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1
Jin, Chenglong, Sung-Min Kang, Do-Hee Kim, and Bong-Jin Lee. "Structural and functional analysis of the Klebsiella pneumoniae MazEF toxin–antitoxin system." IUCrJ 8, no. 3 (March 5, 2021): 362–71. http://dx.doi.org/10.1107/s2052252521000452.
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Bacterial toxin–antitoxin (TA) systems correlate strongly with physiological processes in bacteria, such as growth arrest, survival and apoptosis. Here, the first crystal structure of a type II TA complex structure of Klebsiella pneumoniae at 2.3 Å resolution is presented. The K. pneumoniae MazEF complex consists of two MazEs and four MazFs in a heterohexameric assembly. It was estimated that MazEF forms a dodecamer with two heterohexameric MazEF complexes in solution, and a truncated complex exists in heterohexameric form. The MazE antitoxin interacts with the MazF toxin via two binding modes, namely, hydrophobic and hydrophilic interactions. Compared with structural homologs, K. pneumoniae MazF shows distinct features in loops β1–β2, β3–β4 and β4–β5. It can be inferred that these three loops have the potential to represent the unique characteristics of MazF, especially various substrate recognition sites. In addition, K. pneumoniae MazF shows ribonuclease activity and the catalytic core of MazF lies in an RNA-binding pocket. Mutation experiments and cell-growth assays confirm Arg28 and Thr51 as critical residues for MazF ribonuclease activity. The findings shown here may contribute to the understanding of the bacterial MazEF TA system and the exploration of antimicrobial candidates to treat drug-resistant K. pneumoniae.
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Norouzi, Masoumeh, Abbas Maleki, Elham Aboualigalehdari, and Sobhan Ghafourian. "Type II toxin- antitoxin systems in clinical isolates of antibiotic resistant Acinetobacter baumannii." Genetika 54, no. 2 (2022): 625–32. http://dx.doi.org/10.2298/gensr2202625n.
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The over use of antibiotics to treat infections in humans and animals made a phenomenon of the antibiotic-resistant bacteria. While studies focused to find on new antibiotics but, identification of novel antibacterial targets in bacteria is very important. By Toxin antitoxin systems this hypothesis could be done, whereas by the activation of a toxin or inactivation of an antitoxin, the raised toxin kills the bacterium. These systems are attractive target for antimicrobial therapy. However, the most important step for potency of TA system, as an antibacterial target, is to identify a TA system that is prevalent in all resistant clinical isolates. So, the prevalence of different TA systems among clinical isolates of Acinetobacter baumannii in Emam khomeini hospital, Ilam, Iran was evaluated to determine which TA system is prevalent in all antibiotic resistant A. baumannii. So, one hundred A. baumannii clinical isolates were identified during one-year period in Emam khomeini hospital, Ilam, Iran. A. baumannii clinical isolates were isolated from hospitalized patients in ICU and burn patients. All isolates were resistant to at least one antibiotic. Then, the isolates were subjected to evaluation to find mazEF and higBA TA genes by PCR. The results showed the frequency of mazEF and highBA TA genes in all isolates was 72% and 39%, respectively. mazEF or higBA TA systems are not presented in all isolates. So, the potency of these two TA systems are in challenged. Also, all isolates were not positive for one TA gene. So, more research in different geographical area should be done with functionality of TA genes.
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Choi, Wonho, Yoshihiro Yamaguchi, Ji-Young Park, Sang-Hyun Park, Hyeok-Won Lee, Byung-Kwan Lim, Michael Otto, Masayori Inouye, Min-Ho Yoon, and Jung-Ho Park. "Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens." Microorganisms 9, no. 5 (May 20, 2021): 1107. http://dx.doi.org/10.3390/microorganisms9051107.
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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.
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Hosseini, Mandana, Jamileh Nowroozi, and Nour Amirmozafari. "The effect of type II toxin-antitoxin systems on methicillinresistant Staphylococcus aureus persister cell formation and antibiotic tolerance." Acta Biologica Szegediensis 65, no. 1 (August 23, 2021): 113–17. http://dx.doi.org/10.14232/abs.2021.1.113-117.
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Persister cells are defi ned as a subpopulation of bacteria in a dormant state with the ability to reduce bacterial metabolism and they are involved in antibiotic tolerance. Toxin-antitoxin (TA) systems have been previously suggested as important players in persistence. Therefore, this study aimed to study the involvement of TA systems in persister cell formation in methicillin-resistant Staphylococcus aureus following antibiotic exposure. Using TADB and RASTA database, two type II TA systems including MazF/MazE and RelE/RelB were predicted in S. aureus. The presence of these TA genes was determined in 5 methicillin-resistant S. aureus isolates and the standard strain S. aureus subsp. aureus N315 using PCR method. To induce persistence, isolates were exposed to lethal doses of ciprofl oxacin and the expression of the studied TA system genes was measured after 5 h using Real-Time PCR. According to our results, all the studied isolates harbored the TA system genes. S. aureus was highly capable of persister cell formation following exposure to sub-MIC of ciprofl oxacin and RT-qPCR showed a signifi cant increase in the expression of the MazEF and RelBE loci, indicating their potential role in antibiotic tolerance. Considering the importance of antibiotic tolerance, further studies on persister cell formation and TA systems involved in this phenomenon are required to effi ciently target these systems.
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Tsilibaris, Virginie, Geneviève Maenhaut-Michel, Natacha Mine, and Laurence Van Melderen. "What Is the Benefit to Escherichia coli of Having Multiple Toxin-Antitoxin Systems in Its Genome?" Journal of Bacteriology 189, no. 17 (May 18, 2007): 6101–8. http://dx.doi.org/10.1128/jb.00527-07.
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ABSTRACT The Escherichia coli K-12 chromosome encodes at least five proteic toxin-antitoxin (TA) systems. The mazEF and relBE systems have been extensively characterized and were proposed to be general stress response modules. On one hand, mazEF was proposed to act as a programmed cell death system that is triggered by a variety of stresses. On the other hand, relBE and mazEF were proposed to serve as growth modulators that induce a dormancy state during amino acid starvation. These conflicting hypotheses led us to test a possible synergetic effect of the five characterized E. coli TA systems on stress response. We compared the behavior of a wild-type strain and its derivative devoid of the five TA systems under various stress conditions. We were unable to detect TA-dependent programmed cell death under any of these conditions, even under conditions previously reported to induce it. Thus, our results rule out the programmed-cell-death hypothesis. Moreover, the presence of the five TA systems advantaged neither recovery from the different stresses nor cell growth under nutrient-limited conditions in competition experiments. This casts a doubt on whether TA systems significantly influence bacterial fitness and competitiveness during non-steady-state growth conditions.
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Nigam, Akanksha, Adi Oron-Gottesman, and Hanna Engelberg-Kulka. "A Bias in the Reading of the Genetic Code of Escherichia coli is a Characteristic for Genes that Specify Stress-induced MazF-mediated Proteins." Current Genomics 21, no. 4 (August 8, 2020): 311–18. http://dx.doi.org/10.2174/1389202921999200606215305.
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Background: Escherichia coli (E. coli) mazEF, a stress-induced toxin-antitoxin (TA) system, has been studied extensively. The MazF toxin is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a Stress-induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Materials and Methods: Escherichia coli (E. coli) mazEF, a stress-induced toxin-antitoxin (TA) system, has been studied extensively. The MazF toxin is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a Stress-induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Results: Here it is reported that for most of the E. coli proteins mediated by stress-induced MazF, the ACA threonine codon in their mRNAs is not in-frame but rather out-of-frame; in these same RNAs, the three synonymous threonine codons, ACG, ACU, and ACC, are in-frame. In contrast, for proteins translated by the canonical translation system, in the majority of mRNAs, the ACA codon is located in-frame. Conclusion: The described bias in the genetic code is a characteristic of E. coli genes specifying for stress-induced MazF-mediated proteins.
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Alkhalili, Rawana, Joel Wallenius, and Björn Canbäck. "Towards Exploring Toxin-Antitoxin Systems in Geobacillus: A Screen for Type II Toxin-Antitoxin System Families in a Thermophilic Genus." International Journal of Molecular Sciences 20, no. 23 (November 22, 2019): 5869. http://dx.doi.org/10.3390/ijms20235869.
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The toxin-antitoxin (TA) systems have been attracting attention due to their role in regulating stress responses in prokaryotes and their biotechnological potential. Much recognition has been given to type II TA system of mesophiles, while thermophiles have received merely limited attention. Here, we are presenting the putative type II TA families encoded on the genomes of four Geobacillus strains. We employed the TA finder tool to mine for TA-coding genes and manually curated the results using protein domain analysis tools. We also used the NCBI BLAST, Operon Mapper, ProOpDB, and sequence alignment tools to reveal the geobacilli TA features. We identified 28 putative TA pairs, distributed over eight TA families. Among the identified TAs, 15 represent putative novel toxins and antitoxins, belonging to the MazEF, MNT-HEPN, ParDE, RelBE, and XRE-COG2856 TA families. We also identified a potentially new TA composite, AbrB-ParE. Furthermore, we are suggesting the Geobacillus acetyltransferase TA (GacTA) family, which potentially represents one of the unique TA families with a reverse gene order. Moreover, we are proposing a hypothesis on the xre-cog2856 gene expression regulation, which seems to involve the c-di-AMP. This study aims for highlighting the significance of studying TAs in Geobacillus and facilitating future experimental research.
