Relevance of charged and polar amino acids for functionality of membrane toxin TisB.

Bacterial dormancy is marked by reduced cellular activity and the suspension of growth. It represents a valuable strategy to survive stressful conditions, as exemplified by the long-term tolerance towards antibiotics that is attributable to a fraction of dormant cells, so-called persisters. Here, we investigate the membrane toxin TisB (29 amino acids) from the chromosomal toxin-antitoxin system tisB/istR-1 in Escherichia coli. TisB depolarizes the inner membrane in response to DNA damage, which eventually promotes a stress-tolerant state of dormancy within a small fraction of the population. Using a plasmid-based system for moderate tisB expression and single amino acid substitutions, we dissect the importance of charged and polar amino acids. We observe that the central amino acids lysine 12 and glutamine 19 are of major importance for TisB functionality, which is further validated for lysine 12 in the native context upon treatment with the DNA-damaging antibiotic ciprofloxacin. Finally, we apply a library-based approach to test additional TisB variants in higher throughput, revealing that at least one positive charge at the C-terminus (either lysine 26 or 29) is mandatory for TisB-mediated dormancy. Our study provides insights into the molecular basis for TisB functionality and extends our understanding of bacterial membrane toxins.

Cyclic di-GMP as an antitoxin regulates bacterial genome stability and antibiotic persistence in biofilms.

Biofilms are complex bacterial communities characterized by a high persister prevalence, which contributes to chronic and relapsing infections. Historically, persister formation in biofilms has been linked to constraints imposed by their dense structures. However, we observed an elevated persister frequency accompanying the stage of cell adhesion, marking the onset of biofilm development. Subsequent mechanistic studies uncovered a comparable type of toxin-antitoxin (TA) module (TA-like system) triggered by cell adhesion, which is responsible for this elevation. In this module, the toxin HipH acts as a genotoxic deoxyribonuclease, inducing DNA double strand breaks and genome instability. While the second messenger c-di-GMP functions as the antitoxin, exerting control over HipH expression and activity. The dynamic interplay between c-di-GMP and HipH levels emerges as a crucial determinant governing genome stability and persister generation within biofilms. These findings unveil a unique TA system, where small molecules act as the antitoxin, outlining a biofilm-specific molecular mechanism influencing genome stability and antibiotic persistence, with potential implications for treating biofilm infections.

Bacteria renew an OLD protein to cleave host tRNAs and block phage translation.

Anti-phage defenses must rapidly sense and respond to diverse viruses. A recent pair of papers in Nature reveal via structural and functional assays how the PARIS defense system, a recently discovered toxin-antitoxin system, senses phage-associated molecular patterns (PhAMPs), thereby activating an endonuclease toxin that cleaves tRNA to block phage replication.

Long Dynamic ß1-ß2 Loops in M. tb MazF Toxins Affect the Interaction Modes and Strengths of the Toxin-Antitoxin Pairs.

Tuberculosis is a worldwide plague caused by the pathogen Mycobacterium tuberculosis (M. tb). Toxin-antitoxin (TA) systems are genetic elements abundantly present in prokaryotic organisms and regulate important cellular processes. MazEF is a TA system implicated in the formation of "persisters cells" of M. tb, which contain more than 10 such members. However, the exact function and inhibition mode of each MazF are not fully understood. Here we report crystal structures of MazF-mt3 in its apo form and in complex with the C-terminal half of MazE-mt3. Structural analysis suggested that two long but disordered ß1-ß2 loops would interfere with the binding of the cognate MazE-mt3 antitoxin. Similar loops are also present in the MazF-mt1 and -mt9 but are sustainably shortened in other M. tb MazF members, and these TA pairs behave distinctly in terms of their binding modes and their RNase activities. Systematic crystallographic and biochemical studies further revealed that the biochemical activities of M. tb toxins were combined results between the interferences from the characteristic loops and the electrostatic interactions between the cognate TA pairs. This study provides structural insight into the binding mode and the inhibition mechanism of the MazE/F TA pairs, which facilitate the structure-based peptide designs.

