Toxin-antitoxin Toxin Research Articles

A type II toxin-antitoxin system, ECs3274-ECs3275, in enterohemorrhagic Escherichia coli O157.

The toxin-antitoxin (TA) genetic module controls various bacterial events. Novel toxins with different functions are still being discovered. This study aimed to determine whether the ECs3274-ECs3275 gene pair encoded by enterohemorrhagic Escherichia coli O157 functions as a TA system. To characterize this putative TA system, we analyzed the growth of E. coli expressing ECs3274, ECs3275, or both; the interaction between ECs3274 and ECs3275 using bacterial adenylate cyclase two-hybrid assays; and the DNA-binding ability of ECs3274 using gel-mobility shift assays. We observed that the ECs3274 antitoxin interacted with the ECs3275 toxin, was destabilized by Lon protease, and repressed its promoter activity via its helix-turn-helix (HTH) motif. These properties are consistent with those of typical type II TA antitoxins. Interestingly, ECs3275 has an HTH motif not observed in other TA toxins and is necessary for ECs3275 toxicity, suggesting that ECs3275 may exert its toxicity by regulating the expression of specific genes.

Evaluation of different strategies to produce Vibrio cholerae ParE2 toxin

Toxin-antitoxin (TA) systems are small operons that are omnipresent in bacteria and archaea with suggested roles in stabilization of mobile genetic elements, bacteriophage protection, stress response and possibly persister formation. A major bottleneck in the study of TA toxins is the production of sufficient amounts of well-folded, functional protein. Here we examine alternative approaches for obtaining the VcParE2 toxin from Vibrio cholerae. VcParE2 can be successfully produced via bacterial expression in presence of its cognate antitoxin VcParD2, followed by on-column unfolding and refolding. Alternatively, the toxin can be expressed in Spodoptera frugiperda (Sf9) insect cells. The latter requires disruption of the VcparE2 gene via introduction of an insect cell intron. Both methods provide protein with similar structural and functional characteristics.

Large-scale Purification of Type III Toxin-antitoxin Ribonucleoprotein Complex and its Components from Escherichia coli for Biophysical Studies.

Toxin-antitoxin (TA) systems are widespread bacterial immune systems that confer protection against various environmental stresses. TA systems have been classified into eight types (I-VIII) based on the nature and mechanism of action of the antitoxin. Type III TA systems consist of a noncoding RNA antitoxin and a protein toxin, forming a ribonucleoprotein (RNP) TA complex that plays crucial roles in phage defence in bacteria. Type III TA systems are present in the human gut microbiome and several pathogenic bacteria and, therefore, could be exploited for a novel antibacterial strategy. Due to the inherent toxicity of the toxin for E. coli, it is challenging to overexpress and purify free toxins from E. coli expression systems. Therefore, protein toxin is typically co-expressed and co-purified with antitoxin RNA as an RNP complex from E. coli for structural and biophysical studies. Here, we have optimized the co-expression and purification method for ToxIN type III TA complexes from E. coli that results in the purification of TA RNP complex and, often, free antitoxin RNA and free active toxin in quantities required for the biophysical and structural studies. This protocol can also be adapted to purify isotopically labelled (e.g., uniformly 15N- or 13C-labelled) free toxin proteins, free antitoxin RNAs, and TA RNPs, which can be studied using multidimensional nuclear magnetic resonance (NMR) spectroscopy methods. Key features Detailed protocol for the large-scale purification of ToxIN type III toxin-antitoxin complexes from E. coli. The optimized protocol results in obtaining milligrams of TA RNP complex, free toxin, and free antitoxin RNA. Commercially available plasmid vectors and chemicals are used to complete the protocol in five days after obtaining the required DNA clones. The purified TA complex, toxin protein, and antitoxin RNA are used for biophysical experiments such as NMR, ITC, and X-ray crystallography.

The novel type II toxin-antitoxin PacTA modulates Pseudomonas aeruginosa iron homeostasis by obstructing the DNA-binding activity of Fur.

