VapC20 of Mycobacterium tuberculosis cleaves the Sarcin–Ricin loop of 23S rRNA

Winther, Kristoffer Skovbo; Brodersen, D.E.; Brown, Alistair K.; Gerdes, Kenn

doi:10.1038/ncomms3796

Cited by 114 publications

(146 citation statements)

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“…The HigB toxin targets tmRNA, IdeR and Zur-regulated transcripts, and its activity is regulated by a SecB-like chaperone, Rv1957 (refs 33,34). VapC toxins regulate Mtb growth via mRNA or 23S rRNA cleavage, or by regulating glycerol consumption rates in vitro [35][36][37] . Some of these VapC homologues are redundant in nature and Msm with no functional TA system exhibits a severe growth defect in complex media 35,38 .…”

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MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs

et al. 2015

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Toxin-antitoxin (TA) systems are highly conserved in members of the Mycobacterium tuberculosis (Mtb) complex and have been proposed to play an important role in physiology and virulence. Nine of these TA systems belong to the mazEF family, encoding the intracellular MazF toxin and its antitoxin, MazE. By overexpressing each of the nine putative MazF homologues in Mycobacterium bovis BCG, here we show that Rv1102c (MazF3), Rv1991c (MazF6) and Rv2801c (MazF9) induce bacteriostasis. The construction of various single-, double-and triple-mutant Mtb strains reveals that these MazF ribonucleases contribute synergistically to the ability of Mtb to adapt to conditions such as oxidative stress, nutrient depletion and drug exposure. Moreover, guinea pigs infected with the triple-mutant strain exhibits significantly reduced bacterial loads and pathological damage in infected tissues in comparison with parental strain-infected guinea pigs. The present study highlights the importance of MazF ribonucleases in Mtb stress adaptation, drug tolerance and virulence.

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MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs

et al. 2015

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“…For example, VapC20 of M. tuberculosis cleaves the Sarcin-Ricin loop of 23S rRNA in the intact ribosome (Winther et al 2013), and VapC (MvpT) of Shigella flexneri cleaves the anticodon loop of the initiator tRNA (Winther and Gerdes 2011). To examine whether TtVapC has such substrate-specificities, we first evaluated its activity toward intact ribosomes from E. coli and T. thermophilus.…”

Section: Ttvapc Degrades Free Rna But Not Trna or Rrna In Intact Ribomentioning

confidence: 99%

“…The PIN domain consists of three highly conserved negatively-charged amino acids and is predicted to have RNase activity (Arcus et al 2011). VapC toxins from different organisms were reported to have different target specificities; for example, VapC20 of M. tuberculosis inhibits translation by cleavage of 23S ribosomal RNA (Winther et al 2013), and both VapC of Shigella flexneri and VapCLT2 of Salmonella enterica site-specifically cleave initiator tRNA (Winther and Gerdes 2011); on the other hand, VapC-mt4 of M. tuberculosis degrades mRNA (Sharp et al 2012).…”

Section: Introductionmentioning

confidence: 99%

“…Under normal growth conditions, the antitoxin neutralizes toxin activity by forming a stable protein complex. When an environmental stressor is present, the antitoxin protein is degraded via proteolysis such that the toxin is released from the protein complex and becomes active to target intracellular molecules, such as mRNA at the ribosomal A site (Christensen and Gerdes 2003), initiator tRNA fMet (Winther and Gerdes 2011), Sarcin-Ricin loop of 23S rRNA (Winther et al 2013), and glutamyltRNA synthetase (Germain et al 2013), to inhibit cell growth or cause cell death. Most type II toxins function as RNases for degrading mRNA, either in a ribosome-dependent or independent manner (Unterholzner et al 2014), such as MazF, Kid, ChpBK, MqsR, VapC, and HicA.…”

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confidence: 99%

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Identification of a VapBC toxin–antitoxin system in a thermophilic bacterium Thermus thermophilus HB27

