Bacterial Toxin-antitoxin Systems Research Articles

Toxic small alarmone synthetase FaRel2 inhibits translation by pyrophosphorylating tRNAGly and tRNAThr.

Translation-targeting toxic small alarmone synthetases (toxSAS) are effectors of bacterial toxin-antitoxin systems that pyrophosphorylate the 3'-CCA end of transfer RNA (tRNA) to prevent aminoacylation. toxSAS are implicated in antiphage immunity: Phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNAGly and tRNAThr. The first, second, fourth, and fifth base pairs of the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRelSJ46 differ in tRNA specificity from FaRel2 and rationalize this through structural modeling: While the universal 3'-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defenses, we hypothesize that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

Conformational change as a mechanism for toxin activation in bacterial toxin-antitoxin systems.

Toxin/antitoxin (TA) systems are present in nearly every prokaryotic genome and play the important physiological roles of phage inhibition by reducing metabolism (this includes persistence for the extreme case of complete cessation of metabolism), genetic element stabilization, and biofilm formation. TA systems have also been incorporated into other cell systems, such as CRISPR-Cas and phage quorum sensing. For the simplest and best-studied case, proteinaceous toxins and antitoxins (i.e., type II), toxin activity is masked by direct binding of the antitoxin. A long-standing, unresolved question in the TA field is how toxins are activated when bound to antitoxins at nanomolar affinity. The current paradigm envisions preferential degradation of the antitoxin by a protease, but this is highly unlikely in that a protease cannot discriminate between bound toxin and bound antitoxin because both are highly structured. Strikingly, recent results from several studies show one likely mechanism for toxin activation is conformational changes in the TA complex that result in the release or activation of the toxin as a result of a protein trigger, such as that from phages, and as a result of thermally-driven refolding dynamics.

Characterization of a membrane toxin-antitoxin system, tsaAT, from Staphylococcus aureus.

Bacterial toxin-antitoxin (TA) systems consist of a toxin that inhibits essential cellular processes, such as DNA replication, transcription, translation, or ATP synthesis, and an antitoxin neutralizing their cognate toxin. These systems have roles in programmed cell death, defense against phage, and the formation of persister cells. Here, we characterized the previously identified Staphylococcus aureus TA system, tsaAT, which consists of two putative membrane proteins: TsaT and TsaA. Expression of the TsaT toxin caused cell death and disrupted membrane integrity, whereas TsaA did not show any toxicity and neutralized the toxicity of TsaT. Furthermore, subcellular fractionation analysis demonstrated that both TsaA and TsaT localized to the cytoplasmic membrane of S. aureus expressing either or both 3xFLAG-tagged TsaA and 3xFLAG-tagged TsaT. Taken together, these results demonstrate that the TsaAT TA system consists of two membrane proteins, TsaA and TsaT, where TsaT disrupts membrane integrity, ultimately leading to cell death. Although sequence analyses showed that the tsaA and tsaT genes were conserved among Staphylococcus species, amino acid substitutions between TsaT orthologs highlighted the critical role of the 6th residue for its toxicity. Further amino acid substitutions indicated that the glutamic acid residue at position 63 in the TsaA antitoxin and the cluster of five lysine residues in the TsaT toxin are involved in TsaA's neutralization reaction. This study is the first to describe a bacterial TA system wherein both toxin and antitoxin are membrane proteins. These findings contribute to our understanding of S. aureus TA systems and, more generally, give new insight into highly diverse bacterial TA systems.

Use of Bacterial Toxin-Antitoxin Systems as Biotechnological Tools in Plants.

Toxin-antitoxin (TA) systems in bacteria are key regulators of the cell cycle and can activate a death response under stress conditions. Like other bacterial elements, TA modules have been widely exploited for biotechnological purposes in diverse applications, such as molecular cloning and anti-cancer therapies. However, their use in plants has been limited, leaving room for the development of new approaches. In this study, we examined two TA systems previously tested in plants, MazEF and YefM-YoeB, and identified interesting differences between them, likely related to their modes of action. We engineered modifications to these specific modules to transform them into molecular switches that can be activated by a protease, inducing necrosis in the plant cells where they are expressed. Finally, we demonstrated the antiviral potential of the modified TA modules by using, as a proof-of-concept, the potyvirus plum pox virus as an activator of the death phenotype.