Type II toxin–antitoxin (TA) systems are widely distributed in bacterial and archaeal genomes and are involved in diverse critical cellular functions such as defense against phages, biofilm formation, persistence, and virulence. GCN5-related N-acetyltransferase (GNAT) toxin, with an acetyltransferase activity-dependent mechanism of translation inhibition, represents a relatively new and expanding family of type II TA toxins. We here describe a group of GNAT-Xre TA modules widely distributed among Pseudomonas species. We investigated PacTA (one of its members encoded by PA3270/PA3269) from Pseudomonas aeruginosa and demonstrated that the PacT toxin positively regulates iron acquisition in P. aeruginosa. Notably, other than arresting translation through acetylating aminoacyl-tRNAs, PacT can directly bind to Fur, a key ferric uptake regulator, to attenuate its DNA-binding affinity and thus permit the expression of downstream iron-acquisition-related genes. We further showed that the expression of the pacTA locus is upregulated in response to iron starvation and the absence of PacT causes biofilm formation defect, thereby attenuating pathogenesis. Overall, these findings reveal a novel regulatory mechanism of GNAT toxin that controls iron-uptake-related genes and contributes to bacterial virulence.

Molecular mechanism of toxin neutralization in the HipBST toxin-antitoxin system of Legionella pneumophila

Toxin-antitoxin (TA) systems are ubiquitous genetic modules in bacteria and archaea. Here, we perform structural and biochemical characterization of the Legionella pneumophila effector Lpg2370, demonstrating that it is a Ser/Thr kinase. Together with two upstream genes, lpg2370 constitutes the tripartite HipBST TA. Notably, the toxin Lpg2370 (HipTLp) and the antitoxin Lpg2369 (HipSLp) correspond to the C-terminus and N-terminus of HipA from HipBA TA, respectively. By determining crystal structures of autophosphorylated HipTLp, its complex with AMP-PNP, and the structure of HipTLp-HipSLp complex, we identify residues in HipTLp critical for ATP binding and those contributing to its interactions with HipSLp. Structural analysis reveals that HipSLp binding induces a loop-to-helix shift in the P-loop of HipTLp, leading to the blockage of ATP binding and inhibition of the kinase activity. These findings establish the L. pneumophila effector Lpg2370 as the HipBST TA toxin and elucidate the molecular basis for HipT neutralization in HipBST TA.

Cellular Memory of HipA-Induced Growth Arrest: The Length of Cell Growth Arrest Becomes Shorter for Each Successive Induction.

Toxin–antitoxin (TA) systems are genetic modules found commonly in bacterial genomes. HipA is a toxin protein encoded from the hipBA TA system in the genome of Escherichia coli. Ectopic expression of hipA induces cell growth arrest. Unlike the cell growth arrest caused by other TA toxins, cells resume growth from the HipA-induced cell growth arrest phase after a defined period of time. In this article, we describe the change in the length of growth arrest while cells undergo repeated cycles of hipA induction, growth arrest and regrowth phases. In the multiple conditions tested, we observed that the length of growth arrest became successively shorter for each round of induction. We verified that this was not due to the appearance of HipA-resistant mutants. Additionally, we identified conditions, such as the growth phase of the starting culture and growth vessels, that alter the length of growth arrest. Our results showed that the length of HipA-induced growth arrest was dependent on environmental factors—in particular, the past growth environment of cells, such as a previous hipA induction. These effects lasted even after multiple rounds of cell divisions, indicating the presence of cellular “memory” that impacts cells’ response to HipA-induced toxicity.

Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile

Toxin-antitoxin (TA) systems are widespread on mobile genetic elements and in bacterial chromosomes. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA. The first type I TA were recently identified in the human enteropathogen Clostridioides difficile. Here we report the characterization of five additional type I TA within phiCD630-1 (CD0977.1-RCd11, CD0904.1-RCd13 and CD0956.3-RCd14) and phiCD630-2 (CD2889-RCd12 and CD2907.2-RCd15) prophages of C. difficile strain 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest that is neutralized by antitoxin RNA co-expression. We show that type I TA located within the phiCD630-1 prophage contribute to its stability and heritability. We have made use of a type I TA toxin gene to generate an efficient mutagenesis tool for this bacterium that allowed investigation of the role of these widespread TA in prophage maintenance.

Repertoire and Diversity of Toxin - Antitoxin Systems of Crohn's Disease-Associated Adherent-Invasive Escherichia coli. New Insight of T his Emergent E. coli Pathotype.

