Search Results (12)

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 β1–β2 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. Full article

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. Full article

Toxin–antitoxin systems are preserved by nearly every prokaryote. The type II toxin MazF acts as a sequence-specific endoribonuclease, cleaving ribonucleotides at specific sequences that vary from three to seven bases, as has been reported in different host organisms to date. The present study characterized the MazEF module (MazEF-sth) conserved in the Symbiobacterium thermophilum IAM14863 strain, a Gram-negative syntrophic bacterium that can be supported by co-culture with multiple bacteria, including Bacillus subtilis. Based on a method combining massive parallel sequencing and the fluorometric assay, MazF-sth was determined to cleave ribonucleotides at the UACAUA motif, which is markedly similar to the motifs recognized by MazF from B. subtilis (MazF-bs), and by several MazFs from Gram-positive bacteria. MazF-sth, with mutations at conserved amino acid residues Arg29 and Thr52, lost most ribonuclease activity, indicating that these residues that are crucial for MazF-bs also play significant roles in MazF-sth catalysis. Further, cross-neutralization between MazF-sth and the non-cognate MazE-bs was discovered, and herein, the neutralization mechanism is discussed based on a protein-structure simulation via AlphaFold2 and multiple sequence alignment. The conflict between the high homology shared by these MazF amino acid sequences and the few genetic correlations among their host organisms may provide evidence of horizontal gene transfer. Full article

Toxin–antitoxin (TA) systems are widely present in bacterial genomes. Mycolicibacterium smegmatis, a common model organism for studying Mycobacterium tuberculosis physiology, has eight TA loci, including mazEF and vapBC. This study aims to investigate the physiological significance of these TA systems. Proteomic profiling was conducted on a culture overexpressing the VapC toxin, and the involvement of VapC in M. smegmatis stress responses to heat shock and antibiotic treatment was examined. While deciphering the underlying mechanisms of the altered stress resistance, we assessed the antibiotic susceptibility of vapBC, mazEF, and double vapBC-mazEF deletion mutants. Additionally, the mRNA levels of vapC and mazF were measured following tetracycline supplementation. The results reveal changes in the abundance of metabolic enzymes and stress response proteins associated with VapC overexpression. This activation of the general stress response leads to reduced thermosensitivity in M. smegmatis, but does not affect susceptibility to ciprofloxacin and isoniazid. Under tetracycline treatment, both vapC and mazF expression levels are increased, and the fate of the cell depends on the interaction between the corresponding TA systems. Full article

Methicillin resistance in Staphylococcus aureus has become prevalent globally. Moreover, biofilm-formation makes it more difficult to eradicate bacteria by antibiotics. The mazEF toxin-antitoxin system encodes for mazF, which acts as an endoribonuclease that cleaves cellular mRNAs at specific sequence motifs (ACA), and mazE, which opposes the mazF action. Our goal was to detect mazEF expression in methicillin-resistant S. aureus (MRSA) isolates compared with methicillin-sensitive S. aureus (MSSA) isolates and determine its relation to methicillin susceptibility as well as biofilm-formation. According to their susceptibility to cefoxitin disks, 100 S. aureus isolates obtained from patients admitted to Cairo University Hospitals were categorized into 50 MSSA and 50 MRSA according to their susceptibility to cefoxitin disks (30 µg). Antimicrobial susceptibility and biofilm-formation were investigated using the disk diffusion method and tissue culture plate method, respectively. Finally, using real-time PCR, mazEF expression was estimated and correlated to methicillin susceptibility and biofilm formation. Both MRSA and MSSA isolates showed the best sensitivity results with linezolid and gentamicin, where about 88% of MRSA isolates and 96% of MSSA isolates were sensitive to linezolid while 76% of MRSA isolates and 84% of MSSA isolates were sensitive to gentamicin. MRSA isolates were significantly more able to form biofilm than MSSA isolates (p-value = 0.037). The mazEF expression was significantly correlated to methicillin resistance in S. aureus (p-value < 0.001), but not to biofilm-formation. Full article