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Valizadeh, Nasrin, Firuzeh Valian, Nourkhoda Sadeghifard, Shahriar Karami, Iraj Pakzad, Hossein Kazemian, and Sobhan Ghafourian. "The Role of Peganum harmala Ethanolic Extract and Type II Toxin Antitoxin System in Biofilm Formation." Drug Research 67, no. 07 (March 20, 2017): 385–87. http://dx.doi.org/10.1055/s-0043-102060.
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AbstractToxin antitoxin system is a regulatory system that antitoxin inhibits the toxin. We aimed to determine the role of TA loci in biofilm formation in K. pneumoniae clinical and environmental isolates; also inhibition of biofilm formation by Peganum harmala. So, 40 K. pneumoniae clinical and environmental isolates were subjected for PCR to determine the frequency of mazEF, relEB, and mqsRA TA loci. Biofilm formation assay subjected for all isolates. Then, P. harmala was tested against positive biofilm formation strains. Our results demonstrated that relBE TA loci were dominant TA loci; whereas mqsRA TA loci were negative in all isolates. The most environmental isolates showed weak and no biofilm formation while strong and moderate biofilm formation observed in clinical isolates. Biofilm formations by K. pneumoniae in 9 ug/ml concentration were inhibited by P. harmala. In vivo study suggested to be performed to introduce Peganum harmala as anti-biofilm formation in K. pneumoniae.
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Chen, Ran, Jie Tu, Yaoju Tan, Xingshan Cai, Chengwen Yang, Xiangyu Deng, Biyi Su, et al. "Structural and Biochemical Characterization of the Cognate and Heterologous Interactions of the MazEF-mt9 TA System." ACS Infectious Diseases 5, no. 8 (June 21, 2019): 1306–16. http://dx.doi.org/10.1021/acsinfecdis.9b00001.
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Chen, Ran, Jie Tu, Yaoju Tan, Xingshan Cai, Chengwen Yang, Xiangyu Deng, Biyi Su, et al. "Correction to Structural and Biochemical Characterization of the Cognate and Heterologous Interactions of the MazEF-mt9 TA System." ACS Infectious Diseases 6, no. 9 (August 28, 2020): 2543. http://dx.doi.org/10.1021/acsinfecdis.0c00577.
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Chen, Ran, Jie Zhou, and Wei Xie. "Mechanistic Insight into the Peptide Binding Modes to Two M. tb MazF Toxins." Toxins 13, no. 5 (April 28, 2021): 319. http://dx.doi.org/10.3390/toxins13050319.
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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.
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Shapira, Shiran, Ilana Boustanai, Dina Kazanov, Marina Ben Shimon, Ahmad Fokra, and Nadir Arber. "Innovative dual system approach for selective eradication of cancer cells using viral-based delivery of natural bacterial toxin–antitoxin system." Oncogene 40, no. 31 (June 25, 2021): 4967–79. http://dx.doi.org/10.1038/s41388-021-01792-8.
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AbstractThe inactivation of p53, a tumor suppressor, and the activation of the RAS oncogene are the most frequent genetic alterations in cancer. We have shown that a unique E. coli MazF-MazE toxin–antitoxin (TA) system can be used for selective and effective eradication of RAS-mutated cancer cells. This out of the box strategy holds great promise for effective cancer treatment and management. We provide proof of concept for a novel platform to selectively eradicate cancer cells using an adenoviral delivery system based on the adjusted natural bacterial system. We generated adenoviral vectors carrying the mazF toxin (pAdEasy-Py4-SV40mP-mCherry-MazF) and the antitoxin mazE (pAdEasy-RGC-SV40mP-MazE-IRES-GFP) under the regulation of RAS and p53, resp. The control vector carries the toxin without the RAS-responsive element (pAdEasy-ΔPy4-SV40mP-mCherry-MazF). In vitro, the mazF-mazE TA system (Py4-SV40mP-mCherry-MazF+RGC-SV40mP-MazE-IRES-GFP) induced massive, dose-dependent cell death, at 69% compared to 19% for the control vector, in a co-infected HCT116 cell line. In vivo, the system caused significant tumor growth inhibition of HCT116 (KRASmut/p53mut) tumors at 73 and 65% compared to PBS and ΔPY4 control groups, resp. In addition, we demonstrate 65% tumor growth inhibition in HCT116 (KRASmut/p53wt) cells, compared to the other two control groups, indicating a contribution of the antitoxin in blocking system leakage in WT RAS cells. These data provide evidence of the feasibility of using mutations in the p53 and RAS pathway to efficiently kill cancer cells. The platform, through its combination of the antitoxin (mazE) with the toxin (mazF), provides effective protection of normal cells from basal low activity or leakage of mazF.
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Donegan, Niles P., and Ambrose L. Cheung. "Regulation of the mazEF Toxin-Antitoxin Module in Staphylococcus aureus and Its Impact on sigB Expression." Journal of Bacteriology 191, no. 8 (January 30, 2009): 2795–805. http://dx.doi.org/10.1128/jb.01713-08.
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ABSTRACT In Staphylococcus aureus, the sigB operon codes for the alternative sigma factor σB and its regulators that enable the bacteria to rapidly respond to environmental stresses via redirection of transcriptional priorities. However, a full model of σB regulation in S. aureus has not yet emerged. Earlier data has suggested that mazEF, a toxin-antitoxin (TA) module immediately upstream of the sigB operon, was transcribed with the sigB operon. Here we demonstrate that the promoter P mazE upstream of mazEF is essential for full σB activity and that instead of utilizing autorepression typical of TA systems, sigB downregulates this promoter, providing a negative-feedback loop for sigB to repress its own transcription. We have also found that the transcriptional regulator SarA binds and activates P mazE . In addition, P mazE was shown to respond to environmental and antibiotic stresses in a way that provides an additional layer of control over sigB expression. The antibiotic response also appears to occur in two other TA systems in S. aureus, indicating a shared mechanism of regulation.
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Shapira, Shiran, Ilana Boustanai, Dina Kazanov, Ahmad Fokra, Ezra Bernstein, Ido Wolf, and Nadir Arber. "Innovative dual system for selective eradication of cancer cells using exosomes carrying natural bacterial toxin-antitoxin (TA)." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e14635-e14635. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e14635.
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e14635 Background: Inactivation of P53 and activation of ras are frequent genetic alterations in cancer. We have shown in vitro and in vivo, that the TA system can selectively and effectively eradicate RAS-mutated cancer cells. Aim: Selective killing of cancer cells while sparing the normal cells based on tumor genetic signature. Methods: A “first generation” ΔE1/ΔE3 human type-5 adenoviral-vectors for gene delivery were designed and constructed to specifically target cancer cells. They are designated as "PY4-mazF-mCherry" (PY4, ras responsive element), "ΔPY4-mazF-mCherry" (control viruses) and "RGC-mazE-IRES-GFP" (RGC, P53 responsive element). Their potency was tested in vitro, by the enzymatic MTT assay, microscopic observation, colony formation assay and FACS analysis, and in a xenograft model of CRC. Next, we generated, small natural vesicles, exosomes, that directly targeted cancer through specific small antibody fragments against CD24 that is expressed in most cancer cells and rarely on normal cells. Results: The TA system ("PY4-mazF-mCherry"+"RGC-mazE-IRES-GFP") induced a massive cell death, in a dose-dependent manner in vitro, 69% as compared to 19% in control co-infected ("ΔPY4-mazF-mCherry"+"RGC-mazE-IRES-GFP") HCT116 CRC cells (mutated RAS and p53). In vivo, growth of HCT116-/- ( KRASmutand P53mut) and HCT116+/+ ( KRASmut and P53wt) tumors were significantly inhibited (70% and 65%, respectively). Conclusions: 1. Abusing the P53 genetic status and the activated Ras pathway holds promising effective and safe strategy to target tumor cells while sparing normal tissues. 2. It is a proof of concept for personalized cancer therapy based on the tumor genetic profile.
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Donegan, Niles P., Earl T. Thompson, Zhibiao Fu, and Ambrose L. Cheung. "Proteolytic Regulation of Toxin-Antitoxin Systems by ClpPC in Staphylococcus aureus." Journal of Bacteriology 192, no. 5 (December 28, 2009): 1416–22. http://dx.doi.org/10.1128/jb.00233-09.