Regulation of an RNA toxin-antitoxin system, SdsR-RyeA, by a small RNA GcvB.

The toxin-antitoxin (TA) system regulates many physiological processes in free-living bacteria. One such TA system in Escherichia coli comprises an RNA toxin SdsR and an antitoxin RyeA. An overabundance of SdsR is toxic to the cells. RyeA normalizes SdsR abundance and helps the cells to adapt to altered conditions. The current study showed that a novel small RNA (sRNA) regulator GcvB directly interacts with RyeA to maintain its abundance in the cells under normal or low pH conditions. The deletion of the gcvB allele in the E. coli chromosome resulted in a ∼3-fold decrease in intrabacterial RyeA accumulation. An ectopic expression of GcvB in ΔgcvB strain reinstated RyeA abundance to its normal level. Induction of GcvB in the cells upon exposure to low pH resulted in a simultaneous increase in intracellular RyeA. While GcvB increases RyeA abundance in the cells, SdsR accumulation is divergently regulated by GcvB. The absence of the gcvB gene in E. coli leads to upregulation of SdsR and vice versa. The GcvB-mediated decrease of SdsR accumulation stems from the increased RyeA-driven normalization of SdsR. This study delineates a novel mechanism for the regulation of the expression of an RNA toxin SdsR by another sRNA regulator GcvB through a feed-forward control.

Addictive manipulation: a perspective on the role of reproductive parasitism in the evolution of bacteria-eukaryote symbioses.

Wolbachia bacteria encompass noteworthy reproductive manipulators of their arthropod hosts. which influence host reproduction to favour their own transmission, also exploiting toxin-antitoxin systems. Recently, multiple other bacterial symbionts of arthropods have been shown to display comparable manipulative capabilities. Here, we wonder whether such phenomena are truly restricted to arthropod hosts. We focused on protists, primary models for evolutionary investigations on eukaryotes due to their diversity and antiquity, but still overall under-investigated. After a thorough re-examination of the literature on bacterial-protist interactions with this question in mind, we conclude that such bacterial 'addictive manipulators' of protists do exist, are probably widespread, and have been overlooked until now as a consequence of the fact that investigations are commonly host-centred, thus ineffective to detect such behaviour. Additionally, we posit that toxin-antitoxin systems are crucial in these phenomena of addictive manipulation of protists, as a result of recurrent evolutionary repurposing. This indicates intriguing functional analogy and molecular homology with plasmid-bacterial interplays. Finally, we remark that multiple addictive manipulators are affiliated with specific bacterial lineages with ancient associations with diverse eukaryotes. This suggests a possible role of addictive manipulation of protists in paving the way to the evolution of bacteria associated with multicellular organisms.

Identification and characterization of a novel type II toxin-antitoxin system in Aeromonas veronii.

The bacterial type II toxin-antitoxin (TA) system is a rich genetic element that participates in various physiological processes. Aeromonas veronii is the main bacterial pathogen threatening the freshwater aquaculture industry. However, the distribution of type II TA system in A. veronii was seldom documented and its roles in the life activities of A. veronii were still unexplored. In this study, a novel type II TA system AvtA-AvtT was predicted in a fish pathogen Aeromonas veronii biovar sobria with multi-drug resistance using TADB 2.0. Through an Escherichia coli host killing and rescue assay, we demonstrated that AvtA and AvtT worked as a genuine TA system, and the predicted toxin AvtT actually functioned as an antitoxin while the predicted antitoxin AvtA actually functioned as a toxin. The binding ability of AvtA with AvtT proteins were confirmed by dot blotting analysis and co-immunoprecipitation assay. Furthermore, we found that the toxin and antitoxin labelled with fluorescent proteins were co-localized. In addition, it was found that the transcription of AvtAT bicistronic operon was repressed by the AvtAT protein complex. Deletion of avtA gene and avtT gene had no obvious effect on the drug susceptibility. This study provides first characterization of type II TA system AvtA-AvtT in aquatic pathogen A. veronii.