Backbone assignment of CcdB_G100T toxin from E.coli in complex with the toxin binding C-terminal domain of its cognate antitoxin CcdA.

The CcdAB system expressed in the E.coli cells is a prototypical example of the bacterial toxin-antitoxin (TA) systems that ensure the survival of the bacterial population under adverse environmental conditions. The solution and crystal structures of CcdA, CcdB and of CcdB in complex with the toxin-binding C-terminal domain of CcdA have been reported. Our interest lies in the dynamics of CcdB-CcdA complex formation. Solution NMR studies have shown that CcdB_G100T, in presence of saturating concentrations of CcdA-c, a truncated C-terminal fragment of CcdA exists in equilibrium between two major populations. Sequence specific backbone resonance assignments of both equilibrium forms of the ~ 27kDa complex, have been obtained from a suite of triple resonance NMR experiments acquired on 2H, 13C, 15N enriched samples of CcdB_G100T. Analysis of 1H, 13Cα, 13Cβ secondary chemical shifts, shows that both equilibrium forms of CcdB_G100T have five beta-strands and one alpha-helix as the major secondary structural elements in the tertiary structure. The results of these studies are presented below.

Toxic Small Alarmone Synthetase FaRel2 inhibits translation by pyrophosphorylating tRNAGly and tRNAThr.

Translation-targeting toxic Small Alarmone Synthetases (toxSAS) are effectors of bacterial Toxin-Antitoxin systems that pyrophosphorylate the 3'-CCA end of tRNA to prevent aminoacylation. toxSAS are implicated in antiphage immunity: phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNAGly and tRNAThr. The 1st, 2nd, 4th and 5th base pairs the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRelSJ46 differ in tRNA specificity from FaRel2, and rationalise this through structural modelling: while the universal 3'-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defences, we hypothesise that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

TAC-TIC, a high-throughput genetics method to identify triggers or blockers of bacterial toxin-antitoxin systems.

Toxin-antitoxin systems (TAs) are abundant in bacterial chromosomes and can arrest growth under stress, but usually remain inactive. TAs have been increasingly implicated in halting the growth of infected bacteria from bacteriophages or foreign genetic elements1,2 to protect the population (abortive infection, Abi). The vast diversity and abundance of TAs and other Abi systems3 suggest they play an important immunity role, yet what allows them to sense attack remains largely enigmatic. Here, we describe a method called toxin activation-inhibition conjugation (TAC-TIC), which we used to identify gene products that trigger or block the toxicity of phage-defending tripartite retron-TAs4. TAC-TIC employs high-density arrayed mobilizable gene-overexpression libraries, which are transferred into cells carrying the full TA system or only its toxic component, on inducible vectors. The double-plasmid transconjugants are then pinned on inducer-containing agar plates and their colony fitness is quantified to identify gene products that trigger a TA to inhibit growth (TAC), or that block it from acting (TIC). TAC-TIC is optimized for the Singer ROTOR pinning robot, but can also be used with other robots or manual pinners, and allows screening tens of thousands of genes against any TA or Abi (with toxicity) within a week. Finally, we present a dual conjugation donor/cloning strain (Escherichia coli DATC), which accelerates the construction of TAC-TIC gene-donor libraries from phages, enabling the use of TAC-TIC for identifying TA triggers and antidefense mechanisms in phage genomes.

Specificity of DNA ADP-Ribosylation Reversal by NADARs.

Recent discoveries establish DNA and RNA as bona fide substrates for ADP-ribosylation. NADAR ("NAD- and ADP-ribose"-associated) enzymes reverse guanine ADP-ribosylation and serve as antitoxins in the DarT-NADAR operon. Although NADARs are widespread across prokaryotes, eukaryotes, and viruses, their specificity and broader physiological roles remain poorly understood. Using phylogenetic and biochemical analyses, we further explore de-ADP-ribosylation activity and antitoxin functions of NADAR domains. We demonstrate that different subfamilies of NADAR proteins from representative E. coli strains and an E. coli-infecting phage retain biochemical activity while displaying specificity in providing protection from toxic guanine ADP-ribosylation in cells. Furthermore, we identify a myxobacterial enzyme within the YbiA subfamily that functions as an antitoxin for its associated DarT-unrelated ART toxin, which we termed YarT, thus presenting a hitherto uncharacterised ART-YbiA toxin-antitoxin pair. Our studies contribute to the burgeoning field of DNA ADP-ribosylation, supporting its physiological relevance within and beyond bacterial toxin-antitoxin systems. Notably, the specificity and confinement of NADARs to non-mammals infer their potential as highly specific targets for antimicrobial drugs with minimal off-target effects.