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ABSTRACT Bacterial toxin-antitoxin (TA) systems typically consist of a small, labile antitoxin that inactivates a specific longer-lived toxin. In Escherichia coli, such antitoxins are proteolytically regulated by the ATP-dependent proteases Lon and ClpP. Under normal conditions, antitoxin synthesis is sufficient to replace this loss from proteolysis, and the bacterium remains protected from the toxin. However, if TA production is interrupted, antitoxin levels decrease, and the cognate toxin is free to inhibit the specific cellular component, such as mRNA, DnaB, or gyrase. To date, antitoxin degradation has been studied only in E. coli, so it remains unclear whether similar mechanisms of regulation exist in other organisms. To address this, we followed antitoxin levels over time for the three known TA systems of the major human pathogen Staphylococcus aureus, mazEF, axe1-txe1, and axe2-txe2. We observed that the antitoxins of these systems, MazE sa , Axe1, and Axe2, respectively, were all degraded rapidly (half-life [t 1/2], ∼18 min) at rates notably higher than those of their E. coli counterparts, such as MazE (t 1/2, ∼30 to 60 min). Furthermore, when S. aureus strains deficient for various proteolytic systems were examined for changes in the half-lives of these antitoxins, only strains with clpC or clpP deletions showed increased stability of the molecules. From these studies, we concluded that ClpPC serves as the functional unit for the degradation of all known antitoxins in S. aureus.
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Kang, Sung-Min, Ji Sung Koo, Chang-Min Kim, Do-Hee Kim, and Bong-Jin Lee. "mRNA Interferase Bacillus cereus BC0266 Shows MazF-Like Characteristics Through Structural and Functional Study." Toxins 12, no. 6 (June 8, 2020): 380. http://dx.doi.org/10.3390/toxins12060380.
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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.
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Kim, Younghoon, Xiaoxue Wang, Qun Ma, Xue-Song Zhang, and Thomas K. Wood. "Toxin-Antitoxin Systems in Escherichia coli Influence Biofilm Formation through YjgK (TabA) and Fimbriae." Journal of Bacteriology 191, no. 4 (December 5, 2008): 1258–67. http://dx.doi.org/10.1128/jb.01465-08.
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ABSTRACT The roles of toxin-antitoxin (TA) systems in bacteria have been debated. Here, the role of five TA systems in regard to biofilm development was investigated (listed as toxin/antitoxin: MazF/MazE, RelE/RelB, ChpB, YoeB/YefM, and YafQ/DinJ). Although these multiple TA systems were reported previously to not impact bacterial fitness, we found that deletion of the five TA systems decreased biofilm formation initially (8 h) on three different surfaces and then increased biofilm formation (24 h) by decreasing biofilm dispersal. Whole-transcriptome profiling revealed that the deletion of the five TA systems induced expression of a single gene, yjgK, which encodes an uncharacterized protein; quantitative real-time PCR (qRT-PCR) confirmed consistent induction of this gene (at 8, 15, and 24 h). Corroborating the complex phenotype seen upon deleting the TA systems, overexpression of YjgK decreased biofilm formation at 8 h and increased biofilm formation at 24 h; deletion of yjgK also affected biofilm formation in the expected manner by increasing biofilm formation after 8 h and decreasing biofilm formation after 24 h. In addition, YjgK significantly reduced biofilm dispersal. Whole-transcriptome profiling revealed YjgK represses fimbria genes at 8 h (corroborated by qRT-PCR and a yeast agglutination assay), which agrees with the decrease in biofilm formation upon deleting the five TA systems at 8 h, as well as that seen upon overexpressing YjgK. Sand column assays confirmed that deleting the five TA systems reduced cell attachment. Furthermore, deletion of each of the five toxins increased biofilm formation at 8 h, and overexpression of the five toxins repressed biofilm formation at 8 h, a result that is opposite that of deleting all five TA systems; this suggests that complex regulation occurs involving the antitoxins. Also, the ability of the global regulator Hha to reduce biofilm formation was dependent on the presence of these TA systems. Hence, we suggest that one role of TA systems is to influence biofilm formation.
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SINGH, RANDHIR, and XIUPING JIANG. "Expression of Stress and Virulence Genes in Escherichia coli O157:H7 Heat Shocked in Fresh Dairy Compost." Journal of Food Protection 78, no. 1 (January 1, 2015): 31–41. http://dx.doi.org/10.4315/0362-028x.jfp-13-529.
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The purpose of this study was to determine the gene expression of Escherichia coli O157:H7 heat shocked in dairy compost. A two-step real-time PCR assay was used to evaluate the expression of stress and virulence genes in E. coli O157:H7 heat shocked in compost at 47.5°C for 10 min. Heat-shocked E. coli O157:H7 in compost was isolated by using an immunomagnetic bead separation method, followed by total RNA extraction, which was then converted to cDNA by using a commercial kit. E. coli O157:H7 heat shocked in broth served as the media control. In compost, heat shock genes (clpB, dnaK, and groEL) and the alternative sigma factor (rpoH) of E. coli O157:H7 were upregulated (P < 0.05), whereas the expression of trehalose synthesis genes did not change. Virulence genes, such as stx1 and fliC, were upregulated, while genes stx2, eaeA, and hlyA were down-regulated. In the toxin-antitoxin (TA) system, toxin genes, mazF, hipA, and yafQ were upregulated, whereas among antitoxin genes, only dinJ was upregulated (P < 0.05). In tryptic soy broth, all heat shock genes (rpoH, clpB, dnaK, and groEL) were upregulated (P < 0.05), and most virulence genes (stx1, stx2, hlyA, and fliC) and TA genes (mazF-mazE, hipA-hipB, and yafQ-dinJ and toxin gene chpS) were down-regulated. Our results revealed various gene expression patterns when E. coli O157:H7 inoculated in compost was exposed to a sublethal temperature. Clearly, induction of the heat shock response is one of the important protective mechanisms that prolongs the survival of pathogens during the composting process. In addition, other possible mechanisms (such as the TA system) operating along with heat shock response may be responsible for the extended survival of pathogens in compost.
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Tandon, Himani, Akhila Melarkode Vattekatte, Narayanaswamy Srinivasan, and Sankaran Sandhya. "Molecular and Structural Basis of Cross-Reactivity in M. tuberculosis Toxin–Antitoxin Systems." Toxins 12, no. 8 (July 29, 2020): 481. http://dx.doi.org/10.3390/toxins12080481.
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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.
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Ewalds-Kvist, S. Béatrice M., Ritva-Kajsa Selander, and N. Kenneth Sandnabba. "Sex-Related Coping Responses in Mice Selectively Bred for Aggression." Perceptual and Motor Skills 84, no. 3 (June 1997): 911–14. http://dx.doi.org/10.2466/pms.1997.84.3.911.
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Sex differences in strategies of coping with novel situations were studied in three strains of mice with regard to metabolism and open-field and maze activity as well as learning-induced adjustment. The 140 mice were selectively bred for high (Turku Aggressive [TA]) and low (Turku Nonaggressive [TNA]) levels of aggressiveness and originated from a Swiss albino stock normally distributed [N] for aggressiveness. The results indicated that TNA sex differences are more similar to those of the control N mice as compared to those of TA mice. In maze learning, however, the sex differences of TA mice are more in agreement with those of the N strain. Recordings of metabolism and open-field as well as maze activity were correlates of both gender and strain. Sex differences in learning-induced open-field coping behavior were unrelated to strain.
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Selander, Ritva-Kajsa, and S. Béatrice M. Kvist. "Open-Field Parameters and Maze Learning in Aggressive and Nonaggressive Male Mice." Perceptual and Motor Skills 73, no. 3 (December 1991): 811–24. http://dx.doi.org/10.2466/pms.1991.73.3.811.
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Significant differences were observed in thigmotaxis, ambulation, and latency to move (time to start ambulating) between highly aggressive (TA) and low aggressive (TNA) male mice. The former displayed more thigmotaxis, ambulated more, and had a shorter latency to move than the TNA animals. Also they voided a greater number of urinary spots and defecated less than TNA. Further they were superior to the TNA mice in maze-learning capacity. The tendency to enter inner partitions of the field as well as total ambulation increased after learning by TA mice. The training toward nonaggressiveness of TA mice suppressed aggressive responses, thigmotaxis, and the number of urinary spots but enhanced defecation All measures returned to their initial levels after one month of rest. The attacking behaviour of TA animals increased both thigmotaxis and ambulation.
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Wang, Xiaoxue, and Thomas K. Wood. "Toxin-Antitoxin Systems Influence Biofilm and Persister Cell Formation and the General Stress Response." Applied and Environmental Microbiology 77, no. 16 (June 17, 2011): 5577–83. http://dx.doi.org/10.1128/aem.05068-11.
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ABSTRACTIn many genomes, toxin-antitoxin (TA) systems have been identified; however, their role in cell physiology has been unclear. Here we examine the evidence that TA systems are involved in biofilm formation and persister cell formation and that these systems may be important regulators of the switch from the planktonic to the biofilm lifestyle as a stress response by their control of secondary messenger 3′,5′-cyclic diguanylic acid. Specifically, upon stress, the sequence-specific mRNA interferases MqsR and MazF mediate cell survival. In addition, we propose that TA systems are not redundant, as they may have developed to respond to specific stresses.
