Enteric Virulence Associated Protein Vapc Inhibits Translation by Cleavage of Initiator Trna

Enteric virulence associated protein VapC inhibits translation by cleavage of initiator tRNA Kristoffer S. Winther and Kenn Gerdes1 Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle NE2 4HH, United Kingdom Edited* by Susan Gottesman, National Cancer Institute, Bethesda, MD, and approved March 22, 2011 (received for review December 29, 2010) Eukaryotic PIN (PilT N-terminal) domain proteins are ribonucleases Typhimurium LT2, encoded by bona fide vapBC loci, are site- involved in quality control, metabolism and maturation of mRNA specific tRNases that cleave initiator tRNA between the antico- and rRNA. The majority of prokaryotic PIN-domain proteins are don stem and loop. encoded by the abundant vapBC toxin—antitoxin loci and inhibit translation by an unknown mechanism. Here we show that enteric Results VapCs are site-specific endonucleases that cleave tRNAfMet in the VapC Inhibits Translation In Vitro. We showed previously that over- anticodon stem-loop between nucleotides þ38 and þ39 in vivo production of VapC inhibits global cellular translation (21). To and in vitro. Consistently, VapC inhibited translation in vivo and identify VapC’s target within the translational machinery, we pur- in vitro. Translation-reactions could be reactivated by the addition ified VapC and VapCLT2. Addition of purified, native VapC to an of VapB and extra charged tRNAfMet. Similarly, ectopic production in vitro translation reaction abolished translation (Fig. 1A, lanes 1 of tRNAfMet counteracted VapC in vivo. Thus, tRNAfMet is the only and 2). Preincubation with VapB antitoxin neutralized VapC cellular target of VapC. Depletion of tRNAfMet by vapC induction activity, showing that VapCs inhibition of translation was specific was bacteriostatic and stimulated ectopic translation initiation (lane 3). Thus, the preparation of VapC was active in vitro and its at elongator codons. Moreover, addition of chloramphenicol to activity could be counteracted by VapB. For reasons unknown, cells carrying vapBC induced VapC activity. Thus, by cleavage of VapCLT2 was not active in our in vitro assays and was therefore tRNAfMet, VapC simultaneously may regulate global cellular trans- analyzed in vivo only. lation and reprogram translation initiation. VapC Inhibits Translation by Degradation of Initiator tRNAfMet. Ectopic toxin-antitoxin ∣ tRNase ∣ riboendonuclease ∣ tRNA ∣ RNase production of VapC in vivo did not affect the degradation- patterns of lpp, dksA,orompA mRNAs, ribosomal RNAs, or rokaryotic toxin—antitoxin (TA) loci code for two compo- tmRNA (21). Consistently, VapC exhibited only weak RNase Pnents, a toxin that inhibits cell growth and an antitoxin that activity towards MS2 RNA in vitro (Fig. S2). Moreover, VapC counteracts the toxin. In Type I TA loci, the antitoxins are small did not associate specifically with 70S, 50S, or 30S ribosomal sub- antisense RNAs that repress translation of the toxin genes (1, 2) units (Fig. S3). Therefore, we considered that VapC might target while in Type II loci, the antitoxins are proteins that combine with tRNAs. VapC completely degraded purified E. coli tRNAfMet in a and neutralize the toxins (3). Type III TA loci encode small RNAs 15′ reaction (Fig. 1C). In contrast, tRNAVal and tRNAPhe were that combine with and neutralize the toxins (4). Based on toxin not degraded. Cleavage of tRNAfMet was counteracted by the sequence similarities, Type II loci have been divided into gene prior addition of VapB to VapC, again showing specificity. EDTA families (3, 5). Many of these gene families are present in both also inhibited the reaction, revealing that the reaction required bacteria and archaea, predicting that the cellular targets of the divalent cations (Fig. 1C). The in vitro translation reaction could toxins are of a general nature. Consistently, the RelE family of only be reactivated by the addition of both VapB and fMet- toxins, which is present in both prokaryotic domains, cleave tRNAfMet (Fig. 1B). The ability of fresh fMet-tRNAfMet to reac- mRNAs at codons positioned at the ribosomal A-site (6, 7). RelE tivate the reaction after quenching of VapC activity (compare toxins from archaea cleave mRNAs at A-site codons in Escher- lanes 3 and 4), shows that VapC does not have other targets with- ichia coli (8) and RelE from E. coli cleave A-site codons of mam- in the translational machinery. That tRNAfMet is the sole cellular malian and mitochondrial ribosomes (9, 10). The most abundant target of VapC was further substantiated by the observation that Type II TA loci are vapBC that encode PIN-domain toxins. Inter- ectopic overexpression of tRNAfMet counteracted the toxic effect estingly vapBC are highly abundant in some organisms. For of a vapC pulse (Fig. S4). example, the major human pathogen Mycobacterium tuberculosis and the hyperthermophilic chrenarchaeote Sulfolobus solfataricus VapC Cleaves Initiator tRNA Between the Anticodon Stem and Loop. have at least 45 and 30 