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 2.2 Å 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.     

Structural conservation of the PIN domain active site across all domains of life

The PIN (PilT N‐terminus) domain is a compact RNA‐binding protein domain present in all domains of life. This 120‐residue domain consists of a central and parallel β sheet surrounded by α helices, which together organize 4–5 acidic residues in an active site that binds one or more divalent metal ions and in many cases has endoribonuclease activity. In bacteria and archaea, the PIN domain is primarily associated with toxin–antitoxin loci, consisting of a toxin (the PIN domain nuclease) and an antitoxin that inhibits the function of the toxin under normal growth conditions. During nutritional or antibiotic stress, the antitoxin is proteolytically degraded causing activation of the PIN domain toxin leading to a dramatic reprogramming of cellular metabolism to cope with the new situation. In eukaryotes, PIN domains are commonly found as parts of larger proteins and are involved in a range of processes involving RNA cleavage, including ribosomal RNA biogenesis and nonsense‐mediated mRNA decay. In this review, we provide a comprehensive overview of the structural characteristics of the PIN domain and compare PIN domains from all domains of life in terms of structure, active site architecture, and activity.     

The yefM-yoeB toxin-antitoxin systems of Escherichia coli and Streptococcus 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.     

A cis‐acting antitoxin domain within the chromosomal toxin–antitoxin module EzeT of Escherichia coli quenches toxin activity

Toxin–antitoxin (TA) systems are widespread genetic modules in the genomes of bacteria and archaea emerging as key players that modulate bacterial physiology. They consist of two parts, a toxic component that blocks an essential cellular process and an antitoxin that inhibits this toxic activity during normal growth. According to the nature of the antitoxin and the mode of inhibition, TA systems are subdivided into different types. Here, we describe the characterization of a type II‐like TA system in Escherichia coli called EzeT. While in conventional type II systems the antitoxin is expressed in trans to form an inactive protein–protein complex, EzeT consists of two domains combining toxin and cis‐acting antitoxin functionalities in a single polypeptide chain. We show that the C‐terminal domain of EzeT is homologous to zeta toxins and is toxic in vivo. The lytic phenotype could be attributed to UDP‐N‐acetylglucosamine phosphorylation, so far only described for type II epsilon/zeta systems from Gram‐positive streptococci. Presence of the N‐terminal domain inhibits toxicity in vivo and strongly attenuates kinase activity. Autoinhibition by a cis‐acting antitoxin as described here for EzeT‐type TA systems can explain the occurrence of single or unusually large toxins, further expanding our understanding of the TA system network.     

A novel family of Escherichia coli toxin-antitoxin gene pairs

Bacterial toxin-antitoxin protein pairs (TA pairs) encode a toxin protein, which poisons cells by binding and inhibiting an essential enzyme, and an antitoxin protein, which binds the toxin and restores viability. We took an approach that did not rely on sequence homology to search for unidentified TA pairs in the genome of Escherichia coli K-12. Of 32 candidate genes tested, ectopic expression of 6 caused growth inhibition. In this report, we focus on the initial characterization of yeeV, ykfI, and ypjF, a novel family of toxin proteins. Coexpression of the gene upstream of each toxin restored the growth rate to that of the uninduced strain. Unexpectedly, we could not detect in vivo protein-protein interactions between the new toxin and antitoxin pairs. Instead, the antitoxins appeared to function by causing a large reduction in the level of cellular toxin protein.     

VapC-1 of nontypeable Haemophilus influenzae is a ribonuclease

Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.     

Substrate specificity of bacterial endoribonuclease toxins

Bacterial endoribonuclease toxins belong to a protein family that inhibits bacterial growth by degrading mRNA or rRNA sequences. The toxin genes are organized in pairs with its cognate antitoxins in the chromosome and thus the activities of the toxins are antagonized by antitoxin proteins or RNAs during active translation. In response to a variety of cellular stresses, the endoribonuclease toxins appear to be released from antitoxin molecules via proteolytic cleavage of antitoxin proteins or preferential degradation of antitoxin RNAs and cleave a diverse range of mRNA or rRNA sequences in a sequence-specific or codon-specific manner, resulting in various biological phenomena such as antibiotic tolerance and persister cell formation. Given that substrate specificity of each endoribonuclease toxin is determined by its structure and the composition of active site residues, we summarize the biology, structure, and substrate specificity of the updated bacterial endoribonuclease toxins.     

