Crystal structure of Mycobacterium tuberculosis YefM antitoxin reveals that it is not an intrinsically unstructured protein

Toxin-antitoxin modules are present on chromosomes of almost all free-living prokaryotes. Some are implicated to act as stress-responsive elements, among their many functional roles. The YefM-YoeB toxin-antitoxin system is present in many bacterial species, where YefM belongs to the Phd family antidote of phage P1, whereas YoeB is a homolog of the RelE toxin of the RelBE system, rather than the Doc system of phage P1. YoeB, a ribonuclease, is believed to be conformationally stable, whereas YefM has been proposed to be a member of intrinsically disordered proteins. The ribonucleolytic activity of YoeB is neutralized by YefM upon formation of the YefM-YoeB complex. We report here the crystal structure of Mycobacterium tuberculosis YefM from two crystal isoforms. Our crystallographic and biophysical studies reveal that YefM is not an intrinsically unfolded protein and instead forms a well-defined structure with significant secondary and tertiary structure conformations. The residues involved in core formation of the folded structure are evolutionarily conserved among many bacterial species, supporting our observation. The C-terminal end of its polypeptide is highly pliable, which adopts different conformations in different monomers. Since at the physiological level YefM controls the activity of YoeB through intricate protein-protein interactions, the conformational heterogeneity in YefM revealed by our structure suggests that these might act a master switch in controlling YoeB activity.     

Conformational change in the catalytic site of the ribonuclease YoeB toxin by YefM antitoxin

The eubacterial chromosome encodes various addiction modules that control global levels of translation through RNA degradation. Crystal structures of the Escherichia coli YefM2 (antitoxin)-YoeB (toxin) complex and the free YoeB toxin have been determined. The structure of the heterotrimeric complex reveals an asymmetric disorder-to-order recognition strategy, in which one C terminus of the YefM homodimer exclusively interacts with an atypical microbial ribonuclease (RNase) fold of YoeB. Comparison with the YefM-free YoeB structure indicates a conformational rearrangement of the RNase catalytic site of YoeB, induced by interaction with YefM. Complementary biochemical experiments demonstrate that the YoeB toxin has an in vitro RNase activity that preferentially cleaves at the 3' end of purine ribonucleotides.     

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.     

The YoeB toxin is a folded protein that forms a physical complex with the unfolded YefM antitoxin. Implications for a structural-based differential stability of toxin-antitoxin systems

The chromosomal YoeB-YefM toxin-antitoxin module common to numerous strains of bacteria is presumed to have a significant role in survival under stringent conditions. Recently we showed that the purified YefM antitoxin is a natively unfolded protein, as we previously reported for the Phd antitoxin in the P1 phage Doc-Phd toxin-antitoxin system. Here we report the purification and structural properties of the YoeB toxin and present physical evidence for the existence of a tight YoeB.YefM polypeptide complex in solution. YoeB and YefM proteins co-eluted as single peaks in sequential Ni-affinity FPLC and Q-Sepharose ion-exchange chromatography implying the formation of a YoeB.YefM complex. The unstable antitoxin was removed from the mixture by natural proteolysis, and the residual YoeB protein was purified using ion exchange chromatography. Fluorescence anisotropy studies of the purified YoeB and YefM proteins showed a 2:1 stoichiometry of the complex, providing direct evidence for a physical complex between the proteins. Near- and far-UV circular dichroism spectroscopy of the purified toxin revealed that, similar to the Doc toxin, YoeB is a well-folded protein. Thermal denaturation experiments confirmed the conformational stability of the YoeB toxin, which underwent reversible thermal unfolding at temperatures up to 56 degrees C. The thermodynamic features of the toxin-antitoxin complex were similar. Taken together, our results support the notion of a correlation between differential physiological and structural stability in toxin-antitoxin modules.     

Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM-yoeB toxin-antitoxin system

In Escherichia coli, the Lon ATP-dependent protease is responsible for degradation of several regulatory proteins and for the elimination of abnormal proteins. Previous studies have shown that the overproduction of Lon is lethal. Here, we showed that Lon overproduction specifically inhibits translation through at least two different pathways. We have identified one of the pathways as being the chromosomal yefM-yoeB toxin-antitoxin system. The existence of a second pathway is demonstrated by the observation that the deletion of the yefM-yoeB system did not completely suppress lethality and translation inhibition. We also showed that the YoeB toxin induces cleavage of translated mRNAs and that Lon overproduction specifically activates YoeB-dependent mRNAs cleavage. Indeed, none of the other identified chromosomal toxin-antitoxin systems (relBE, mazEF, chpB and dinJ-yafQ) was involved in Lon-dependent lethality, translation inhibition and mRNA cleavage even though the RelB and MazE antitoxins are known to be Lon substrates. Based on our results and other studies, translation inhibition appears to be the key element that triggers chromosomal toxin-antitoxin systems. We propose that under Lon overproduction conditions, translation inhibition is mediated by Lon degradation of a component of the YoeB-independent pathway, in turn activating the YoeB toxin by preventing synthesis of its unstable YefM antidote.     

副溶血弧菌染色体上毒素-抗毒素系统YefM-YoeB的鉴定

【背景】副溶血弧菌是一种重要的食源性病原菌,给公众健康带来严重危害。毒素-抗毒素系统广泛存在于细菌和古生菌基因组中,具有重要的生物学功能。【目的】在副溶血弧菌中鉴定新的毒素-抗毒素系统,为从毒素-抗毒素系统角度探讨该菌致病性和耐药性的分子机制奠定基础。【方法】通过在线工具预测副溶血弧菌染色体上的假定II型毒素-抗毒素系统;通过生长曲线分析和稀释点板实验检测假定毒素对大肠杆菌的毒性作用及相应抗毒素的抗毒性作用;通过反转录PCR确定毒素和抗毒素基因是否共转录;通过生物信息学分析确定新鉴定毒素-抗毒素系统的同源蛋白;通过LacZ报告实验确定抗毒素及毒素-抗毒素复合物对自身启动子的调控作用。【结果】副溶血弧菌染色体中编码6个假定II型毒素-抗毒素系统;基因vp1820的表达产物(VP1820)对大肠杆菌具有杀菌活性,vp1821的表达产物(VP1821)能中和VP1820的毒性;基因vp1821和vp1820共转录;vp1821-vp1820编码YefM-YoeB毒素-抗毒素系统;抗毒素YefM正调控启动子,YefM-YoeB复合物负调控启动子。【结论】在副溶血弧菌中鉴定了一个新的II型毒素-抗毒素系统,即YefM-YoeB,为进一步研究该系统对副溶血弧菌致病性和耐药性的影响奠定了基础。     

The YefM antitoxin defines a family of natively unfolded proteins: implications as a novel antibacterial target

Although natively unfolded proteins are being observed increasingly, their physiological role is not well understood. Here, we demonstrate that the Escherichia coli YefM protein is a natively unfolded antitoxin, lacking secondary structure even at low temperature or in the presence of a stabilizing agent. This conformation of the protein is suggested to have a key role in its physiological regulatory activity. Because of the unfolded state of the protein, a linear determinant rather than a conformational one is presumably being recognized by its toxin partner, YoeB. A peptide array technology allowed the identification and validation of such a determinant. This recognition element may provide a novel antibacterial target. Indeed, a pair-constrained bioinformatic analysis facilitated the definite determination of novel YefM-YoeB toxin-antitoxin systems in a large number of bacteria including major pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis. Taken together, the YefM protein defines a new family of natively unfolded proteins. The existence of a large and conserved group of proteins with a clear physiologically relevant unfolded state serves as a paradigm to understand the structural basis of this state.     