vapBC loci, respectively (11, 12). PIN-do- VapC degraded full-length tRNAfMet into smaller fragment(s) mains (PilT N-terminal) consist of approximately 140 amino acids (Fig. 1C), indicating endonucleolytic cleavage. The cleavage was characterized by a quartet of conserved, negatively charged ami- mapped to occur between þ38 and þ39, at the anticodon stem- no acid residues configured in an RNase H-like fold (Fig. S1) loop boundary (Fig. S5). Thus, VapC is a site-specific tRNAfMet (13). In eukaryotes, PIN-domain proteins function in several endo-nuclease in vitro. The 3′-terminus of the 5′-product of types of RNA metabolism, such as nonsense-mediated mRNA tRNAfMet could not be ligated to [5′-32P]Cytidine 3’,5’-bispho- decay (14, 15), rRNA maturation (16), and mRNA turnover sphate (pCp) with T4 RNA ligase, consistent with a 2′,3′-cyclic (17–19). Ectopic production of VapC from both Gram positive and negative bacteria efficiently inhibits cell growth by inhibiting the global rate of translation (11, 20, 21). As with other Type II Author contributions: K.S.W. and K.G. designed research; K.S.W. performed research; TA loci, VapB antitoxins neutralize cognate VapCs by direct pro- K.S.W. and K.G. analyzed data; and K.S.W. and K.G. wrote the paper. tein—protein interaction (21). Even though several studies The authors declare no conflict of interest. showed that VapC has nonspecific ribo- or deoxyribonuclease *This Direct Submission article had a prearranged editor. activity in vitro, the specific target of VapC within the translation Freely available online through the PNAS open access option. apparatus has remained elusive (11, 13, 21, 22). Here we show 1To whom correspondence should be addressed. E-mail: [email protected]. that VapC (MvpT) of Shigella flexneri 2a virulence plasmid This article contains supporting information online at www.pnas.org/lookup/suppl/ BIOCHEMISTRY pMYSH6000 (23) and VapCLT2 of Salmonella enterica serovar doi:10.1073/pnas.1019587108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1019587108 PNAS ∣ May 3, 2011 ∣ vol. 108 ∣ no. 18 ∣ 7403–7407 Downloaded by guest on September 26, 2021 Thus, VapC cleavage of tRNAfMet was highly specific. It should be noted that tRNAfMet isoforms of E. coli, S. flexneri, and S. enterica are identical, validating the use of E. coli as the host organism in these experiments. To confirm this inference, we fMet showed that tRNA of S. enterica was also cleaved after vapCLT2 induction (Fig. S7). The complete cleavage seen both in vitro and in vivo indicates that VapC can cleave both charged and uncharged tRNAfMet (Figs. 1C and 2A). The in vivo VapC cleavage site was mapped by isolating tRNA fragments using a biotinylated tRNAfMet-specific probe. Cells fMet expressing VapC or VapCLT2 produced tRNA -derived RNAs that had sizes identical to those obtained in vitro (Fig. 2B). The slightly smaller RNA fragments also observed in vivo most likely reflect nibbling of the RNA ends after the initial cleavage. Again, neither chloramphenicol nor induction of relE produced detect- able cleavage products in vivo (Fig. 2B). A sequence alignment of tRNAfMet and tRNAMet (Fig. 2C) showed that cleavage occurred between the anticodon loop and the tRNAfMet specific GC nucleotide basepair stem that acts as a discriminator for tRNAfMet loading into the ribosomal P-site (25). The GC stem is conserved in initiator tRNAs in all three domains of life. Change of a Predicted Catalytic, Highly Conserved aa Residue Abolishes VapC Activity. The catalytic centers of PIN-domain proteins consist of quartets of conserved, acidic aa residues (13). The sequence alignment in Fig. S1 predicted that the conserved aspartate at þ7 of VapC is required for catalytic activity. To test this possibility, we changed the aspartate to alanine. As described in the following, VapCD7A indeed was inactive in vivo. Depletion of tRNAfMet by VapC Is Reversible and Bacteriostatic. It has been debated whether toxins encoded by TA loci are bacterioci- dal or bacteriostatic. We followed the cellular content of fMet Fig. 1. VapC inhibits translation in vitro by cleavage of tRNAfMet.(A) Native tRNA in a physiological growth experiment. After induction VapC inhibits translation in vitro and can be neutralized by VapB. Each reac- of vapC or vapCLT2, cell-growth ceased rapidly (Fig. 3A). μ μ μ D7A tion contained the following: 6 L Premix, 4.5 L S30 Extract, 1.5 L amino In contrast, induction of vapCLT2 , that encodes a predicted acid mix without methionine, 0.5 μL 35S-methionine, and 4.5 pmol VapC with catalytically inactive VapC, did not inhibit cell growth. Thirty min- and without preincubation with 30 pmol VapB. The reaction was incubated utes later, vapC expression was terminated and cognate vapB μ ∼0 7 for 5 min at 37 °C before addition of 1 L MS2 RNA ( . pmol) (Roche). The genes were induced. As seen, cell growth resumed ≈30 min after reactions were incubated for 1 h at 37 °C before termination of the reaction ≈90 by acetone precipitation.

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