VapC toxins from the two paralogous NTHi vapBC TA systems. RNA sequencing and Northern blot analysis revealed that VapC1 and VapC2 cleave tRNAfMet in the anticodon loop. Overexpression of tRNAfMet suppresses VapC toxicity, suggesting that translation inhibition results from the depletion of tRNAfMet These experiments also identified base pairs in the tRNAfMet anticodon stem that play a key role in VapC-specific cleavage of the tRNA. Together these findings suggest the potential for NTHi VapC1 and VapC2 to induce dormancy by sequence-specific cleavage of tRNAfMetIMPORTANCE Bacterial persistence is a significant concern in regard to pathogenic organisms, such as nontypeable Haemophilus influenzae, as it can result in recurrent and chronic infections. Toxin-antitoxin systems can lead to persistence by causing bacteria to enter a slow-growing state that renders them antibiotic tolerant. Type II toxin components affect a wide variety of bacterial targets in order to elicit dormancy, and for many toxin-antitoxin systems, these mechanisms are not well understood. Thus, in order to understand how vapBC toxin-antitoxin systems cause dormancy, it is crucial to investigate the substrate specificity of VapC toxins. This study identifies the target of the VapC1 and VapC2 toxins from NTHi and takes important steps toward understanding the specificity of these toxins for their tRNA target.

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Growth and translation inhibition through sequence-specific RNA binding by Mycobacterium tuberculosis VapC toxin.

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