2016

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Abbreviations AbstractThere are 12 putative toxin-antitoxin (TA) loci in the Thermus thermophilus HB27 genome, including four VapBC and three HicBA families. Expression of these seven putative toxin genes in Escherichia coli demonstrated that one putative VapC toxin TTC0125 and two putative HicA toxins, TTC1395 and TTC1705, inhibited cell growth, and co-expression with cognate antiotoxin genes rescued growth, indicating that these genes function as TA loci. In vitro analysis with the purified TTC0125 and total RNA/mRNA from E. coli and T. thermophilus showed that TTC0125 has RNase activity to rRNA and mRNA; this activity was inhibited by the addition of the purified TTC0126. Translation inhibition assays showed that TTC0125 inhibited protein synthesis by degrading mRNA but not by inactivating ribosomes. Amino acid substitutions of 14 predicted catalytic and conserved residues in VapC toxins to Ala or Asp in TTC0125 indicated that nine residues are important for its in vivo toxin activity and in vitro RNase activity. These data demonstrate that TTC0125-TTC0126 functions as a VapBC TA module and causes growth inhibition by degrading free RNA. This is the first study to identify the function of TA systems in T. thermophilus.

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“…VapC toxins from the enteric bacteria Salmonella enterica and Shigella flexneri cleave initiator tRNA fMet (26). VapC20 from Mycobacterium tuberculosis cleaves 23S rRNA at the sarcin-ricin loop (27). VapC1 and VapC29 from M. tuberculosis cleave single-stranded RNAs in GC-rich sequences (28,29), and VapC4 cleaves specific tRNA isoacceptors (30).…”

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Structural Determinants for Antitoxin Identity and Insulation of Cross Talk between Homologous Toxin-Antitoxin Systems

Walling

Butler

2016

J Bacteriol

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Toxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Under normal growth conditions, the antitoxin neutralizes the toxin. However, under conditions of stress, such as nutrient starvation or antibiotic treatment, cellular proteases degrade the antitoxin, and the toxin functions to arrest bacterial growth. We characterized the specificity determinants of the interactions between VapB antitoxins and VapC toxins from nontypeable Haemophilus influenzae (NTHi) in an effort to gain a better understanding of how antitoxins control toxin activity and bacterial persistence. We studied truncated and full-length antitoxins with single amino acid mutations in the toxin-binding domain. Coexpressing the toxin and antitoxin in Escherichia coli and measuring bacterial growth by dilution plating assayed the ability of the mutant antitoxins to neutralize the toxin. Our results identified two single amino acid residues (W48 and F52) in the C-terminal region of the VapB2 antitoxin necessary for its ability to neutralize its cognate VapC2 toxin. Additionally, we attempted to alter the specificity of VapB1 by making a mutation that would allow it to neutralize its noncognate toxin. A mutation in VapB1 to contain the tryptophan residue identified herein as important in the VapB2-VapC2 interaction resulted in a VapB1 mutant (the T47W mutant) that binds to and neutralizes both its cognate VapC1 and noncognate VapC2 toxins. This represents the first example of a single mutation causing relaxed specificity in a type II antitoxin. IMPORTANCEToxin-antitoxin systems are of particular concern in pathogenic organisms, such as nontypeable Haemophilus influenzae, as they can elicit dormancy and persistence, leading to chronic infections and failure of antibiotic treatment. Despite the importance of the TA interaction, the specificity determinants for VapB-VapC complex formation remain uncharacterized. Thus, our understanding of how antitoxins control toxin-induced dormancy and bacterial persistence requires thorough investigation of antitoxin specificity for its cognate toxin. This study characterizes the crucial residues of the VapB2 antitoxin from NTHi necessary for its interaction with VapC2 and provides the first example of a single amino acid change altering the toxin specificity of an antitoxin.T oxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Originally discovered as addictive elements involved in postsegregational killing during cell division, they have since been identified as playing a critical role in responding to stress (1-3). TA systems are typically encoded by a single operon that produces a stable protein toxin and its cognate labile antitoxin, with the gene encoding the antitoxin located upstream of the toxin in most cases (4). Under normal growth conditions, the antitoxin, which may be a protein (types II, IV, and V) or an RNA...

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VapC20 of Mycobacterium tuberculosis cleaves the Sarcin–Ricin loop of 23S rRNA

Cited by 114 publications

References 41 publications

MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs

MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs

Identification of a VapBC toxin–antitoxin system in a thermophilic bacterium Thermus thermophilus HB27

Structural Determinants for Antitoxin Identity and Insulation of Cross Talk between Homologous Toxin-Antitoxin Systems

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