Toxin-antitoxin Interactions Research Articles

Co-evolution of interacting proteins through non-contacting and non-specific mutations.

SummaryProteins often accumulate neutral mutations that do not affect current functions but can profoundly influence future mutational possibilities and functions. Understanding such hidden potential has major implications for protein design and evolutionary forecasting, but has been limited by a lack of systematic efforts to identify potentiating mutations. Here, through the comprehensive analysis of a bacterial toxin-antitoxin system, we identified all possible single substitutions in the toxin that enable it to tolerate otherwise interface-disrupting mutations in its antitoxin. Strikingly, the majority of enabling mutations in the toxin do not contact, and promote tolerance non-specifically to, many different antitoxin mutations, despite covariation in homologs occurring primarily between specific pairs of contacting residues across the interface. In addition, the enabling mutations we identified expand future mutational paths that both maintain old toxin-antitoxin interactions and form new ones. These non-specific mutations are missed by widely used covariation and machine learning methods. Identifying such enabling mutations will be critical for ensuring continued binding of therapeutically relevant proteins, such as antibodies, aimed at evolving targets.

Antitoxin ε Reverses Toxin ζ-Facilitated Ampicillin Dormants.

Toxin-antitoxin (TA) modules are ubiquitous in bacteria, but their biological importance in stress adaptation remains a matter of debate. The inactive ζ-ε2-ζ TA complex is composed of one labile ε2 antitoxin dimer flanked by two stable ζ toxin monomers. Free toxin ζ reduces the ATP and GTP levels, increases the (p)ppGpp and c-di-AMP pool, inactivates a fraction of uridine diphosphate-N-acetylglucosamine, and induces reversible dormancy. A small subpopulation, however, survives toxin action. Here, employing a genetic orthogonal control of ζ and ε levels, the fate of bacteriophage SPP1 infection was analyzed. Toxin ζ induces an active slow-growth state that halts SPP1 amplification, but it re-starts after antitoxin expression rather than promoting abortive infection. Toxin ζ-induced and toxin-facilitated ampicillin (Amp) dormants have been revisited. Transient toxin ζ expression causes a metabolic heterogeneity that induces toxin and Amp dormancy over a long window of time rather than cell persistence. Antitoxin ε expression, by reversing ζ activities, facilitates the exit of Amp-induced dormancy both in rec+ and recA cells. Our findings argue that an unexploited target to fight against antibiotic persistence is to disrupt toxin-antitoxin interactions.

Molecular and Structural Basis of Cross-Reactivity in M. tuberculosis Toxin-Antitoxin Systems.

Mycobacterium tuberculosis genome encodes over 80 toxin–antitoxin (TA) systems. While each toxin interacts with its cognate antitoxin, the abundance of TA systems presents an opportunity for potential non-cognate interactions. TA systems mediate manifold interactions to manage pathogenicity and stress response network of the cell and non-cognate interactions may play vital roles as well. To address if non-cognate and heterologous interactions are feasible and to understand the structural basis of their interactions, we have performed comprehensive computational analyses on the available 3D structures and generated structural models of paralogous M. tuberculosis VapBC and MazEF TA systems. For a majority of the TA systems, we show that non-cognate toxin–antitoxin interactions are structurally incompatible except for complexes like VapBC15 and VapBC11, which show similar interfaces and potential for cross-reactivity. For TA systems which have been experimentally shown earlier to disfavor non-cognate interactions, we demonstrate that they are structurally and stereo-chemically incompatible. For selected TA systems, our detailed structural analysis identifies specificity conferring residues. Thus, our work improves the current understanding of TA interfaces and generates a hypothesis based on congenial binding site, geometric complementarity, and chemical nature of interfaces. Overall, our work offers a structure-based explanation for non-cognate toxin-antitoxin interactions in M. tuberculosis.