Neurite extension by neuroblastoma cells on substratum-bound fibronectin''s cell-binding fragment but not on the heparan sulfate-binding protein, platelet factor-4

Human and rat neuroblastoma cells extend neurites over plasma fibronectin (pFN)-coated substrata. For resolution of which fibronectin binding activities (the cell-binding domain (CBD), the heparan sulfate-binding domains, or a combination of the two) are responsible for neurite outgrowth, CBD was prepared free of heparan sulfate-binding activity as described by Pierschbacher et al. (Cell 26 (1981) 259-267). Neuroblastoma cells attached and extended neurites as stably and as effectively on CBD-coated substrata as on intact pFN, while cytoplasmic spreading was more extensive on pFN-coated substrata. The structures of growth cones on CBD or pFN were virtually identical. On substrata coated with the model heparan sulfate-binding protein, platelet factor 4 (PF4), cells attached and spread somewhat but never extended neurites. When cells were challenged with substrata coated with various ratios of CBD and PF4, PF4 was found to be an effective inhibitor of CBD-mediated neurite extension. Similarly, cells grown on substrata coated at different locations with CBD or PF4 in order to evaluate topographical dependence of growth cone formation extended neurites only onto the CBD-coated region or along the interface between these two proteins, but never onto the PF4 side of cells that bridged the interface. These studies indicate that (a) the CBD activity of pFN, and not its heparan sulfate-binding activity, is the critical determinant in neurite extension of these neural tumor cells from the central nervous system; (b) under some circumstances, heparan sulfate-binding activity can be antagonistic to neurite extension; (c) the chemical nature of the substratum controls the direction of neurite extension; (d) these neuroblastoma cells respond to these binding proteins very differently than fibroblasts or neurons from the peripheral nervous system.     

IGF-II and IGFBP-6 regulate cellular contractility and proliferation in Dupuytren's disease

Dupuytren's disease (DD) is a common and heritable fibrosis of the palmar fascia that typically manifests as permanent finger contractures. The molecular interactions that induce the development of hyper-contractile fibroblasts, or myofibroblasts, in DD are poorly understood. We have identified IGF2 and IGFBP6, encoding insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-6 respectively, as reciprocally dysregulated genes and proteins in primary cells derived from contracture tissues (DD cells). Recombinant IGFBP-6 inhibited the proliferation of DD cells, patient-matched control (PF) cells and normal palmar fascia (CT) cells. Co-treatments with IGF-II, a high affinity IGFBP-6 ligand, were unable to rescue these effects. A non-IGF-II binding analog of IGFBP-6 also inhibited cellular proliferation, implicating IGF-II-independent roles for IGFBP-6 in this process. IGF-II enhanced the proliferation of CT cells, but not DD or PF cells, and significantly enhanced DD and PF cell contractility in stressed collagen lattices. While IGFBP-6 treatment did not affect cellular contractility, it abrogated the IGF-II-induced contractility of DD and PF cells in stressed collagen lattices. IGF-II also significantly increased the contraction of DD cells in relaxed lattices, however this effect was not evident in relaxed collagen lattices containing PF cells. The disparate effects of IGF-II on DD and PF cells in relaxed and stressed contraction models suggest that IGF-II can enhance lattice contractility through more than one mechanism. This is the first report to implicate IGFBP-6 as a suppressor of cellular proliferation and IGF-II as an inducer of cellular contractility in this connective tissue disease.     

Axe-Txe,a broad-spectrum proteic toxin-antitoxin system specified by a multidrug-resistant,clinical isolate of Enterococcus faecium

Enterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections. However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria. Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium. A novel proteic-based toxin-antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species. Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli. The toxic effect of Txe was alleviated by co-expression of the antitoxin protein, Axe. Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria. These homologues (yefM-yoeB) present in the E. coli chromosome function as a toxin-antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo. Axe-Txe is one of the first functional proteic toxin-antitoxin systems to be accurately described for Gram-positive bacteria.     

Chromosomal bacterial type II toxin-antitoxin systems

Most prokaryotic chromosomes contain a number of toxin-antitoxin (TA) modules consisting of a pair of genes that encode 2 components, a stable toxin and its cognate labile antitoxin. TA systems are also known as addiction modules, since the cells become "addicted" to the short-lived antitoxin product (the unstable antitoxin is degraded faster than the more stable toxin) because its de novo synthesis is essential for their survival. While toxins are always proteins, antitoxins are either RNAs (type I, type III) or proteins (type II). Type II TA systems are widely distributed throughout the chromosomes of almost all free-living bacteria and archaea. The vast majority of type II toxins are mRNA-specific endonucleases arresting cell growth through the mechanism of RNA cleavage, thus preventing the translation process. The physiological role of chromosomal type II TA systems still remains the subject of debate. This review describes the currently known type II toxins and their characteristics. The different hypotheses that have been proposed to explain their role in bacterial physiology are also discussed.     

Solution NMR structure of the helicase associated domain BVU_0683(627–691) from Bacteroides vulgatus provides first structural coverage for protein domain family PF03457 and indicates domain binding to DNA

A high-quality NMR structure of the helicase associated (HA) domain comprising residues 627–691 of the 753-residue protein BVU_0683 from Bacteroides vulgatus exhibits an all α-helical fold. The structure presented here is the first representative for the large protein domain family PF03457 (currently 742 members) of HA domains. Comparison with structurally similar proteins supports the hypothesis that HA domains bind to DNA and that binding specificity varies greatly within the family of HA domains constituting PF03457.     

Toxin-antitoxin systems in bacteria: Apoptotic tools or metabolic regulators?