Architecture and activation mechanism of the bacterial PARIS defence system.

Bacteria and their viruses (bacteriophages or phages) are engaged in an intense evolutionary arms race1-5. While the mechanisms of many bacterial antiphage defence systems are known1, how these systems avoid toxicity outside infection yet activate quickly after infection is less well understood. Here we show that the bacterial phage anti-restriction-induced system (PARIS) operates as a toxin-antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefence protein Ocr. Using cryo-electron microscopy, we show that AriA is related to SMC-family ATPases but assembles into a distinctive homohexameric complex through two oligomerization interfaces. In uninfected cells, the AriA hexamer binds to up to three monomers of AriB, maintaining them in an inactive state. After Ocr binding, the AriA hexamer undergoes a structural rearrangement, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease, the activation of which arrests cell growth and inhibits phage propagation by globally inhibiting protein translation through specific cleavage of a lysine tRNA. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defence system triggered by a phage counterdefence protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor.

A type II toxin-antitoxin system is responsible for the cell death at low temperature in Pseudomonas syringae Lz4W lacking RNase R.

RNase R (encoded by the rnr gene) is a highly processive 3' → 5' exoribonuclease essential for the growth of the psychrotrophic bacterium Pseudomonas syringae Lz4W at low temperature. The cell death of a rnr deletion mutant at low temperature has been previously attributed to processing defects in 16S rRNA, defective ribosomal assembly, and inefficient protein synthesis. We recently showed that RNase R is required to protect P. syringae Lz4W from DNA damage and oxidative stress, independent of its exoribonuclease activity. Here, we show that the processing defect in 16S rRNA does not cause cell death of the rnr mutant of P. syringae at low temperature. Our results demonstrate that the rnr mutant of P. syringae Lz4W, complemented with a RNase R deficient in exoribonuclease function (RNase RD284A), is defective in 16S rRNA processing but can grow at 4 °C. This suggested that the processing defect in ribosomal RNAs is not a cause of the cold sensitivity of the rnr mutant. We further show that the rnr mutant accumulates copies of the indigenous plasmid pLz4W that bears a type II toxin-antitoxin (TA) system (P. syringae antitoxin-P. syringae toxin). This phenotype was rescued by overexpressing antitoxin psA in the rnr mutant, suggesting that activation of the type II TA system leads to cold sensitivity of the rnr mutant of P. syringae Lz4W. Here, we report a previously unknown functional relationship between the cold sensitivity of the rnr mutant and a type II TA system in P. syringae Lz4W.

Characterization of mycobacteriophage Adephagia cytotoxic proteins.

Mycobacterium phage Adephagia is a cluster K phage that infects Mycobacterium smegmatis and some strains of Mycobacterium pathogens. Adephagia has a siphoviral virion morphology and is temperate. Its genome is 59,646 bp long and codes for one tRNA gene and 94 predicted protein-coding genes; most genes not associated with virion structure and assembly are functionally ill-defined. Here, we determined the Adephagia gene expression patterns in lytic and lysogenic growth and used structural predictions to assign additional gene functions. We characterized 66 nonstructural genes for their toxic phenotypes when expressed in M. smegmatis, and we show that 25 of these (38%) are either toxic or strongly inhibit growth, resulting in either reduced viability or small colony sizes. Some of these genes are predicted to be involved in DNA metabolism or regulation, but others are of unknown function. We also characterize the HicAB-like toxin-antitoxin (TA) system encoded by Adephagia (gp91 and gp90, respectively) and show that the gp90 antitoxin is lysogenically expressed, abrogates gp91 toxicity, and is required for normal lytic and lysogenic growth.

Decoding the TAome and computational insights into parDE toxin-antitoxin systems in Pseudomonas aeruginosa.