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Shapira, Shiran, Daniela Eisurovich, Dina Kazanov, and Nadir Arber. "Abstract 1707: Establishment of a targeted exosome drug delivery system containing natural bacterial toxins and antitoxins." Cancer Research 83, no. 7_Supplement (April 4, 2023): 1707. http://dx.doi.org/10.1158/1538-7445.am2023-1707.
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Abstract Background: The inactivation of p53, a tumor suppressor, and the activation of the Ras oncogene are the most frequent genetic alterations in cancer. We have shown that a unique E. coli MazF-MazE toxin-antitoxin (TA) system, controlled by Ras and p53, delivered via adenoviruses can be used to selectively and effectively eradicate Ras mutated cancer cells (Shapira et al., oncogene, 2021). Because of their low immunogenicity and known advantage as ideal cell transporters, exosomes offer great promise as cancer immunotherapy agents. CD24 is a GPI-anchored protein that highly expressed in most of human malignancies and not normal cells. Aim: Development of a drug delivery platform for effective and selective eradication of cancer cells while sparing normal cells based on the Trojan horse strategy and genetic signature. Methods: Two delivery systems have been designed and developed; targeted lentivirus and exosomes. Trex293 cells were used to establish stable scFv-CD24-expressing clones, which were adapted to suspension culture in a chemically-defined media. Exosomes were purified by polymer precipitation method and characterized by several methods including nano-particle tracking analysis, Western blot and EXO-ELISA. CD24 stable transfected HCT116-/- (P53 null) and HCT116+/+ (WTP53) clones were established, and evaluated for CD24 and P53 expression. HCT116+/+-CD24 cells were treated with exosomes loaded with GFP or MazF expressing plasmids and analyzed for GFP expression and cell viability. Results: Targeted lentiviruses carrying the TA system effiently shrinked CRC-derived xenografts tumors in mice. Then, a new genetically encoded pseudotyping platform, based on exosomes, was developed. A homogenic and pure population of exosomes was purified from 3D culture of the highest expressing clone (TREx-scFv). Stable and high expression of CD24 were shown, by Western blot and flow cytometry, in both, HCT116-/- and HCT116+/+ cells. HCT116+/+-CD24 cells treated with 5FU showed active P53. The ability of loading DNA into the purified exosomes and deliver them to the target cells were evaluated and showed high and intensive GFP expression. mazF was successfully loaded into the scFv targeted-exosomes and induced a massive cell death, in a dose-dependent manner. Conclusions: By exploiting the P53 genetic status as well as the Ras pathway, an effective and safe treatment could be developed that targets tumor cells specifically while sparing normal tissues. The effectiveness of this out-of-the-box approach holds great promise for the treatment and management of cancer. Citation Format: Shiran Shapira, Daniela Eisurovich, Dina Kazanov, Nadir Arber. Establishment of a targeted exosome drug delivery system containing natural bacterial toxins and antitoxins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1707.
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Tasneem, Maisha, Shipan Das Gupta, Monira Binte Momin, Kazi Modasser Hossain, Tasnim Binta Osman, and Md Fazley Rabbi. "In silico annotation of a hypothetical protein from Listeria monocytogenes EGD-e unfolds a toxin protein of the type II secretion system." Genomics & Informatics 21, no. 1 (March 31, 2023): e7. http://dx.doi.org/10.5808/gi.22071.
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The gram-positive bacterium Listeria monocytogenes is an important foodborne intracellular pathogen that is widespread in the environment. The functions of hypothetical proteins (HP) from various pathogenic bacteria have been successfully annotated using a variety of bioinformatics strategies. In this study, a HP Imo0888 (NP_464414.1) from the Listeria monocytogenes EGD-e strain was annotated using several bioinformatics tools. Various techniques, including CELLO, PSORTb, and SOSUIGramN, identified the candidate protein as cytoplasmic. Domain and motif analysis revealed that the target protein is a PemK/MazF-like toxin protein of the type II toxin-antitoxin system (TA) which was consistent with BLASTp analysis. Through secondary structure analysis, we found the random coil to be the most frequent. The Alpha Fold 2 Protein Structure Prediction Database was used to determine the three-dimensional (3D) structure of the HP using the template structure of a type II TA PemK/MazF family toxin protein (DB ID_AFDB: A0A4B9HQB9) with 99.1% sequence identity. Various quality evaluation tools, such as PROCHECK, ERRAT, Verify 3D, and QMEAN were used to validate the 3D structure. Following the YASARA energy minimization method, the target protein's 3D structure became more stable. The active site of the developed 3D structure was determined by the CASTp server. Most pathogens that harbor TA systems create a crucial risk to human health. Our aim to annotate the HP Imo088 found in Listeria could offer a chance to understand bacterial pathogenicity and identify a number of potential targets for drug development.
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Maleki, Abbas, Sobhan Ghafourian, Iraj Pakzad, Behzad Badakhsh, and Nourkhoda Sadeghifard. "mazE Antitoxin of Toxin Antitoxin System and fbpA as Reliable Targets to Eradication of Neisseria meningitidis." Current Pharmaceutical Design 24, no. 11 (June 27, 2018): 1204–10. http://dx.doi.org/10.2174/1381612824666171213094730.
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Background: Neisseria meningitidis is considered as a dangerous pathogen threatening human health. Nowadays, the new drug target is focused. Toxin antitoxin (TA) system is recently identified as an antimicrobial drug target. Also, in N. meningitidis, iron-uptake system could be an interesting target for drug discovery. Methods: In this study, fbpA and mazE genes were chosen as new antimicrobial targets and treated with antisense peptide nucleic acid (PNA). Firstly, they were evaluated by bioinformatics and then analyzed by experimental procedures. Secondly, the functionality was evaluated by stress conditions. Results: Our results interestingly demonstrated that when fbpA and mazE loci of N. meningitidis were targeted by antisense PNA, 8 µM concentration of fbpA-PNA as well as 30 µM concentration of mazE-PNA inhibited the growth of N. meningitides and were found to be bacteriostatic, whereas 10 μM concentration of fbpA-PNA showed bacteriocidal activity. Conclusion: Our findings demonstrated the bactriocidal activity of fbpA-PNA and bacteriostatic activity of mazEPNA. Therefore, mazE and fbpA genes should be potent antimicrobial targets but further analysis including in vivo analysis should be performed.
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Klimina, Ksenia M., Artem S. Kasianov, Elena U. Poluektova, Kirill V. Emelyanov, Viktoriya N. Voroshilova, Natalia V. Zakharevich, Anna V. Kudryavtseva, Vsevolod J. Makeev, and Valery N. Danilenko. "Employing toxin-antitoxin genome markers for identification of Bifidobacterium and Lactobacillus strains in human metagenomes." PeerJ 7 (March 4, 2019): e6554. http://dx.doi.org/10.7717/peerj.6554.
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Recent research has indicated that in addition to the unique genotype each individual may also have a unique microbiota composition. Difference in microbiota composition may emerge from both its species and strain constituents. It is important to know the precise composition especially for the gut microbiota (GM), since it can contribute to the health assessment, personalized treatment, and disease prevention for individuals and groups (cohorts). The existing methods for species and strain composition in microbiota are not always precise and usually not so easy to use. Probiotic bacteria of the genus Bifidobacterium and Lactobacillus make an essential component of human GM. Previously we have shown that in certain Bifidobacterium and Lactobacillus species the RelBE and MazEF superfamily of toxin-antitoxin (TA) systems may be used as functional biomarkers to differentiate these groups of bacteria at the species and strain levels. We have composed a database of TA genes of these superfamily specific for all lactobacilli and bifidobacteria species with complete genome sequence and confirmed that in all Lactobacillus and Bifidobacterium species TA gene composition is species and strain specific. To analyze composition of species and strains of two bacteria genera, Bifidobacterium and Lactobacillus, in human GM we developed TAGMA (toxin antitoxin genes for metagenomes analyses) software based on polymorphism in TA genes. TAGMA was tested on gut metagenomic samples. The results of our analysis have shown that TAGMA can be used to characterize species and strains of Lactobacillus and Bifidobacterium in metagenomes.
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Zhu, Ling, Koichi Inoue, Satoshi Yoshizumi, Hiroshi Kobayashi, Yonglong Zhang, Ming Ouyang, Fuminori Kato, Motoyuki Sugai, and Masayori Inouye. "Staphylococcus aureus MazF Specifically Cleaves a Pentad Sequence, UACAU, Which Is Unusually Abundant in the mRNA for Pathogenic Adhesive Factor SraP." Journal of Bacteriology 191, no. 10 (February 27, 2009): 3248–55. http://dx.doi.org/10.1128/jb.01815-08.