Discovery of Antimicrobial Agents Based on Structural and Functional Study of the Klebsiella pneumoniae MazEF Toxin-Antitoxin System.

Klebsiella pneumoniae causes severe human diseases, but its resistance to current antibiotics is increasing. Therefore, new antibiotics to eradicate K. pneumoniae are urgently needed. Bacterial toxin-antitoxin (TA) systems are strongly correlated with physiological processes in pathogenic bacteria, such as growth arrest, survival, and apoptosis. By using structural information, we could design the peptides and small-molecule compounds that can disrupt the binding between K. pneumoniae MazE and MazF, which release free MazF toxin. Because the MazEF system is closely implicated in programmed cell death, artificial activation of MazF can promote cell death of K. pneumoniae. The effectiveness of a discovered small-molecule compound in bacterial cell killing was confirmed through flow cytometry analysis. Our findings can contribute to understanding the bacterial MazEF TA system and developing antimicrobial agents for treating drug-resistant K. pneumoniae.

Jatrophihabitans cynanchi sp. nov., isolated from rhizosphere soil of Cynanchum wilfordii.

A novel actinobacterial strain, SB3-54T was isolated from rhizosphere soil of Cynanchum wilfodill, Jaecheon, Chungcheongbuk-do, Republic of Korea. Cells of strain SB3-54T were Gram-stain-positive, aerobic, rod-shaped, and flagellated which formed pale yellow colonies on Reasoner's 2A (R2A) agar. Growth occurred at 15-30°C (optimum 25°C), pH 5-8 (optimum pH 7), and 0-2.5% NaCl (optimum 0%). Phylogenetic and phylogenomic analyses showed that strain SB3-54T formed a separate lineage in the genus Jatrophihabitans with Jatrophihabitans telluris N237T. Strain SB3-54T was positive for catalase activity. Genomic analysis showed that SB3-54T has plant-beneficial function contributing (referred to as PBFC) genes such as root colonization and plant protection from oxidative stress. Furthermore, genome of SB3-54T contained gene clusters related to cytokinin biosynthesis, auxin response, tryptophan biosynthesis, siderophore biosynthesis and bacterial toxin-antitoxin systems. Strain SB3-54T contained iso-C16:0 as the major fatty acid and MK-9(H4) and MK-9(H6) as the predominant quinones. The organism had meso-diaminopimelic acid as the diagnostic diamino acid in the peptidoglycan. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol polymannosides, two unidentified aminoglycophospholipids and three unidentified phospholipids. Based on phylogenetic, physiological and chemotaxonomic characteristics, strain SB3-54T represents a novel species of the genus Jatrophihabitans. The type strain is SB3-54T (= KCTC 49134T = NBRC 114108T).

Arbitrium communication controls phage lysogeny through non-lethal modulation of a host toxin-antitoxin defence system.

Temperate Bacillus phages often utilize arbitrium communication to control lysis/lysogeny decisions, but the mechanisms by which this control is exerted remains largely unknown. Here we find that the arbitrium system of Bacillus subtilis phage ϕ3T modulates the host-encoded MazEF toxin-antitoxin system to this aim. Upon infection, the MazF ribonuclease is activated by three phage genes. At low arbitrium signal concentrations, MazF is inactivated by two phage-encoded MazE homologues: the arbitrium-controlled AimX and the later-expressed YosL proteins. At high signal, MazF remains active, promoting lysogeny without harming the bacterial host. MazF cleavage sites are enriched on transcripts of phage lytic genes but absent from the phage repressor in ϕ3T and other Spβ-like phages. Combined with low activation levels of MazF during infections, this pattern explains the phage-specific effect. Our results show how a bacterial toxin-antitoxin system has been co-opted by a phage to control lysis/lysogeny decisions without compromising host viability.

Structural Variations and Rearrangements in Bacterial Type II Toxin-Antitoxin Systems.