The results of recent (10–12 years) research in the functions of two-gene chromosomal modules are considered and generalized. One of the genes encodes a toxin protein; the product of the other gene is an antitoxin protein. In the course of balanced bacterial growth, the toxin is constantly neutralized by the antitoxin; however, certain metabolic changes (amino acid starvation, etc.) disturb the balance and then the toxin “poisons” the cell (in most cases, by destroying mRNA). As a result, bacterial growth ceases. In accordance with one group of the data, long-term inhibition of growth of most cells results in their programmed death and destruction, corresponding to apoptosis; this allows a minor part of the population to survive due to an additional nutrient source. The results of other works show that growth inhibition is mostly reversible and the functions of the relevant gene modules are restricted to the regulation of cell metabolism, i.e., transition of bacteria to the hypometabolic state. There is also a compromise point of view. The possibilities of biotechnological applications for “toxin-antitoxin” systems are discussed.     

A novel extracellular metallopeptidase domain shared by animal host-associated mutualistic and pathogenic microbes

The mucosal microbiota is recognised as an important factor for our health, with many disease states linked to imbalances in the normal community structure. Hence, there is considerable interest in identifying the molecular basis of human-microbe interactions. In this work we investigated the capacity of microbes to thrive on mucosal surfaces, either as mutualists, commensals or pathogens, using comparative genomics to identify co-occurring molecular traits. We identified a novel domain we named M60-like/PF13402 (new Pfam entry PF13402), which was detected mainly among proteins from animal host mucosa-associated prokaryotic and eukaryotic microbes ranging from mutualists to pathogens. Lateral gene transfers between distantly related microbes explained their shared M60-like/PF13402 domain. The novel domain is characterised by a zinc-metallopeptidase-like motif and is distantly related to known viral enhancin zinc-metallopeptidases. Signal peptides and/or cell surface anchoring features were detected in most microbial M60-like/PF13402 domain-containing proteins, indicating that these proteins target an extracellular substrate. A significant subset of these putative peptidases was further characterised by the presence of associated domains belonging to carbohydrate-binding module family 5/12, 32 and 51 and other glycan-binding domains, suggesting that these novel proteases are targeted to complex glycoproteins such as mucins. An in vitro mucinase assay demonstrated degradation of mammalian mucins by a recombinant form of an M60-like/PF13402-containing protein from the gut mutualist Bacteroides thetaiotaomicron. This study reveals that M60-like domains are peptidases targeting host glycoproteins. These peptidases likely play an important role in successful colonisation of both vertebrate mucosal surfaces and the invertebrate digestive tract by both mutualistic and pathogenic microbes. Moreover, 141 entries across various peptidase families described in the MEROPS database were also identified with carbohydrate-binding modules defining a new functional context for these glycan-binding domains and providing opportunities to engineer proteases targeting specific glycoproteins for both biomedical and industrial applications.     

Structure–function studies of Escherichia coli RnlA reveal a novel toxin structure involved in bacteriophage resistance

Escherichia coli RnlA–RnlB is a newly identified toxin–antitoxin (TA) system that plays a role in bacteriophage resistance. RnlA functions as a toxin with mRNA endoribonuclease activity and the cognate antitoxin RnlB inhibits RnlA toxicity in E. coli cells. Interestingly, T4 phage encodes the antitoxin Dmd, which acts against RnlA to promote its own propagation, suggesting that RnlA‐Dmd represents a novel TA system. Here, we have determined the crystal structure of RnlA refined to 2.10 Å. RnlA is composed of three independent domains: NTD (N ‐t erminal d omain), NRD (N r epeated d omain) and DBD (D md‐b inding d omain), which is an organization not previously observed among known toxin structures. Small‐angle X‐ray scattering (SAXS) analysis revealed that RnlA forms a dimer in solution via interactions between the DBDs from both monomers. The in vitro and in vivo functional studies showed that among the three domains, only the DBD is responsible for recognition and inhibition by Dmd and subcellular location of RnlA. In particular, the helix located at the C‐terminus of DBD plays a vital role in binding Dmd. Our comprehensive studies reveal the key region responsible for RnlA toxicity and provide novel insights into its structure–function relationship.     

Escherichia coli MazF Leads to the Simultaneous Selective Synthesis of Both “Death Proteins” and “Survival Proteins”

The Escherichia coli mazEF module is one of the most thoroughly studied toxin–antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. MazF is an endoribonuclease that leads to the inhibition of protein synthesis by cleaving mRNAs at ACA sequences. Here, using 2D-gels, we show that in E. coli, although MazF induction leads to the inhibition of the synthesis of most proteins, the synthesis of an exclusive group of proteins, mostly smaller than about 20 kDa, is still permitted. We identified some of those small proteins by mass spectrometry. By deleting the genes encoding those proteins from the E. coli chromosome, we showed that they were required for the death of most of the cellular population. Under the same experimental conditions, which induce mazEF-mediated cell death, other such proteins were found to be required for the survival of a small sub-population of cells. Thus, MazF appears to be a regulator that induces downstream pathways leading to death of most of the population and the continued survival of a small sub-population, which will likely become the nucleus of a new population when growth conditions become less stressful.