Adherent-invasive Escherichia coli (AIEC) corresponds to an E. coli pathovar proposed as a possible agent trigger associated to Crohn’s disease. It is characterized for its capacity to adhere and to invade epithelial cells, and to survive and replicate inside macrophages. Mechanisms that allow intestinal epithelium colonization, and host factors that favor AIEC persistence have been partly elucidated. However, bacterial factors involved in AIEC persistence are currently unknown. Toxin–antitoxin (TA) systems are recognized elements involved in bacterial persistence, in addition to have a role in stabilization of mobile genetic elements and stress response. The aim of this study was to elucidate the repertoire and diversity of TA systems in the reference AIEC NRG857c strain and to compare it with AIEC strains whose genomes are available at databases. In addition, toxin expression levels under in vitro stress conditions found by AIEC through the intestine and within the macrophage were measured. Our results revealed that NRG857c encodes at least 33 putative TA systems belonging to types I, II, IV, and V, distributed around all the chromosome, and some in close proximity to genomic islands. A TA toxin repertoire marker of the pathotype was not found and the repertoire of 33 TA toxin genes described here was exclusive of the reference strains, NRG857c and LF82. Most toxin genes were upregulated in the presence of bile salts and acidic pH, as well as within the macrophage. However, different transcriptional responses were detected between reference strains (NRG857c and HM605), recalling the high diversity associated to this pathotype. To our knowledge this is the first analysis of TA systems associated to AIEC and it has revealed new insight associated to this emergent E. coli pathotype.

The Variety in the Common Theme of Translation Inhibition by Type II Toxin-Antitoxin Systems.

Type II Toxin–antitoxin (TA) modules are bacterial operons that encode a toxic protein and its antidote, which form a self-regulating genetic system. Antitoxins put a halter on toxins in many ways that distinguish different types of TA modules. In type II TA modules, toxin and antitoxin are proteins that form a complex which physically sequesters the toxin, thereby preventing its toxic activity. Type II toxins inhibit various cellular processes, however, the translation process appears to be their favorite target and nearly every step of this complex process is inhibited by type II toxins. The structural features, enzymatic activities and target specificities of the different toxin families are discussed. Finally, this review emphasizes that the structural folds presented by these toxins are not restricted to type II TA toxins or to one particular cellular target, and discusses why so many of them evolved to target translation as well as the recent developments regarding the role(s) of these systems in bacterial physiology and evolution.

The RES domain toxins of RES-Xre toxin-antitoxin modules induce cell stasis by degrading NAD+

Type II toxin-antitoxin (TA) modules, which are important cellular regulators in prokaryotes, usually encode two proteins, a toxin that inhibits cell growth and a nontoxic and labile inhibitor (antitoxin) that binds to and neutralizes the toxin. Here, we demonstrate that the res-xre locus from Photorhabdus luminescens and other bacterial species function as bona fide TA modules in Escherichia coli. The 2.2Å crystal structure of the intact Pseudomonas putida RES-Xre TA complex reveals an unusual 2:4 stoichiometry in which a central RES toxin dimer binds two Xre antitoxin dimers. The antitoxin dimers each expose two helix-turn-helix DNA-binding domains of the Cro repressor type, suggesting the TA complex is capable of binding the upstream promoter sequence on DNA. The toxin core domain shows structural similarity to ADP-ribosylating enzymes such as diphtheria toxin but has an atypical NAD+ -binding pocket suggesting an alternative function. We show that activation of the toxin in vivo causes a depletion of intracellular NAD+ levels eventually leading to inhibition of cell growth in E. coli and inhibition of global macromolecular biosynthesis. Both structure and activity are unprecedented among bacterial TA systems, suggesting the functional scope of bacterial TA toxins is much wider than previously appreciated.

GNAT toxins of bacterial toxin-antitoxin systems: acetylation of charged tRNAs to inhibit translation.

GCN5-related N-acetyltransferase (GNAT) is a huge superfamily of proteins spanning the prokaryotic and eukaryotic domains of life. GNAT proteins usually transfer an acetyl group from acetyl-CoA to a wide variety of substrates ranging from aminoglycoside antibiotics to large macromolecules. Type II toxin-antitoxin (TA) modules are typically bicistronic and widespread in bacterial and archael genomes with diverse cellular functions. Recently, a novel family of type II TA toxins was described, which presents a GNAT-fold and functions by acetylating charged tRNA thereby precluding translation. These GNAT toxins are usually associated with a corresponding ribbon-helix-helix-fold (RHH) antitoxin. In this issue, Qian et al. describes a unique GNAT-RHH TA system, designated KacAT, from a multidrug resistant strain of the pathogen, Klebsiella pneumoniae. As most type II TA loci, kacAT is transcriptionally autoregulated with the KacAT complex binding to the operator site via the N-terminus region of KacA to repress kacAT transcription. The crystal structure of the KacT toxin is also presented giving a structural basis for KacT toxicity. These findings expand our knowledge on this newly discovered family of TA toxins and the potential role that they may play in antibiotic tolerance and persistence of bacterial pathogens.