Bioinformatic and mutational studies of related toxin–antitoxin pairs in Mycobacterium tuberculosis predict and identify key functional residues

Mycobacterium tuberculosis possesses an unusually large representation of type II toxin-antitoxin (TA) systems, whose functions and targets are mostly unknown. To better understand the basis of their unique expansion and to probe putative functional similarities among these systems, here we computationally and experimentally investigated their sequence relationships. Bioinformatic and phylogenetic investigations revealed that 51 sequences of the VapBC toxin family group into paralogous sub-clusters. On the basis of conserved sequence fingerprints within paralogues, we predicted functional residues and residues at the putative TA interface that are useful to evaluate TA interactions. Substitution of these likely functional residues abolished the toxin's growth-inhibitory activity. Furthermore, conducting similarity searches in 101 mycobacterial and ∼4500 other prokaryotic genomes, we assessed the relative conservation of the M. tuberculosis TA systems and found that most TA orthologues are well-conserved among the members of the M. tuberculosis complex, which cause tuberculosis in animal hosts. We found that soil-inhabiting, free-living Actinobacteria also harbor as many as 12 TA pairs. Finally, we identified five novel putative TA modules in M. tuberculosis. For one of them, we demonstrate that overexpression of the putative toxin, Rv2514c, induces bacteriostasis and that co-expression of the cognate antitoxin Rv2515c restores bacterial growth. Taken together, our findings reveal that toxin sequences are more closely related than antitoxin sequences in M. tuberculosis Furthermore, the identification of additional TA systems reported here expands the known repertoire of TA systems in M. tuberculosis.

Structural, functional and biological insights into the role of Mycobacterium tuberculosis VapBC11 toxin-antitoxin system: targeting a tRNase to tackle mycobacterial adaptation.

Toxin–antitoxin (TA) systems are involved in diverse physiological processes in prokaryotes, but their exact role in Mycobacterium tuberculosis (Mtb) virulence and in vivo stress adaptation has not been extensively studied. Here, we demonstrate that the VapBC11 TA module is essential for Mtb to establish infection in guinea pigs. RNA-sequencing revealed that overexpression of VapC11 toxin results in metabolic slowdown, suggesting that modulation of the growth rate is an essential strategy for in vivo survival. Interestingly, overexpression of VapC11 resulted in the upregulation of chromosomal TA genes, suggesting the existence of highly coordinated crosstalk among TA systems. In this study, we also present the crystal structure of the VapBC11 heterooctameric complex at 1.67 Å resolution. Binding kinetic studies suggest that the binding affinities of toxin–substrate and toxin–antitoxin interactions are comparable. We used a combination of structural studies, molecular docking, mutational analysis and in vitro ribonuclease assays to enhance our understanding of the mode of substrate recognition by the VapC11 toxin. Furthermore, we have also designed peptide-based inhibitors to target VapC11 ribonuclease activity. Taken together, we propose that the structure-guided design of inhibitors against in vivo essential ribonucleases might be a novel strategy to hasten clearance of intracellular Mtb.

Toxin inhibition in C. crescentus VapBC1 is mediated by a flexible pseudo-palindromic protein motif and modulated by DNA binding.

Expression of bacterial type II toxin-antitoxin (TA) systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo-palindromic sequences on operator DNA. In this context, the toxin functions as a co-repressor by stimulating DNA binding through direct interaction with the antitoxin. Here, we determine crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to its cognate DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions in which C-terminal extended structures of the antitoxin VapB1 swap positions to interlock the complex in the DNA-bound state. We further show that a pseudo-palindromic protein sequence in the antitoxin is responsible for this interaction and required for binding and inactivation of the VapC1 toxin dimer. Sequence analysis of 4127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread and unique to bacterial and archaeal VapB antitoxins suggesting a general principle governing regulation of VapBC TA systems. Finally, a structure of C-terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for toxin activation in vivo.