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ABSTRACT Escherichia coli mRNA interferases, such as MazF and ChpBK, are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems present in its genome. A MazF homologue in Staphylococcus aureus (MazFSa) has been shown to inhibit cell growth when induced in E. coli. Here, we determined the cleavage site for MazFSa with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. MazFSa specifically cleaves the RNA at a pentad sequence, U↓ACAU. Bioinformatics analysis revealed that this pentad sequence is significantly abundant in several genes, including the sraP gene in the S. aureus N315 strain. This gene encodes a serine-rich protein, which is known to play an important role in adhesion of the pathogen to human tissues and thus in endovascular infection. We demonstrated that the sraP mRNA became extremely unstable in comparison with the ompA mRNA only when MazFSa was induced in E. coli. Further bioinformatics analysis indicated that the pentad sequence is also significantly abundant in the mRNAs for all the pathogenic factors in S. aureus. This observation suggests a possible regulatory relationship between the MazEFSa TA module and the pathogenicity in S. aureus.
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Jørgensen, Mikkel G., Deo P. Pandey, Milena Jaskolska, and Kenn Gerdes. "HicA of Escherichia coli Defines a Novel Family of Translation-Independent mRNA Interferases in Bacteria and Archaea." Journal of Bacteriology 191, no. 4 (December 5, 2008): 1191–99. http://dx.doi.org/10.1128/jb.01013-08.
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ABSTRACT Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins, have been identified. Recently, Makarova et al. (K. S. Makarova, N. V. Grishin, and E. V. Koonin, Bioinformatics 22:2581-2584, 2006) suggested that the hicAB loci constitute a novel TA gene family. Using the hicAB locus of Escherichia coli K-12 as a model system, we present evidence that supports this inference: expression of the small HicA protein (58 amino acids [aa]) induced cleavage in three model mRNAs and tmRNA. Concomitantly, the global rate of translation was severely reduced. Using tmRNA as a substrate, we show that HicA-induced cleavage does not require the target RNA to be translated. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. These results suggest that HicB neutralizes HicA and therefore functions as an antitoxin. As with other antitoxins (RelB and MazF), HicB could resuscitate cells inhibited by HicA, indicating that ectopic production of HicA induces a bacteriostatic rather than a bactericidal condition. Nutrient starvation induced strong hicAB transcription that depended on Lon protease. Mining of 218 prokaryotic genomes revealed that hicAB loci are abundant in bacteria and archaea.
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Calcuttawala, Fatema, Rahul Shaw, Arpita Sarbajna, Moumita Dutta, Saptarshi Sinha, and Sujoy K. Das Gupta. "Apoptosis like symptoms associated with abortive infection of Mycobacterium smegmatis by mycobacteriophage D29." PLOS ONE 17, no. 5 (May 17, 2022): e0259480. http://dx.doi.org/10.1371/journal.pone.0259480.
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Mycobacteriophages are phages that infect mycobacteria resulting in their killing. Although lysis is the primary mechanism by which mycobacteriophages cause cell death, others such as abortive infection may also be involved. We took recourse to perform immunofluorescence and electron microscopic studies using mycobacteriophage D29 infected Mycobacterium smegmatis cells to investigate this issue. We could observe the intricate details of the infection process using these techniques such as adsorption, the phage tail penetrating the thick mycolic acid layer, formation of membrane pores, membrane blebbing, and phage release. We observed a significant increase in DNA fragmentation and membrane depolarization using cell-biological techniques symptomatic of programmed cell death (PCD). As Toxin-Antitoxin (TA) systems mediate bacterial PCD, we measured their expression profiles with and without phage infection. Of the three TAs examined, MazEF, VapBC, and phd/doc, we found that in the case of VapBC, a significant decrease in the antitoxin (VapB): toxin (VapC) ratio was observed following phage infection, implying that high VapC may have a role to play in the induction of mycobacterial apoptotic cell death following phage infection. This study indicates that D29 infection causes mycobacteria to undergo morphological and molecular changes that are hallmarks of apoptotic cell death.
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Chandra, Soumyanetra, Gopinath Chattopadhyay, and Raghavan Varadarajan. "Rapid Identification of Secondary Structure and Binding Site Residues in an Intrinsically Disordered Protein Segment." Frontiers in Genetics 12 (November 2, 2021). http://dx.doi.org/10.3389/fgene.2021.755292.
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Mycobacterium tuberculosis harbours nine toxin-antitoxin (TA) systems of the MazEF family. MazEF TA modules are of immense importance due to the perceived role of the MazF toxin in M. tuberculosis persistence and disease. The MazE antitoxin has a disordered C-terminal domain that binds the toxin, MazF and neutralizes its endoribonuclease activity. However, the structure of most MazEF TA complexes remains unsolved till date, obscuring structural and functional information about the antitoxins. We present a facile method to identify toxin binding residues on the disordered antitoxin. Charged residue scanning mutagenesis was used to screen a yeast surface displayed MazE6 antitoxin library against its purified cognate partner, the MazF6 toxin. Binding residues were deciphered by probing the relative reduction in binding to the ligand by flow cytometry. We have used this to identify putative antitoxin interface residues and local structure attained by the antitoxin upon interaction in the MazEF6 TA system and the same methodology is readily applicable to other intrinsically disordered protein regions.
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Nim, Jogendra Singh, Mohit Yadav, Lalit Kumar Gautam, Chaitali Ghosh, Shakti Sahi, and Jitendra Singh Rathore. "Novel Toxin-antitoxin System Xn-mazEF from Xenorhabdus nematophi-la: Identification, Characterization and Functional Exploration." Current Computer-Aided Drug Design 16 (June 25, 2020). http://dx.doi.org/10.2174/1573409916666200625135850.
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Background: Xenorhabdus nematophila maintains species-specific mutual interaction with nematodes of Steinernema genus. Type II Toxin Antitoxin (TA) systems, the mazEF TA system controls stress and programmed cell death in bacteria. Objective: This study elucidates the functional characterization of Xn-mazEF, a mazEF homolog in X. nematophila by computational and in vitro approaches. Methods: 3 D- structural models for Xn-MazE toxin and Xn-MazF antitoxin were generated, validated and characterized for protein - RNA interaction analysis. Further biological and cellular functions of Xn-MazF toxin were also predicted. Molecular dynamics simulations of 50ns for Xn-MazF toxin complexed with nucleic acid units (DU, RU, RC, and RU) were performed. The MazF toxin and complete MazEF operon were endogenously expressed and monitored for the killing of Escherichia coli host cells under arabinose induced tightly regulated system. Results: Upon induction, E. coli expressing toxin showed rapid killing within four hours and attained up to 65% growth inhibition, while the expression of the entire operon did not show significant killing. The observation suggests that the Xn-mazEF TA system control transcriptional regulation in X. nematophila and helps to manage stress or cause toxicity leading to programmed death of cells. Conclusion: The study provides insights into structural and functional features of novel toxin, XnMazF and provides an initial inference on control of X. nematophila growth regulated by TA systems.
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Dai, Jingli, Zijing Chen, Jinfeng Hou, Yudong Wang, Miao Guo, Jiajia Cao, Liangyan Wang, Hong Xu, Bing Tian, and Ye Zhao. "MazEF Toxin-Antitoxin System-Mediated DNA Damage Stress Response in Deinococcus radiodurans." Frontiers in Genetics 12 (February 19, 2021). http://dx.doi.org/10.3389/fgene.2021.632423.
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Deinococcus radiodurans shows marked resistance to various types of DNA-damaging agents, including mitomycin C (MMC). A type II toxin-antitoxin (TA) system that responds to DNA damage stress was identified in D. radiodurans, comprising the toxin MazF-dr and the antitoxin MazE-dr. The cleavage specificity of MazF-dr, an endoribonuclease, was previously characterized. Here, we further investigated the regulatory role of the MazEF system in the response to DNA damage stress in D. radiodurans. The crystal structure of D. radiodurans MazF (MazF-dr) was determined at a resolution of 1.3 Å and is the first structure of the toxin of the TA system of D. radiodurans. MazF-dr forms a dimer mediated by the presence of interlocked loops. Transcriptional analysis revealed 650 downregulated genes in the wild-type (WT) strain, but not in the mazEF mutant strain, which are potentially regulated by MazEF-dr in response to MMC treatment. Some of these genes are involved in membrane trafficking and metal ion transportation. Subsequently, compared with the WT strain, the mazEF mutant strain exhibited much lower MMC-induced intracellular iron concentrations, reactive oxygen species (ROS), and protein carbonylation levels. These results provide evidence that MazEF-mediated cell death in D. radiodurans might be caused by an increase in ROS accumulation upon DNA damage stress.
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Sultan, Amira M., and Nawal S. Gouda. "The Association of the mazEF Toxin-antitoxin System and Vancomycin Resistance in Clinical Isolates of Vancomycin Resistant Enterococcus faecalis." Journal of Pure and Applied Microbiology, May 31, 2022. http://dx.doi.org/10.22207/jpam.16.2.46.