Toxin-antitoxin (TA) modules are widely found in the genomes of pathogenic bacteria. They regulate vital cellular functions like transcription, translation, and DNA replication, and are therefore essential to the survival of bacteria under stress. With a focus on the type II parDE modules, this study thoroughly examines TAome in Pseudomonas aeruginosa, a bacterium well-known for its adaptability and antibiotic resistance. We explored the TAome in three P. aeruginosa strains: ATCC 27,853, PAO1, and PA14, and found 15 type II TAs in ATCC 27,853, 12 in PAO1, and 13 in PA14, with significant variation in the associated mobile genetic elements. Five different parDE homologs were found by further TAome analysis in ATCC 27,853, and their relationships were confirmed by sequence alignments and precise genomic positions. After comparing these ParDE modules' sequences to those of other pathogenic bacteria, it was discovered that they were conserved throughout many taxa, especially Proteobacteria. Nucleic acids were predicted as potential ligands for ParD antitoxins, whereas ParE toxins interacted with a wide range of small molecules, indicating a diverse functional repertoire. The interaction interfaces between ParDE TAs were clarified by protein-protein interaction networks and docking studies, which also highlighted important residues involved in binding. This thorough examination improves our understanding of the diversity, evolutionary dynamics, and functional significance of TA systems in P. aeruginosa, providing insights into their roles in bacterial physiology and pathogenicity.

Chaperones help TACkle phage infection.

Bacteria have evolved anti-viral defenses, but the mechanisms of sensing and stopping infection are still under investigation. In this issue of Cell Host & Microbe, Mets, Kurata, Ernits et al. describe how direct sensing of a phage protein by a bacterial toxin-antitoxin-associated chaperone unleashes toxin activity to prevent infection.

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.

A simple BLASTn-based approach generates novel insights into the regulation and biological function of type I toxin-antitoxins.

Bacterial chromosomal type I toxin-antitoxin systems consist of a small protein, typically under 60 amino acids, and a small RNA (sRNA) that represses toxin translation. These gene pairs have gained attention over the last decade for their contribution to antibiotic persistence and phage tolerance in bacteria. However, biological functions for many remain elusive as gene deletions often fail to produce an observable phenotype. For many pairs, it is still unknown when the toxin and/or antitoxin gene are natively expressed within the bacterium. We examined sequence conservation of three type I toxin-antitoxin systems, tisB/istR-1, shoB/ohsC, and zor/orz, in over 2,000 Escherichia coli strains, including pathogenic and commensal isolates. Using our custom database, we found that these gene pairs are widespread across E. coli and have expression potential via BLASTn. We identified an alternative, dominant sequence variant of TisB and confirmed that it is toxic upon overproduction. Additionally, analyses revealed a highly conserved sequence in the zorO mRNA untranslated region that is required for full toxicity. We further noted that over 30% of E. coli genomes contain an orz antitoxin gene only and confirmed its expression in a representative strain: the first confirmed report of a type I antitoxin without its cognate toxin. Our results add to our understanding of these systems, and our methodology is applicable for other type I loci to identify critical regulatory and functional features.IMPORTANCEChromosomal type I toxin-antitoxins are a class of genes that have gained increasing attention over the last decade for their roles in antibiotic persistence which may contribute to therapeutic failures. However, the control of many of these genes and when they function have remained elusive. We demonstrate that a simple genetic conservation-based approach utilizing free, publicly available data yields known and novel insights into the regulation and function of three chromosomal type I toxin-antitoxins in Escherichia coli. This study also provides a framework for how this approach could be applied to other genes of interest.

Unraveling the role of toxin-antitoxin systems in Burkholderia pseudomallei: exploring bacterial pathogenesis and interactions within the HigBA families.