Characterization of YjjJ toxin of Escherichia coli.

Reminiscent of eukaryotic apoptotic programmed cell death, bacteria also contain a large number of suicide genes, which are in general co-expressed with their cognate antitoxin genes. These systems called the toxin-antitoxin (TA) systems are associated with cellular dormancy, and play major roles in biofilm formation and persistent multidrug resistance of many human pathogens. In recent years, the study on TA system toxins has become a hot topic due to the health implications of these toxins by virtue of their role in bacterial pathogenicity. Here we report functional characterization of a hitherto uncharacterized Escherichia coli TA toxin, YjjJ. YjjJ exhibits several uncommon properties: (i) unlike the genes encoding most type II TA system toxins, the gene encoding YjjJ is present as a single gene and not in an operon, (ii) despite being a homolog of the well-characterized toxin HipA, YjjJ seems to have different cellular target(s), and (iii) HipB, the cognate antitoxin of HipA, also acts as an antitoxin for YjjJ. This forms a basis for an interesting next step in the study of TA systems with respect to cross-regulation between various TA systems and the evolutionary as well as clinical significance of these observations.

AtaT blocks translation initiation by N-acetylation of the initiator tRNAfMet.

Toxin-antitoxin (TA) loci are prevalent in bacterial genomes. They are suggested to play a central role in dormancy and persister states. Under normal growth conditions, TA toxins are neutralized by their cognate antitoxins, and under stress conditions, toxins are freed and inhibit essential cellular processes using a variety of mechanisms. Here we characterize ataR-ataT, a novel TA system, from enterohemorrhagic Escherichia coli. We show that the toxin AtaT is a GNAT family enzyme that transfers an acetyl group from acetyl coenzyme A to the amine group of the methionyl aminoacyl moiety of initiator tRNA. AtaT specifically modifies Met-tRNAfMet, but no other aminoacyl-tRNAs, including the elongator Met-tRNAMet. We demonstrate that once acetylated, AcMet-tRNAfMet fails to interact with initiation factor-2 (IF2), resulting in disruption of the translation initiation complex. This work reveals a new mechanism of translation inhibition and confirms Met-tRNAfMet as a prime target to efficiently block cell growth.

Heterologous expression of the Streptococcus pneumoniae yoeB and pezT toxin genes is lethal in Chlorella vulgaris

Chlorella vulgaris is a eukaryotic microalga with potential for the production of biofuels. However, its thick and rigid cell wall is an impediment to cost-effective, large-scale harvesting of biofuels from these cells. Bacterial toxin-antitoxin (TA) systems, comprising of a stable proteic toxin and its labile cognate antitoxin, have no known homologs in eukaryotic cells. Several bacterial TA toxins have been found to be lethal when expressed in eukaryotes such as yeasts, animal and human cell lines. In this study, the functionality of the yoeBSpn and pezT toxin genes from the Gram-positive bacterium Streptococcus pneumoniae in C. vulgaris was investigated using a two-component inducible expression system. The yoeBSpn and pezT toxin genes were each cloned as green fluorescent protein (GFP) fusion constructs and introduced into C. vulgaris by Agrobacterium tumefaciens-mediated co-transformation with recombinant activator and responder vectors. Following induction for the expression of the toxin-GFP fusion transgenes, GFP fluorescence was observed in the transformed C. vulgaris cells which also showed signs of cellular damage and lysis. This is the first report of the lethal expression of bacterial TA toxins in eukaryotic microalgae, which can form the basis of a novel method for harvesting of microalgal cellular contents.

AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental Contexts.

Toxin-antitoxin (TA) systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors.

Toxin-Antitoxin Systems of Staphylococcus aureus.

Toxin-antitoxin (TA) systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA) and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery.