Exploiting yoeB-yefM toxin-antitoxin system of Streptococcus pneumoniae on the selective killing of miR-21 overexpressing breast cancer cell line (MCF-7)

Toxin-antitoxin (TA) systems are two-component genetic modules widespread in bacterial and archaeal genomes, in which the toxin module is rendered inactive under resting conditions by its antitoxin counterpart. Under stress conditions, however, the antitoxin is degraded, freeing the toxin to exert its lethal effects. Although not evolved to function in eukaryotes, some studies have established the lethal activity of these bacterial toxins by inducing apoptosis in mammalian cells, an effect that can be neutralized by its cognate antitoxin. Inspired by the way the toxin can become active in eukaryotes cells, we produced an engrained yoeB-yefM TA system to selectively kill human breast cancer cells expressing a high level of miR-21. Accordingly, we generated an engineered yefM antitoxin gene with eight miR-21 target sites placed in its 3′untranslated region. The resulting TA system acts autonomously in human cells, distinguishing those that overexpress miR-21, killed by YoeB, from those that do not, remaining protected by YefM. Thus, we indicated that microRNA-control of the antitoxin protein of bacterial TA systems constitutes a novel strategy to enhance the selective killing of human cancer cells by the toxin module. The present study provides significant insights for developing novel anticancer strategies avoiding off-target effects, a challenge that has been pursued by many investigators over the years.     

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.     

Function inferences from a molecular structural model of bacterial ParE toxin

Toxin-antitoxin (TA) systems contribute to plasmid stability by a mechanism that relies on the differential stabilities of the toxin and antitoxin proteins and leads to the killing of daughter bacteria that did not receive a plasmid copy at the cell division. ParE is the toxic component of a TA system that constitutes along with RelE an important class of bacterial toxin called RelE/ParE superfamily. For ParE toxin, no crystallographic structure is available so far and rare in vitro studies demonstrated that the target of toxin activity is E. coli DNA gyrase. Here, a 3D Model for E. coli ParE toxin by molecular homology modeling was built using MODELLER, a program for comparative modeling. The Model was energy minimized by CHARMM and validated using PROCHECK and VERIFY3D programs. Resulting Ramachandran plot analysis it was found that the portion residues failing into the most favored and allowed regions was 96.8%. Structural similarity search employing DALI server showed as the best matches RelE and YoeB families. The Model also showed similarities with other microbial ribonucleases but in a small score. A possible homologous deep cleft active site was identified in the Model using CASTp program. Additional studies to investigate the nuclease activity in members of ParE family as well as to confirm the inhibitory replication activity are needed. The predicted Model allows initial inferences about the unexplored 3D structure of the ParE toxin and may be further used in rational design of molecules for structure-function studies.     

Characterization of PfiT/PfiA toxin–antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction

Toxin–antitoxin (TA) systems are small genetic modules usually consisting of two elements—a toxin and an antitoxin. The abundance of TA systems among various bacterial strains may indicate an important evolutionary role. Pseudomonas aeruginosa, which can be found in a variety of niches in nature, is an opportunistic pathogen for various hosts. While P. aeruginosa strains are very versatile and diverse, only a few TA systems were characterized in this species. Here, we describe a newly characterized TA system in P. aeruginosa that is encoded within the filamentous Pf4 prophage. This system, named PfiT/PfiA, is a homologue of the ParE/YefM TA system. It is a type II TA system, in which the antitoxin is a protein that binds the toxic protein and eliminates the toxic effect. PfiT/PfiA carries several typical type II characteristics. Specifically, it constitutes two small genes expressed in a single operon, PfiT inhibits growth and PfiA eliminates this effect, PfiA binds PfiT, and PfiT expression results in elongated cells. Finally, we assigned a novel function to this TA system, where an imbalance between PfiT and PfiA, favouring the toxin, resulted in cell elongation and an increase in virion production.     

Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa

Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf 4 i nhibition t oxin and Rorf0727 be named PfiA for Pf iT a ntitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5′-AATTC N₅GTTAA -3′) overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.