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Vancomycin resistant enterococci are challenging bacteria as they are difficult to be eradicated. Toxin-antitoxin (TA) systems are genetic elements located in most prokaryotic genomes. The mazEF TA system is harbored by a plasmid among Enterococcus faecalis (E. faecalis). To explore the relation between the existence of mazEF TA system and vancomycin resistance among clinical isolates of E. faecalis. Samples were collected from patients showing clinical picture of infection. Isolates of E. faecalis were identified by standard microbiological methods and their antimicrobial susceptibility patterns were detected by disk diffusion method. In addition, the E-test was used to confirm vancomycin resistant isolates. All the E. faecalis isolates were screened for the mazEF TA system by PCR. A total of 180 E. faecalis strains were identified with a vancomycin resistance rate of 30.6%. Vancomycin resistance was significantly associated with prolonged hospital stay (P= 0.04) and ICU setting (P= 0.001). The mazEF TA system was detected among 100% of vancomycin resistant isolates, while only 33.6% of the vancomycin sensitive isolates carried the system with a significant difference (P= 0.002). In addition, there was a significant association between the mazEF TA system-positive strains and the ICU setting (P= 0.02). A significant association was found between vancomycin resistance and the presence of the mazEF TA system among E. faecalis isolates. This association supports the current efforts to utilize the mazEF TA system as a possible target for novel antibacterial agents; however, further studies on a wider scale are necessary.
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Wei, Yanxia, Yang Li, Fan Yang, Qiong Wu, Dianbin Liu, Xiangyang Li, Hui Hua, et al. "Physical and Functional Interplay between MazF1Bif and Its Noncognate Antitoxins from Bifidobacterium longum." Applied and Environmental Microbiology 83, no. 9 (February 17, 2017). http://dx.doi.org/10.1128/aem.03232-16.
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ABSTRACT Bifidobacterium longum strain JDM301, a widely used commercial strain in China, encodes at least two MazEF-like modules and one RelBE-like toxin-antitoxin (TA) system in its chromosome, designated MazE1F1 Bif, MazE2F2 Bif, and RelBEBif, respectively. Bacterial TA systems play an important role in several stress responses, but the relationship between these TA systems is largely unknown. In this study, the interactions between MazF1 Bif and MazE2 Bif or RelBBif were assessed in B. longum strain JDM301. MazF1 Bif caused the degradation of tufA Bif mRNA, and its toxicity was inhibited by forming a protein complex with its cognate antitoxin, MazE1 Bif. Notably, MazF1 Bif toxicity was also partially neutralized when jointly expressed with noncognate antitoxin MazE2 Bif or RelBBif. Our results show that the two noncognate antitoxins also inhibited mRNA degradation caused by MazF1 Bif toxin. Furthermore, the physical interplay between MazF1 Bif and its noncognate antitoxins was confirmed by immunoprecipitation. These results suggest that MazF1 Bif can arrest cell growth and that MazF1 Bif toxicity can be neutralized by its cognate and noncognate antitoxins. These results imply that JDM301 uses a sophisticated toxin-antitoxin interaction network to alter its physiology when coping with environmental stress. IMPORTANCE Although toxin-antitoxin (TA) systems play an important role in several stress responses, the regulatory mechanisms of multiple TA system homologs in the bacterial genome remain largely unclear. In this study, the relationships between MazE1F1 Bif and the other two TA systems of Bifidobacterium longum strain JDM301 were explored, and the interactions between MazF1 Bif and MazE2 Bif or RelBBif were characterized. In addition, the mRNA degradation activity of MazF1 Bif was demonstrated. In particular, the interaction of the toxin with noncognate antitoxins was shown, even between different TA families (MazF1 Bif toxin and RelBBif antitoxin) in JDM301. This work provides insight into the regulatory mechanisms of TA systems implicated in the stress responses of bifidobacteria.
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Kumari, Khushboo, and Siddhartha P. Sarma. "Structural and mutational analysis of MazE6-operator DNA complex provide insights into autoregulation of toxin-antitoxin systems." Communications Biology 5, no. 1 (September 15, 2022). http://dx.doi.org/10.1038/s42003-022-03933-5.
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AbstractOf the 10 paralogs of MazEF Toxin-Antitoxin system in Mycobacterium tuberculosis, MazEF6 plays an important role in multidrug tolerance, virulence, stress adaptation and Non Replicative Persistant (NRP) state establishment. The solution structures of the DNA binding domain of MazE6 and of its complex with the cognate operator DNA show that transcriptional regulation occurs by binding of MazE6 to an 18 bp operator sequence bearing the TANNNT motif (-10 region). Kinetics and thermodynamics of association, as determined by NMR and ITC, indicate that the nMazE6-DNA complex is of high affinity. Residues in N-terminal region of MazE6 that are key for its homodimerization, DNA binding specificity, and the base pairs in the operator DNA essential for the protein-DNA interaction, have been identified. It provides a basis for design of chemotherapeutic agents that will act via disruption of TA autoregulation, leading to cell death.
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Alexander, Cyrus, Ankeeta Guru, Pinkilata Pradhan, Sunanda Mallick, Nimai Charan Mahanandia, Bharat Bhusan Subudhi, and Tushar Kant Beuria. "MazEF-rifampicin interaction suggests a mechanism for rifampicin induced inhibition of persisters." BMC Molecular and Cell Biology 21, no. 1 (October 27, 2020). http://dx.doi.org/10.1186/s12860-020-00316-8.
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Abstract Background Persistence is a natural phenomenon whereby a subset of a population of isogenic bacteria either grow slow or become dormant conferring them with the ability to withstand various stresses including antibiotics. In a clinical setting bacterial persistence often leads to the recalcitrance of various infections increasing the treatment time and cost. Additionally, some studies also indicate that persistence can also pave way for the emergence of resistant strains. In a laboratory setting this persistent phenotype is enriched in nutritionally deprived environments. Consequently, in a batch culture the late stationary phase is enriched with persistent bacteria. The mechanism of persister cell formation and its regulation is not well understood. Toxin-antitoxin (TA) systems have been implicated to be responsible for bacterial persistence and rifampicin is used to treat highly persistent bacterial strains. The current study tries to explore a possible interaction between rifampicin and the MazEF TA system that furthers the former’s success rate in treating persistent bacteria. Results In the current study we found that the population of bacteria in the death phase of a batch culture consists of metabolically inactive live cells resembling persisters, which showed higher membrane depolarization as compared to the log phase bacteria. We also observed an increase in the expression of the MazEF TA modules in this phase. Since rifampicin is used to kill the persisters, we assessed the interaction of rifampicin with MazEF complex. We showed that rifampicin moderately interacts with MazEF complex with 1:1 stoichiometry. Conclusion Our study suggests that the interaction of rifampicin with MazEF complex might play an important role in inhibition of persisters.
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Al-Hadban, Wedean, Maysaa Adil Ali, and Nuha Kandala. "The Correlation Between the Persistence of Methicillin Resistant Staphylococcus Aureus Isolates to Mupirocine and Toxin-Antitoxin Type II Genes." Iraqi Journal of Science, May 25, 2022, 1930–40. http://dx.doi.org/10.24996/ijs.2022.63.5.7.
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Under high concentrations of antibiotics, a fraction of the bacterial population exhibits a phenomenon known as persistence. Toxin- system (TA system) has been reported to be involved in the formation of E. coli, Mycobacterium, and S. aureus persisters. In this study, the ability of thirty Iraqi isolates of MRSA to form in vitro persister cells after exposure to three different antibiotics (Ceftriaxone 30 µg, Mecillinam 10 µg, and Mupirocin 20 µg) was examined by TD test. Additionally, efflux pump inhibitor [Fluphenazine 0.25 mg/ml] was combined with the antibiotic that triggered persister formation. The distribution of mazEF and yefM-yoeB (Type II TA system) in the tested isolates was detected by PCR. 91% of Mupirocin susceptible isolates formed persister cells.42% of the persistent level was reduced when Mupirocin was combined with the Fluphenazine. Genes for homologs of the yefM-yoeB and mazEF TA system were present in 100% of the tested isolates. The prevalence of these genes in the tested isolates suggested a link between persistence and the TA system. Further investigation is required to study the expression of these genes under stress conditions.
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Jain, Sonia, Arghya Bhowmick, Bohyun Jeong, Taeok Bae, and Abhrajyoti Ghosh. "Unravelling the physiological roles of mazEF toxin–antitoxin system on clinical MRSA strain by CRISPR RNA-guided cytidine deaminase." Journal of Biomedical Science 29, no. 1 (May 7, 2022). http://dx.doi.org/10.1186/s12929-022-00810-5.