The toxin GraT inhibits ribosome biogenesis.

Most bacteria encode numerous chromosomal toxin-antitoxin (TA) systems that are proposed to contribute to stress tolerance, as they are able to shift the cells to a dormant state. Toxins act on a variety of targets with the majority attacking the translational apparatus. Intriguingly, the toxicity mechanisms of even closely related toxins may differ essentially. Here, we report on a new type of TA toxin that inhibits ribosome biogenesis. GraT of the GraTA system has previously been described in Pseudomonas putida as an unusually moderate toxin at optimal growth temperatures. However, GraT causes a severe growth defect at lower temperatures. Here, we demonstrate that GraT causes the accumulation of free ribosomal subunits. Mapping the rRNA 5' ends reveals incomplete processing of the free subunits and quantification of modified nucleosides shows an underrepresentation of late subunit assembly specific modifications. This indicates that GraT inhibits ribosome subunit assembly. Interestingly, GraT effects can be alleviated by modification of the chaperone DnaK, a known facilitator of late stages in ribosome biogenesis. We show that GraT directly interacts with DnaK and suggest two possible models for the role of this interaction in GraT toxicity.

TRNAs taking charge.

Most bacterial toxins derived from chromosomally encoded toxin–antitoxin (TA) systems that have been studied to date appear to protect cells from relatively short pulses of stress by triggering a reversible state of growth arrest. In contrast to many bacterial toxins that are produced as defense mechanisms and secreted from their hosts, TA toxins exert their protective effect from within the cell that produces them. TA toxin-mediated growth arrest is most frequently achieved through their ability to selectively cleave RNA species that participate in protein synthesis. Until very recently, it was thought that the primary conduit for toxin-mediated translation inhibition was cleavage of a single class of RNA, mRNA, thus depleting transcripts and precluding production of essential proteins. This minireview focuses on how the development and implementation of a specialized RNA-seq method to study Mycobacterium tuberculosis TA systems enabled the identification of unexpected RNA targets for toxins, i.e. a handful of tRNAs that are cleaved into tRNA halves. Our result brings to light a new perspective on how these toxins may act in this pathogen and uncovers a striking parallel to signature features of the eukaryotic stress response.

Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase.

Toxin-antitoxin (TA) systems are implicated in the downregulation of bacterial cell growth associated with stress survival and latent tuberculosis infection, yet the activities and intracellular targets of these TA toxins are largely uncharacterized. Here, we use a specialized RNA-seq approach to identify targets of a Mycobacterium tuberculosis VapC TA toxin, VapC-mt4 (also known as VapC4), which have eluded detection using conventional approaches. Distinct from the one other characterized VapC toxin in M. tuberculosis that cuts 23S rRNA at the sarcin-ricin loop, VapC-mt4 selectively targets three of the 45 M. tuberculosis tRNAs (tRNA(Ala2), tRNA(Ser26) and tRNA(Ser24)) for cleavage at, or adjacent to, their anticodons, resulting in the generation of tRNA halves. While tRNA cleavage is sometimes enlisted as a bacterial host defense mechanism, VapC-mt4 instead alters specific tRNAs to inhibit translation and modulate growth. This stress-linked activity of VapC-mt4 mirrors basic features of eukaryotic tRNases that also generate tRNA halves and inhibit translation in response to stress.

Post-transcriptional regulation of gene expression in bacterial pathogens by toxin-antitoxin systems

Toxin-antitoxin (TA) systems are small genetic elements ubiquitous in prokaryotic genomes that encode toxic proteins targeting various vital cellular functions. Typically, toxin activity is controlled by adjacently encoded protein or RNA antitoxins and unleashed as a consequence of genetic fluctuations or stressful conditions. Whereas some TA systems interfere with replication or cell wall synthesis, most of them influence transcriptional and post-transcriptional gene regulation. Antitoxin proteins often act as DNA binding transcriptional regulators and many TA toxins exhibit endoribonuclease activity to selectively degrade different RNA species and thus alter gene expression patterns. Some TA RNases cleave tRNA, tmRNAs or rRNAs, whereas most commonly mRNAs either in association with the ribosome or as free transcripts, are targeted. Examples are provided on how TA toxins differentially shape gene expression in bacterial pathogens by creating specialized ribosomes or by altering the transcriptome and how this may be tied in the control of pathogenicity factors.