Bacteria encode a wide range of survival and immunity systems, including CRISPR-Cas, restriction-modification systems, and toxin-antitoxin systems involved in defence against bacteriophages, as well as survival during challenging growth conditions or exposure to antibiotics. Toxin-antitoxin (TA) systems are small two- or three-gene cassettes consisting of a metabolic regulator (the "toxin") and its associated antidote (the "antitoxin"), which also often functions as a transcriptional regulator. TA systems are widespread in the genomes of pathogens but are also present in commensal bacterial species and on plasmids. For mobile elements such as plasmids, TA systems play a role in maintenance, and increasing evidence now points to roles of chromosomal toxin-antitoxin systems in anti-phage defence. Moreover, the widespread occurrence of toxin-antitoxin systems in the genomes of pathogens has been suggested to relate to survival during host infection as well as in persistence during antibiotic treatment. Upon repeated exposure to antibiotics, TA systems have been shown to acquire point mutations as well as more dramatic rearrangements such as in-frame deletions with potential relevance for bacterial survival and pathogenesis. In this review, we present an overview of the known functional and structural consequences of mutations and rearrangements arising in bacterial toxin-antitoxin systems and discuss their relevance for survival and persistence of pathogenic species.

Antagonistic interactions between phage and host factors control arbitrium lysis–lysogeny decision

Phages can use a small-molecule communication arbitrium system to coordinate lysis–lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin–antitoxin system MazE–MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis–lysogeny by domesticating and fine-tuning a phage-defence mechanism.

Molecular basis for the reversible ADP-ribosylation of guanosine bases

Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems.

Bacterial Toxin-Antitoxin Systems' Cross-Interactions-Implications for Practical Use in Medicine and Biotechnology.

Toxin-antitoxin (TA) systems are widely present in bacterial genomes. They consist of stable toxins and unstable antitoxins that are classified into distinct groups based on their structure and biological activity. TA systems are mostly related to mobile genetic elements and can be easily acquired through horizontal gene transfer. The ubiquity of different homologous and non-homologous TA systems within a single bacterial genome raises questions about their potential cross-interactions. Unspecific cross-talk between toxins and antitoxins of non-cognate modules may unbalance the ratio of the interacting partners and cause an increase in the free toxin level, which can be deleterious to the cell. Moreover, TA systems can be involved in broadly understood molecular networks as transcriptional regulators of other genes' expression or modulators of cellular mRNA stability. In nature, multiple copies of highly similar or identical TA systems are rather infrequent and probably represent a transition stage during evolution to complete insulation or decay of one of them. Nevertheless, several types of cross-interactions have been described in the literature to date. This implies a question of the possibility and consequences of the TA system cross-interactions, especially in the context of the practical application of the TA-based biotechnological and medical strategies, in which such TAs will be used outside their natural context, will be artificially introduced and induced in the new hosts. Thus, in this review, we discuss the prospective challenges of system cross-talks in the safety and effectiveness of TA system usage.

Comparative analysis of five type II TA systems identified in Pseudomonas aeruginosa reveals their contributions to persistence and intracellular survival.

Pseudomonas aeruginosa is a grave nosocomial pathogen that persistently inhabits the lungs of patients with cystic fibrosis (CF) and causes various chronic infections. The bacterial toxin-antitoxin (TA) system is associated with latent and long-term infections, but the underlying mechanisms remain to be fully characterized. We here investigated the diversity and function of five genomic type II TA systems widely distributed among P. aeruginosa clinical isolates. We also examined the distinct structural features of the toxin protein from different TA systems and characterized their contributions to persistence, invasion ability, and intracellular infection caused by P. aeruginosa. ParDE, PA1030/PA1029, and HigBA could modulate persister cell formation under treatment with specific antibiotics. Furthermore, cell-based transcriptional and invasion assays revealed that PA1030/PA1029 and HigBA TA systems were critical for intracellular survival. Our results highlight the prevalence and diverse roles of type II TA systems in P. aeruginosa and evaluate the possibility of using PA1030/PA1029 and HigBA TA pairs as targets for novel antibiotic treatments.

Role of PemI in the Staphylococcus aureus PemIK toxin-antitoxin complex: PemI controls PemK by ating as a PeK loop mimic.