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Abstract Background Curiosity on toxin–antitoxin modules has increased intensely over recent years as it is ubiquitously present in many bacterial genomes, including pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). Several cellular functions of TA systems have been proposed however, their exact role in cellular physiology remains unresolved. Methods This study aims to find out the impact of the mazEF toxin–antitoxin module on biofilm formation, pathogenesis, and antibiotic resistance in an isolated clinical ST239 MRSA strain, by constructing mazE and mazF mutants using CRISPR–cas9 base-editing plasmid (pnCasSA-BEC). Transcriptome analysis (RNA-seq) was performed for the mazE antitoxin mutant in order to identify the differentially regulated genes. The biofilm formation was also assessed for the mutant strains. Antibiogram profiling was carried out for both the generated mutants followed by murine experiment to determine the pathogenicity of the constructed strains. Results For the first time our work showed, that MazF promotes cidA mediated cell death and lysis for biofilm formation without playing any significant role in host virulence as suggested by the murine experiment. Interestingly, the susceptibility to oxacillin, daptomycin and vancomycin was reduced significantly by the activated MazF toxin in the mazE mutant strain. Conclusions Our study reveals that activated MazF toxin leads to resistance to antibiotics like oxacillin, daptomycin and vancomycin. Therefore, in the future, any potential antibacterial drug can be designed to target MazF toxin against the problematic multi-drug resistant bug.
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Ahmed, Shahbaz, Gopinath Chattopadhyay, Kavyashree Manjunath, Munmun Bhasin, Neelam Singh, Mubashir Rasool, Sayan Das, et al. "Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region." Frontiers in Molecular Biosciences 9 (October 7, 2022). http://dx.doi.org/10.3389/fmolb.2022.997653.
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The Mycobacterium tuberculosis genome harbours nine toxin-antitoxin (TA) systems of the mazEF family. These consist of two proteins, a toxin and an antitoxin, encoded in an operon. While the toxin has a conserved fold, the antitoxins are structurally diverse and the toxin binding region is typically intrinsically disordered before binding. We describe high throughput methodology for accurate mapping of interfacial residues and apply it to three MazEF complexes. The method involves screening one partner protein against a panel of chemically masked single cysteine mutants of its interacting partner, displayed on the surface of yeast cells. Such libraries have much lower diversity than those generated by saturation mutagenesis, simplifying library generation and data analysis. Further, because of the steric bulk of the masking reagent, labeling of virtually all exposed epitope residues should result in loss of binding, and buried residues are inaccessible to the labeling reagent. The binding residues are deciphered by probing the loss of binding to the labeled cognate partner by flow cytometry. Using this methodology, we have identified the interfacial residues for MazEF3, MazEF6 and MazEF9 TA systems of M. tuberculosis. In the case of MazEF9, where a crystal structure was available, there was excellent agreement between our predictions and the crystal structure, superior to those with AlphaFold2. We also report detailed biophysical characterization of the MazEF3 and MazEF9 TA systems and measured the relative affinities between cognate and non-cognate toxin–antitoxin partners in order to probe possible cross-talk between these systems.
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Oron-Gottesman, Adi, Martina Sauert, Isabella Moll, and Hanna Engelberg-Kulka. "A Stress-Induced Bias in the Reading of the Genetic Code in Escherichia coli." mBio 7, no. 6 (November 15, 2016). http://dx.doi.org/10.1128/mbio.01855-16.
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ABSTRACT Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a stress-induced translation machinery (STM), composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we further characterized the STM system, finding that MazF cleaves only ACA sites located in the open reading frames of processed mRNAs, while out-of-frame ACAs are resistant. This in-frame ACA cleavage of MazF seems to depend on MazF binding to an extracellular-death-factor (EDF)-like element in ribosomal protein bS1 (bacterial S1), apparently causing MazF to be part of STM ribosomes. Furthermore, due to the in-frame MazF cleavage of ACAs under stress, a bias occurs in the reading of the genetic code causing the amino acid threonine to be encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. IMPORTANCE The genetic code is a universal characteristic of all living organisms. It defines the set of rules by which nucleotide triplets specify which amino acid will be incorporated into a protein. Our results represent the first existing report on a stress-induced bias in the reading of the genetic code. We found that in E. coli , under stress, the amino acid threonine is encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. This is because under stress, MazF generates a stress-induced translation machinery (STM) in which MazF cleaves in-frame ACA sites of the processed mRNAs.
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Nigam, Akanksha, Tamar Ziv, Adi Oron-Gottesman, and Hanna Engelberg-Kulka. "Stress-Induced MazF-Mediated Proteins in Escherichia coli." mBio 10, no. 2 (March 26, 2019). http://dx.doi.org/10.1128/mbio.00340-19.
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ABSTRACT Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. By that means, under stress, the induced MazF generates a stress-induced translation machinery (STM) composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through the chromosomally borne mazF gene. We show that the mRNAs of almost all of them are characterized by the presence of an ACA site up to 100 nucleotides upstream of the AUG initiator. Therefore, under stressful conditions, induced MazF processes mRNAs that are translated by STM. Furthermore, the presence of the ACA sites far upstream (up to 100 nucleotides) of the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual-translation mechanisms enable the bacterium under stress also to prepare proteins for immediate functions while coming back to normal growth conditions. IMPORTANCE The stress response, the strategy that bacteria have developed in order to cope up with all kinds of adverse conditions, is so far understood at the level of transcription. Our previous findings of a uniquely modified stress-induced translation machinery (STM) generated in E. coli under stress by the endoribonucleolytic activity of the toxin MazF opens a new chapter in understanding microbial physiology under stress at the translational level. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated by chromosomally borne MazF through STM.
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Shahi, Fatemeh, Azar Dokht Khosravi, Mohammad Savari, Saeed Khoshnood, and Zahra Farshadzadeh. "Toxin-antitoxin Genes Expression in Multidrug-Resistant Mycobacterium tuberculosis Isolates under Drug Exposure." Infectious Disorders - Drug Targets 23 (May 24, 2023). http://dx.doi.org/10.2174/1871526523666230524144448.
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Introduction: Toxin–antitoxin systems (TAs) are highly conserved in Mycobacterium tuberculosis (Mtb). The TAs role in maintaining and disseminating drug resistance in bacterial populations has been indicated. So, we aimed to analyze the expression level of MazEF-related genes in drug-susceptible and multidrug-resistant (MDR) Mtb isolates under isoniazid (INH) and rifampin (RIF) stress. Methods: We obtained 23 Mtb isolates, including 18 MDR and 5 susceptible isolates, from the Ahvaz Regional TB Laboratory collection. The expression levels of mazF3, mazF6, and mazF9 toxin genes, and mazE3, mazE6, and mazE9 antitoxin genes in MDR and susceptible isolates were evaluated by quantitative real-time PCR (qRT-PCR) after exposure to RIF and INH. Results: The mazF3, F6, and F9 toxin genes were overexpressed in at least two MDR isolates in the presence of RIF and INH, in contrast to mazE antitoxin genes. More MDR isolates were induced to overexpress mazF genes by RIF than INH (72.2% vs. 50%). Compared to the H37Rv strain and susceptible isolates, the expression levels of mazF3,6 by RIF and mazF3,6,9 by INH were significantly upregulated in MDR isolates (p<0.05), but no remarkable difference was detected in the expression level of mazF9 genes by INH between these groups. In susceptible isolates, the expression levels of mazE3,6 by RIF and mazE3,6,9 by INH were induced and enhanced significantly compared to MDR isolates, but there was no difference between MDR and H37Rv strain. Conclusion: Based on the results, we propose that mazF expression under RIF/INH stress may be associated with drug resistance in Mtb in addition to mutations, and the mazE antitoxins may be related to enhanced susceptibility of Mtb to INH and RIF. Further experiments are needed to investigate the exact mechanism underlying the TA system's role in drug resistance.
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Tian, Xiao-Lin, Miao Li, Zachariah Scinocca, Heather Rutherford, and Yung-Hua Li. "ClpP is required for proteolytic regulation of type II toxin–antitoxin systems and persister cell formation in Streptococcus mutans." Access Microbiology 1, no. 8 (October 1, 2019). http://dx.doi.org/10.1099/acmi.0.000054.
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The type II toxin–antitoxin (TA) modules, mazEF and relBE, in Streptococcus mutans have been implicated in stress response, antibiotic tolerance and persister cell formation. However, how S. mutans regulates these systems to prevent unwanted toxin activation and persister cell formation is unclear. In this study, we provide evidence that ClpP is required for the proteolytic regulation of these TA systems and persister cell formation in S. mutans following antibiotic challenge. A persister viability assay showed that S. mutans UA159 (WT) formed a larger quantity of persister cells than its isogenic mutant ΔclpP following antibiotic challenge. However, the lux reporter assay revealed that clpP deletion did not affect the transcriptional levels of mazEF and relBE, since no significant differences (P>0.05) in the reporter activities were detected between the wild-type and ΔclpP background. Instead, all antibiotics tested at a sub-minimum inhibitory concentration (sub-MIC) induced transcriptional levels of mazEF and relBE operons. We then examined the protein profiles of His-tagged MazE and RelB proteins in the UA159 and ΔclpP backgrounds by Western blotting analysis. The results showed that S. mutans strains grown under non-stress conditions expressed very low but detectable levels of MazE and RelB antitoxin proteins. Antibiotics at sub-MICs induced the levels of the MazE and RelB proteins, but the protein levels decreased rapidly in the wild-type background. In contrast, a stable level of MazE and RelB proteins could be detected in the ΔclpP mutant background, suggesting that both proteins accumulated in the ΔclpP mutant. We conclude that ClpP is required for the proteolytic regulation of cellular levels of the MazE and RelB antitoxins in S. mutans , which may play a critical role in modulating the TA activities and persister cell formation of this organism following antibiotic challenge.