Amino acid residues crucial for specificity of toxin–antitoxin interactions in the homologous Axe–Txe and YefM–YoeB complexes

Toxin-antitoxin complexes are ubiquitous in bacteria. The specificity of interactions between toxins and antitoxins from homologous but non-interacting systems was investigated. Based on molecular modeling, selected amino acid residues were changed to assess which positions were crucial in the specificity of toxin-antitoxin interaction in the related Axe-Txe and YefM-YoeB complexes. No cross-interactions between wild-type proteins were detected. However, a single amino acid substitution that converts a Txe-specific residue to a YoeB-specific residue reduced, but did not abolish, Txe interaction with the Axe antitoxin. Interestingly, this alteration (Txe-Asp83Tyr) promoted functional interactions between Txe and the YefM antitoxin. The interactions between Txe-Asp83Tyr and YefM were sufficiently strong to abolish Txe toxicity and to allow effective corepression by YefM-Txe-Asp83Tyr of the promoter from which yefM-yoeB is expressed. We conclude that Asp83 in Txe is crucial for the specificity of toxin-antitoxin interactions in the Axe-Txe complex and that swapping this residue for the equivalent residue in YoeB relaxes the specificity of the toxin-antitoxin interaction.

TheyefM-yoeBToxin-Antitoxin Systems ofEscherichia coliandStreptococcus pneumoniae: Functional and Structural Correlation

Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM(Spn)) and toxin (YoeB(Spn)) products. We showed that overproduction of YoeB(Spn) is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB(Spn)-mediated toxicity could be reversed by the cognate antitoxin, YefM(Spn), but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.

The chromosomal relBE2 toxin–antitoxin locus of Streptococcus pneumoniae: characterization and use of a bioluminescence resonance energy transfer assay to detect toxin–antitoxin interaction

Proteic toxin-antitoxin (TA) loci were first identified in bacterial plasmids, and they were regarded as involved in stable plasmid maintenance by a so-called 'addiction' mechanism. Later, chromosomally encoded TA loci were identified and their function ascribed to survival mechanisms when bacteria were subjected to stress. In the search for chromosomally encoded TA loci in Gram-positive bacteria, we identified various in the pathogen Streptococcus pneumoniae. Two of these cassettes, sharing homology with the Escherichia coli relBE locus were cloned and tested for their activity. The relBE2Spn locus resulted to be a bona fide TA locus. The toxin exhibited high toxicity towards E. coli and S. pneumoniae, although in the latter, the chromosomal copy of the antitoxin relB2Spn gene had to be inactivated to detect full toxicity. Cell growth arrest caused by expression of the relE2Spn toxin gene could be reverted by expression of the cognate antitoxin, relB2Spn, although prolonged exposition to the toxin led to cell death. The pneumococcal relBE2Spn locus is the first instance of a chromosomally encoded TA system from Gram-positive bacteria characterized in its own host. We have developed a bioluminescence resonance energy transfer (BRET) assay to detect the interactions between the RelB2Spn antitoxin and the RelE2Spn toxin in vivo. This technique has shown to be amenable to a high-throughput screening (HTS), opening new avenues in the search of molecules with potential antibacterial activity able to inhibit TA interactions.

The PIN-domain toxin–antitoxin array in mycobacteria

PIN-domains (homologues of the pilT N-terminal domain) are small protein domains of approximately 140 amino acids. They are found in a diverse range of organisms and recent evidence from bioinformatics, biochemistry, structural biology and microbiology suggest that the majority of the prokaryotic PIN-domain proteins are the toxic components of toxin-antitoxin (TA) operons. Several microorganisms have a large cohort of these operons. For example, the genome of Mycobacterium tuberculosis encodes 48 PIN-domain proteins, of which 38 are thought to be involved in TA interactions. This large array of PIN-domain TA operons raises questions as to their evolutionary origin and contemporary functional significance. We suggest that the evolutionary origin of genes encoding mycobacterial PIN-domain TA operons is linked to the mobile gene pool, but that TA operons can become resident within the chromosome of host cells from where they might be recruited to fulfil a variety of roles associated with retardation of cell growth and persistence in stressful environments.