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Enteric virulence associated protein VapC inhibits translation by cleavage of initiator tRNA.

ABSTRACT: Eukaryotic PIN (PilT N-terminal) domain proteins are ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. The majority of prokaryotic PIN-domain proteins are encoded by the abundant vapBC toxin--antitoxin loci and inhibit translation by an unknown mechanism. Here we show that enteric VapCs are site-specific endonucleases that cleave tRNA(fMet) in the anticodon stem-loop between nucleotides +38 and +39 in vivo and in vitro. Consistently, VapC inhibited translation in vivo and in vitro. Translation-reactions could be reactivated by the addition of VapB and extra charged tRNA(fMet). Similarly, ectopic production of tRNA(fMet) counteracted VapC in vivo. Thus, tRNA(fMet) is the only cellular target of VapC. Depletion of tRNA(fMet) by vapC induction was bacteriostatic and stimulated ectopic translation initiation at elongator codons. Moreover, addition of chloramphenicol to cells carrying vapBC induced VapC activity. Thus, by cleavage of tRNA(fMet), VapC simultaneously may regulate global cellular translation and reprogram translation initiation.

SUBMITTER: Winther KS

PROVIDER: S-EPMC3088637 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Enteric virulence associated protein VapC inhibits translation by cleavage of initiator tRNA.

Winther Kristoffer S KS Gerdes Kenn K

Proceedings of the National Academy of Sciences of the United States of America 20110418 18

Eukaryotic PIN (PilT N-terminal) domain proteins are ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. The majority of prokaryotic PIN-domain proteins are encoded by the abundant vapBC toxin--antitoxin loci and inhibit translation by an unknown mechanism. Here we show that enteric VapCs are site-specific endonucleases that cleave tRNA(fMet) in the anticodon stem-loop between nucleotides +38 and +39 in vivo and in vitro. Consistently, VapC inhibited translatio ...[more]

PMID: 21502523

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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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Enteric virulence associated protein VapC inhibits translation by cleavage of initiator tRNA.

Publications

Enteric virulence associated protein VapC inhibits translation by cleavage of initiator tRNA.

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