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications. Full article

Introduction: Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), is a significant global public health threat. Besides extensive multidrug resistance, MTB possesses several properties for long-term viability in the host as well as stress adaptation and resistance in harsh conditions. The role of toxin-antitoxin (TA) systems in disseminating and maintaining antimicrobial resistance in bacterial populations has also been demonstrated. This study aimed to evaluate differences in expression of MazEF (a well-known TA system) related genes (mazE3, mazF3, mazE6, and mazF6) amongst drug-susceptible and resistant MTB isolates in Iran. Material and methods: A total of 20 confirmed clinical isolates of MTB including 10 drug-susceptible and 10 drug-resistant (nine MDR, and one XDR) species were included in this study. M. tuberculosis H37Rv was used as the standard strain. RNA extraction, cDNA synthesis, and relative quantitative real-time PCR were performed according to the standard procedures. Results: Our analysis indicated significant enhanced expression of the mazE6 antitoxin gene in drug-susceptible isolates compared to drug-resistant isolates and the standard strain. The expression of the mazF6 toxin gene was also increased in drug-susceptible isolates compared with the standard strain. In drug-resistant isolates, the expression levels of mazF3 and mazF6 genes were significantly higher than that in the susceptible isolates and the standard strain. Conclusions: In this study, there was significant overexpression of mazE6 in drug-susceptible isolates. As well, mazF3 and F6 were overexpressed in drug-resistant isolates when compared with the standard strain. The changes in expression levels of MazEF6 associated genes were greater than that of MazEF3 in both groups of isolates. Full article

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis (M. tb). It is regarded as a major health threat all over the world, mainly because of its high mortality and drug-resistant nature. Toxin-antitoxin (TA) systems are modules ubiquitously found in prokaryotic organisms, and the well-studied MazEF systems (MazE means “what is it?” in Hebrew) are implicated in the formation of “persister cells” in the M. tb pathogen. Here, we report cocrystal structures of M. tb MazF-mt1 and -mt9, two important MazF members responsible for specific mRNA and tRNA cleavages, respectively, in complexes with truncated forms of their cognate antitoxin peptides. These peptides bind to the toxins with comparable affinities to their full-length antitoxins, which would reduce the RNA-cleavage capacities of the toxins in vitro. After structural analysis of the binding modes, we systemically tested the influence of the substitutions of individual residues in the truncated MazE-mt9 peptide on its affinity. This study provides structural insight into the binding modes and the inhibition mechanisms between the MazE/F-mt TA pairs. More importantly, it contributes to the future design of peptide-based antimicrobial agents against TB and potentially relieves the drug-resistance problems by targeting novel M. tb proteins. Full article

Toxin–antitoxin (TA) systems are prevalent in bacteria and are known to regulate cellular growth in response to stress. As various functions related to TA systems have been revealed, the importance of TA systems are rapidly emerging. Here, we present the crystal structure of putative mRNA interferase BC0266 and report it as a type II toxin MazF. The MazF toxin is a ribonuclease activated upon and during stressful conditions, in which it cleaves mRNA in a sequence-specific, ribosome-independent manner. Its prolonged activity causes toxic consequences to the bacteria which, in turn, may lead to bacterial death. In this study, we conducted structural and functional investigations of Bacillus cereus MazF and present the first toxin structure in the TA system of B. cereus. Specifically, B. cereus MazF adopts a PemK-like fold and also has an RNA substrate-recognizing loop, which is clearly observed in the high-resolution structure. Key residues of B. cereus MazF involved in the catalytic activity are also proposed, and in vitro assay together with mutational studies affirm the ribonucleic activity and the active sites essential for its cellular toxicity. Full article

MazF is an endoribonucleolytic toxin that cleaves intracellular RNAs in sequence-specific manners. It is liberated in bacterial cells in response to environmental changes and is suggested to contribute to bacterial survival by inducing translational regulation. Thus, determining the cleavage specificity provides insights into the physiological functions of MazF orthologues. Nitrospira, detected in a wide range of environments, is thought to have evolved the ability to cope with their surroundings. To investigate the molecular mechanism of its environmental adaption, a MazF module from Nitrospira strain ND1, which was isolated from the activated sludge of a wastewater treatment plant, is examined in this study. By combining a massive parallel sequencing method and fluorometric assay, we detected that this functional RNA-cleaving toxin specifically recognizes the AACU, AACG, and AAUU motifs. Additionally, statistical analysis suggested that this enzyme regulates various specific functions in order to resist environmental stresses. Full article

Nitrosomonas europaea carries numerous toxin-antitoxin systems. However, despite the abundant representation in its chromosome, studies have not surveyed the underlying molecular functions in detail, and their biological roles remain enigmatic. In the present study, we found that a chromosomally-encoded MazF family member, predicted at the locus NE1181, is a functional toxin endoribonuclease, and constitutes a toxin-antitoxin system, together with its cognate antitoxin, MazE. Massive parallel sequencing provided strong evidence that this toxin endoribonuclease exhibits RNA cleavage activity, primarily against the AAU triplet. This sequence-specificity was supported by the results of fluorometric assays. Our results indicate that N. europaea alters the translation profile and regulates its growth using the MazF family of endoribonuclease under certain stressful conditions. Full article

Toxin-antitoxin (TA) systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA) and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1_AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery. Full article