Staphylococcus aureus is a notorious and globally distributed pathogenic bacterium. New strategies to develop novel antibiotics based on intrinsic bacterial toxin-antitoxin (TA) systems have been recently reported. Because TA systems are present only in bacteria and not in humans, these distinctive systems are attractive targets for developing antibiotics with new modes of action. S. aureus PemIK is a type II TA system, comprising the toxin protein PemK and the labile antitoxin protein PemI. Here, we determined the crystal structures of both PemK and the PemIK complex, in which PemK is neutralized by PemI. Our biochemical approaches, including fluorescence quenching and polarization assays, identified Glu20, Arg25, Thr48, Thr49, and Arg84 of PemK as being important for RNase function. Our study indicates that the active site and RNA-binding residues of PemK are covered by PemI, leading to unique conformational changes in PemK accompanied by repositioning of the loop between β1 and β2. These changes can interfere with RNA binding by PemK. Overall, PemK adopts particular open and closed forms for precise neutralization by PemI. This structural and functional information on PemIK will contribute to the discovery and development of novel antibiotics in the form of peptides or small molecules inhibiting direct binding between PemI and PemK.

Tuning the expression of the bacterial relBE toxin–antitoxin system inSaccharomyces cerevisiaeallows characterizing the subsequent growth inhibition

The bacterial toxin-antitoxin systems are each composed of a toxin, which severely inhibits bacterial cells growth, and a specific neutralizing antitoxin. Some toxin-antitoxin systems are functional when expressed in the yeast Saccharomyces cerevisiae. For instance, the expression of the relE toxin gene leads to a strong growth defect in yeast, whereas the expression of the relB antitoxin gene restores growth. Nevertheless, there is no available data regarding the required expression levels of each component of the relBE system leading to these growth phenotypes, neither their effects on cell viability. Here we used a double inducible plasmid-based system to independently modulate the relative amounts of relB and relE, and performed growth and gene expression analyses. These results allow us to correlate growth phenotypes to the expression levels of the toxin and the antitoxin, and to determine the levels necessary to observe either a strong growth inhibition or a normal growth. We also showed that the relE expression produces cell cycle progression defect without affecting cell viability. These results provide a detailed characterization of the functioning of the relBE system in S. cerevisiae, and open applicative perspectives of yeast growth control by bacterial toxin-antitoxin systems.

The role of bacterial toxin-antitoxin systems in phage abortive infections

Bacteria are often infected by large numbers of phages, and host bacteria have evolved diverse molecular strategies in the race with phages, with abortive infection (Abi) being one of them. The toxin-antitoxin system (TA) is expressed in response to bacterial stress, mediating hypometabolism and even dormancy, as well as directly reducing the formation of offspring phages. In addition, some of the toxins' sequences and structures are highly homologous to Cas, and phages even encode antitoxin analogs to block the activity of the corresponding toxins. This suggests that the failure of phage infection due to bacterial death in abortive infections is highly compatible with TA function, whereas TA may be one of the main resistance and defense forces for phage infestation of the host. This review summarized the TA systems involved in phage abortive infections based on classification and function. Moreover, TA systems with abortive functions and future use in antibiotic development and disease treatment were predicted. This will facilitate the understanding of bacterial-phage interactions as well as phage therapy and related synthetic biology research.

Functional analysis of Escherichia coli K12 toxin-antitoxin systems as novel drug targets using a network biology approach

Bacterial resistance to various drugs and antibiotics has become a significant issue in the fight against infectious diseases. Due to the presence of diverse toxin-antitoxin (TA) systems, bacteria undergo adaptive metabolic alterations and can tolerate the effects of drugs and antibiotics. Bacterial TA systems are unique and can be therapeutic targets for developing new antimicrobial agents, owing to their ability to influence bacterial fate. With this background, our study aims to identify novel drug targets against Escherichia coli K12 MG1655 antitoxin using homology modelling approach. In this study, the protein-protein interaction network of 87 E. coli K12 MG1655 TA systems identified through literature mining was screened for the identification of hub proteins. The model evaluation, assessment, and homology modelling of the hub proteins were evaluated. Furthermore, computer-aided mathematical models of selected phytochemicals have been tested against the identified hub proteins. The TA system was functionally enriched in regulation of cell growth, negative regulation of cell growth, regulation of mRNA stability, mRNA catabolic process and RNA phosphodiester bond hydrolysis. RelE, RelB, MazE, MazF, MqsR, MqsA, and YoeB were identified as hub proteins. The robustness and superior quality of the RelB and MazE modelled structure were discovered by model evaluation, quality assessment criteria, and homology modelling of hub proteins. Clorobiocin was found to be a strong inhibitor by docking these modelled structures. Clorobiocin could be utilized as an antibacterial agent against multidrug resistant E. coli which may inactivate antitoxins and cause programmed cell death.