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

Winther, Kristoffer Skovbo; Gerdes, Kenn

doi:10.1073/pnas.1019587108

Cited by 242 publications

(295 citation statements)

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“…Initiator tRNA cleavage has previously been described in Salmonella and shown to be in part mediated via the toxin/anti-toxin system (TA system) VapB/C (Winther and Gerdes, 2011).…”

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confidence: 99%

Dual RNA-seq of pathogen and host

2012

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SummaryThe infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections.However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella.Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and

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“…Initiator tRNA cleavage has previously been described in Salmonella and shown to be in part mediated via the toxin/anti-toxin system (TA system) VapB/C (Winther and Gerdes, 2011).…”

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confidence: 99%

Dual RNA-seq of pathogen and host

2012

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“…The TTC0125 toxin was purified according to Winther and Gerdes (2011). E. coli Rosetta (DE3) harboring pET28-TTC0125-TTC0126 was cultured in LB medium supplemented with 50 μg/mL kanamycin and 20 μg/mL chloramphenicol at 30 °C.…”

Section: Purification Of Ttc0125 and Ttc0126mentioning

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.…”

Section: Introductionmentioning

confidence: 99%

“…TA systems consist of two small genes that encode a low molecular weight toxic protein (toxin) which reacts with intracellular target molecules related to essential cellular processes, such as DNA replication, protein translation, membrane integrity, and cell wall synthesis (Bertram and Schuster 2014;Maisonneuve et al 2013;Page and Peti 2016), and a cognate antitoxin protein or RNA that inhibits toxin activity. Initially, TA loci were reported to play a role in bacterial programmed cell death for maintaining plasmid stability by killing the daughter cells that failed to inherit the plasmids (Winther and Gerdes 2011), or for acting as antiviral factors (Hazan and Engelberg-Kulka 2004;Pecota and Wood, 1996). More recently, it was recognized that the toxins play roles in response to cellular stresses (Bertram and Schuster 2014), such as amino acid starvation and the presence of antibiotics (Fauvart et al 2011;Gerdes et al 2005;Maisonneuve and Gerdes 2014), and triggering bacterial persistence (Yamaguchi and Inouye 2011).…”

Section: Introductionmentioning

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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Toxin–Antitoxin Systems in Bacteria and Archaea

Yamaguchi

Inouye

2016

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria

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

Cited by 242 publications

References 36 publications

Dual RNA-seq of pathogen and host

Dual RNA-seq of pathogen and host

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

Toxin–Antitoxin Systems in Bacteria and Archaea

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