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Tamiya-Ishitsuka, Hiroko, Masako Tsuruga, Naohiro Noda, and Akiko Yokota. "Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity." Frontiers in Microbiology 12 (November 11, 2021). http://dx.doi.org/10.3389/fmicb.2021.748619.
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The toxin-antitoxin (TA) system, inherent to various prokaryotes, plays a critical role in survival and adaptation to diverse environmental stresses. The toxin MazF, belonging to the type II TA system, functions as a sequence-specific ribonuclease that recognizes 3 to 7 bases. In recent studies, crystallographic analysis of MazFs from several species have suggested the presence of amino acid sites important for MazF substrate RNA binding and for its catalytic activity. Herein, we characterized MazF obtained from Candidatus Desulforudis audaxviator (MazF-Da) and identified the amino acid residues necessary for its catalytic function. MazF-Da, expressed using a cell-free protein synthesis system, is a six-base-recognition-specific ribonuclease that preferentially cleaves UACAAA sequences and weakly cleaves UACGAA and UACUAA sequences. We found that MazF-Da exhibited the highest activity at around 60°C. Analysis using mutants with a single mutation at an amino acid residue site that is well conserved across various MazF toxins showed that G18, E20, R25, and P26 were important for the ribonuclease activity of MazF-Da. The recognition sequence of the N36A mutant differed from that of the wild type. This mutant cleaved UACAAG sequences in addition to UACAAA sequences, but did not cleave UACGAA or UACUAA sequences, suggesting that Asn36 affects the loosening and narrowing of MazF-Da cleavage sequence recognition. Our study posits UACAAA as the recognition sequence of MazF-Da and provides insight into the amino acid sites that are key to its unique enzymatic properties.
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Karimaei, Samira, Behrooz Sadeghi Kalani, Nader Shahrokhi, Rahil Mashhadi, and Mohammad Reza Pourmand. "Expression of type II toxin-antitoxin systems and ClpP protease of methicillin-resistant Staphylococcus aureus under thermal and oxidative stress conditions." Iranian Journal of Microbiology, April 14, 2021. http://dx.doi.org/10.18502/ijm.v13i2.5982.
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Background and Objectives: Staphylococcus aureus is a main human pathogen that causes a variety of chronic to persistent infections. Across the diverse factors of pathogenesis in bacteria, Toxin-Antitoxin (TA) systems can be considered as an antibacterial target due to their involvement in cellular physiology counting stress responses. Here, the expression of TA system genes and ClpP protease was investigated under the thermal and oxidative conditions in S. aureus strains.
Materials and Methods: The colony-forming unit (CFU) was used to determine the effects of thermal and oxidative stresses on bacterial survival. Moreover, the expressions of TA system genes in S. aureus strains were evaluated 30 min and 1 h after thermal and oxidative stresses, respectively, by quantitative reverse transcriptase real-time PCR (qRT-PCR).
Results: The cell viability was constant across thermal stress while oxidative stress induction showed a significantly decrease in the growth of Methicillin-Resistant S. aureus (MRSA) strain. Based on the qRT-PCR results, the expression of mazF gene increased under both thermal and oxidative stresses in the MRSA strain.
Conclusion: A putative TA system (namely immA/irrA) most likely has a role under the stress condition of S. aureus. The MRSA strain responds to stress by shifting the expression level of TA genes that has diverse effects on the survival of the pathogen due to the stress conditions. The TA systems may be introduced as potential targets for antibacterial treatment.
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Chattopadhyay, Gopinath, Munmun Bhasin, Shahbaz Ahmed, Tannu Priya Gosain, Srivarshini Ganesan, Sayan Das, Chandrani Thakur, Nagasuma Chandra, Ramandeep Singh, and Raghavan Varadarajan. "Functional and Biochemical Characterization of the MazEF6 Toxin-Antitoxin System of Mycobacterium tuberculosis." Journal of Bacteriology 204, no. 4 (April 19, 2022). http://dx.doi.org/10.1128/jb.00058-22.
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M. tuberculosis harbors a large number of type II toxin-antitoxin (TA) systems, the exact roles for most of which are unclear. Prior studies have reported that overexpression of several of these type II toxins inhibits bacterial growth and contributes to the formation of drug-tolerant populations in vitro .
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Fico, Sarah, and Jacques Mahillon. "TasA-tasB, a new putative toxin-antitoxin (TA) system from Bacillus thuringiensis pGI1 plasmid is a widely distributed composite mazE-doc TA system." BMC Genomics 7, no. 1 (October 13, 2006). http://dx.doi.org/10.1186/1471-2164-7-259.
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Kheradmand, Erfan, Shabnam Razavi, Malihe Talebi, and Mahmood Jamshidian. "Evaluation of Putative Type II Toxin-Antitoxin Systems and Lon Protease Expression in Shigella flexneri Following Infection of Caco-2 Cells." Archives of Clinical Infectious Diseases 15, no. 3 (September 20, 2020). http://dx.doi.org/10.5812/archcid.98625.
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: Shigella flexneri causes bacillary dysentery in developing countries. Due to recent reports regarding antimicrobial resistance in human S. flexneri, finding alternative therapeutics is of vital importance. Toxin-antitoxin (TA) systems have recently been introduced as antimicrobial targets owing to their involvement in bacterial survival in stress conditions and “persister” cell formation. In this study, the presence of four TA loci were studied in S. flexneri ATCC 12022. The presence of genes coding for the identified TA loci and Lon protease were confirmed by the PCR method using specific primers. Caco-2 cell lines were then infected with this standard strain, and 8 and 24 h post-infection, expression levels of genes coding for the studied TA loci, and Lon protease were evaluated using a real-time PCR method. Expression of mazF, GNAT (Gcn5-related N-acetyltransferase), yeeU, pfam13975, and Lon genes showed 5.4, 9.8, 2.3, 2.7, and 13.8-fold increase, respectively, 8 h after bacterial invasion of the Caco-2 cell line. In addition, the expression of the aforementioned genes showed 4.8, 10.8, 2.3, 3.7, and 16.8-fold increase after 24 h. The GNAT and lon genes showed significantly higher expression levels compared to the control (P value < 0.05). However, the increase in the expression level of yeeU was the same at 8 h and 24 h post-infection. In addition, mazF expression level showed a slight decrease at 24 h compared to 8h post-infection. Genes coding for GNAT and Lon protease showed a significantly higher expression after invading the Caco-2 cell line. Therefore, targeting GNAT or Lon protease can be taken into consideration for finding novel antimicrobial drug strategies. The exact functions and mechanisms of TA systems in S. flexneri isolates are suggested to be experimentally determined.
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Garcia, Pamela K., Rosemarie Martinez Borrero, Thirunavukkarasu Annamalai, Esnel Diaz, Steve Balarezo, Purushottam B. Tiwari, and Yuk-Ching Tse-Dinh. "Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4." Frontiers in Microbiology 13 (December 5, 2022). http://dx.doi.org/10.3389/fmicb.2022.1032320.
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Only about half the multi-drug resistant tuberculosis (MDR-TB) cases are successfully cured. Thus, there is an urgent need of new TB treatment against a novel target. Mycobacterium tuberculosis (Mtb) topoisomerase I (TopA) is the only type IA topoisomerase in this organism and has been validated as an essential target for TB drug discovery. Toxin-antitoxin (TA) systems participate as gene regulators within bacteria. The TA systems contribute to the long-term dormancy of Mtb within the host-cell environment. Mtb’s toxin MazF4 (Rv1495) that is part of the MazEF4 TA system has been shown to have dual activities as endoribonuclease and topoisomerase I inhibitor. We have developed a complementary assay using an Escherichia coli strain with temperature-sensitive topA mutation to provide new insights into the MazF4 action. The assay showed that E. coli is not sensitive to the endoribonuclease activity of Mtb MazF4 but became vulnerable to MazF4 growth inhibition when recombinant Mtb TopA relaxation activity is required for growth. Results from the complementation by Mtb TopA mutants with C-terminal deletions showed that the lysine-rich C-terminal tail is required for interaction with MazF4. Site-directed mutagenesis is utilized to identify two lysine residues within a conserved motif in this C-terminal tail that are critical for MazF4 inhibition. We performed molecular dynamics simulations to predict the Mtb TopA-MazF4 complex. Our simulation results show that the complex is stabilized by hydrogen bonds and electrostatic interactions established by residues in the TopA C-terminal tail including the two conserved lysines. The mechanism of Mtb TopA inhibition by MazF4 could be useful for the discovery of novel inhibitors against a new antibacterial target in pathogenic mycobacteria for treatment of both TB and diseases caused by the non-tuberculosis mycobacteria (NTM).