Burkholderia pseudomallei (Bpm) is a Gram-negative intracellular pathogen that causes melioidosis in humans, a neglected, underreported, and lethal disease that can reach a fatal outcome in over 50% of the cases. It can produce both acute and chronic infections, the latter being particularly challenging to eliminate because of the intracellular life cycle of the bacteria and its ability to generate a "persister" dormant state. The molecular mechanism that allows the switch between growing and persister phenotypes is not well understood but it is hypothesized to be due at least in part to the participation of toxin-antitoxin (TA) systems. We have previously studied the link between one of those systems (defined as HigBA) with specific expression patterns associated with levofloxacin antibiotic exposure. Through in silico methods, we predicted the presence of another three pairs of genes encoding for additional putative HigBA systems. Therefore, our main goal was to establish which mechanisms are conserved as well as which pathways are specific among different Bpm TA systems from the same family. We hypothesize that the high prevalence, and sometimes even redundancy of these systems in the Bpm chromosomes indicates that they can interact with each other and not function as only individual systems, as it was traditionally thought, and might be playing an undefined role in Bpm lifecycle. Here, we show that both the toxin and the antitoxin of the different systems contribute to bacterial survival and that toxins from the same family can have a cumulative effect under environmental stressful conditions. IMPORTANCE: Toxin-antitoxin (TA) systems play a significant role in bacterial persistence, a phenomenon where bacterial cells enter a dormant or slow-growing state to survive adverse conditions such as nutrient deprivation, antibiotic exposure, or host immune responses. By studying TA systems in Burkholderia pseudomallei, we can gain insights into how this pathogen survives and persists in the host environment, contributing to its virulence and ability to cause melioidosis chronic infections.

An RNA pseudoknot mediates toxin translation and antitoxin inhibition.

Type I toxin-antitoxin systems (T1TAs) are bipartite bacterial loci encoding a growth-inhibitory toxin and an antitoxin small RNA (sRNA). In many of these systems, the transcribed toxin mRNA is translationally inactive, but becomes translation-competent upon ribonucleolytic processing. The antitoxin sRNA targets the processed mRNA to inhibit its translation. This two-level control mechanism prevents cotranscriptional translation of the toxin and allows its synthesis only when the antitoxin is absent. Contrary to this, we found that the timP mRNA of the timPR T1TA locus does not undergo enzymatic processing. Instead, the full-length timP transcript is both translationally active and can be targeted by the antitoxin TimR. Thus, tight control in this system relies on a noncanonical mechanism. Based on the results from in vitro binding assays, RNA structure probing, and cell-free translation experiments, we suggest that timP mRNA adopts mutually exclusive structural conformations. The active form uniquely possesses an RNA pseudoknot structure which is essential for translation initiation. TimR preferentially binds to the active conformation, which leads to pseudoknot destabilization and inhibited translation. Based on this, we propose a model in which "structural processing" of timP mRNA enables tight inhibition by TimR in nonpermissive conditions, and TimP synthesis only upon TimR depletion.

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.

Mechanism of phage sensing and restriction by toxin-antitoxin-chaperone systems.

Toxin-antitoxins (TAs) are prokaryotic two-gene systems composed of a toxin neutralized by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilizes the antitoxin by recognizing its chaperone addiction (ChAD) element. TACs mediate antiphage defense, but the mechanisms of viral sensing and restriction are unexplored. We identify two Escherichia coli antiphage TAC systems containing host inhibition of growth (HigBA) and CmdTA TA modules, HigBAC and CmdTAC. HigBAC is triggered through recognition of the gpV major tail protein of phage λ. Chaperone HigC recognizes gpV and ChAD via analogous aromatic molecular patterns, with gpV outcompeting ChAD to trigger toxicity. For CmdTAC, the CmdT ADP-ribosyltransferase toxin modifies mRNA to halt protein synthesis and limit phage propagation. Finally, we establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with a HigC chaperone phage sensor. Collectively, these findings reveal the potential of TAC systems in broad-spectrum antiphage defense.

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.

Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages.

Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.