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1.
J Biomed Sci ; 29(1): 28, 2022 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-35524246

RESUMO

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.


Assuntos
Antitoxinas , Toxinas Bacterianas , Daptomicina , Staphylococcus aureus Resistente à Meticilina , Sistemas Toxina-Antitoxina , Animais , Antibacterianos/farmacologia , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Citidina Desaminase , Staphylococcus aureus Resistente à Meticilina/genética , Camundongos , Oxacilina , RNA , Sistemas Toxina-Antitoxina/genética , Vancomicina
2.
Nat Ecol Evol ; 6(5): 590-603, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35361892

RESUMO

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


Assuntos
Antitoxinas , Toxinas Bacterianas , Sequência de Aminoácidos , Antitoxinas/química , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Mutação
3.
Toxins (Basel) ; 14(3)2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35324661

RESUMO

Abrin is a types II ribosome-inactivating protein (RIP) isolated from Abrus precatorious seeds, which comprises a catalytically active A chain and a lectin-like B chain linked by a disulfide bond. Four isotoxins of abrin have been reported with similar amino-acid composition but different cytotoxicity, of which abrin-a is the most potent toxin. High lethality and easy availability make abrin a potential bioterrorism agent. However, there are no antidotes available for managing abrin poisoning, and treatment is only symptomatic. Currently, neutralizing antibodies remain the most effective therapy against biotoxin poisoning. In this study, we prepared, identified, and acquired a high-affinity neutralizing monoclonal antibody (mAb) 10D8 with a potent pre- and post-exposure protective effect against cytotoxicity and animal toxicity induced by abrin-a or abrin crude extract. The mAb 10D8 could rescue the mouse injected intraperitoneally with a 25 × LD50 dose of abrin-a from lethality and prevent tissue damages. Results indicated that 10D8 does not prevent the binding and internalization of abrin-a to cells but inhibits the enzymatic activity of abrin-a and reduces protein synthesis inhibition of cells. The high affinity, good specificity, and potent antitoxic efficiency of 10D8 make it a promising candidate for therapeutic antibodies against abrin.


Assuntos
Abrina , Abrus , Antitoxinas , Abrus/química , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Camundongos
4.
Toxins (Basel) ; 14(3)2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35324669

RESUMO

Anthrax is an acute disease caused by the bacterium Bacillus anthracis, and is a potential biowarfare/bioterrorist agent. Its pulmonary form, caused by inhalation of the spores, is highly lethal and is mainly related to injury caused by the toxins secretion. Antibodies neutralizing the toxins of B. anthracis are regarded as promising therapeutic drugs, and two are already approved by the Federal Drug Administration. We developed a recombinant human-like humanized antibody, 35PA83 6.20, that binds the protective antigen and that neutralized anthrax toxins in-vivo in White New Zealand rabbits infected with the lethal 9602 strain by intranasal route. Considering these promising results, the preclinical and clinical phase one development was funded and a program was started. Unfortunately, after 5 years, the preclinical development was cancelled due to industrial and scientific issues. This shutdown underlined the difficulty particularly, but not only, for an academic laboratory to proceed to clinical development, despite the drug candidate being promising. Here, we review our strategy and some preliminary results, and we discuss the issues that led to the no-go decision of the pre-clinical development of 35PA83 6.20 mAb. Our review provides general information to the laboratories planning a (pre-)clinical development.


Assuntos
Vacinas contra Antraz , Antraz , Antitoxinas , Bacillus anthracis , Administração por Inalação , Animais , Antraz/tratamento farmacológico , Antraz/microbiologia , Anticorpos Antibacterianos , Antígenos de Bactérias , Coelhos , Proteínas Recombinantes , Esporos Bacterianos
5.
J Bacteriol ; 204(4): e0005822, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35357163

RESUMO

The Mycobacterium tuberculosis genome harbors nine toxin-antitoxin (TA) systems that are members of the mazEF family, unlike other prokaryotes, which have only one or two. Although the overall tertiary folds of MazF toxins are predicted to be similar, it is unclear how they recognize structurally different RNAs and antitoxins with divergent sequence specificity. Here, we have expressed and purified the individual components and complex of the MazEF6 TA system from M. tuberculosis. Size exclusion chromatography-multiangle light scattering (SEC-MALS) was performed to determine the oligomerization status of the toxin, antitoxin, and the complex in different stoichiometric ratios. The relative stabilities of the proteins were determined by nano-differential scanning fluorimetry (nano-DSF). Microscale thermophoresis (MST) and yeast surface display (YSD) were performed to measure the relative affinities between the cognate toxin-antitoxin partners. The interaction between MazEF6 complexes and cognate promoter DNA was also studied using MST. Analysis of paired-end RNA sequencing data revealed that the overexpression of MazF6 resulted in differential expression of 323 transcripts in M. tuberculosis. Network analysis was performed to identify the nodes from the top-response network. The analysis of mRNA protection ratios resulted in identification of putative MazF6 cleavage site in its native host, M. tuberculosis. IMPORTANCE 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. To obtain insights into M. tuberculosis MazEF6 type II TA system function, we determined stability, oligomeric states, and binding affinities of cognate partners with each other and with their promoter operator DNA. Using RNA-seq data obtained from M. tuberculosis overexpression strains, we have identified putative MazF6 cleavage sites and targets in its native, cellular context.


Assuntos
Antitoxinas , Mycobacterium tuberculosis , Sistemas Toxina-Antitoxina , Tuberculose , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismo , Sistemas Toxina-Antitoxina/genética
6.
J Chem Inf Model ; 62(5): 1249-1258, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35103473

RESUMO

Nontypeable Haemophilus influenzae (NTHi) are clinically important Gram-negative bacteria that are responsible for various human mucosal diseases, including otitis media (OM). Recurrent OM caused by NTHi is common, and infections that recur less than 2 weeks following antimicrobial therapy are largely attributable to the recurrence of the same strain of bacteria. Toxin-antitoxin (TA) modules encoded by bacteria enable rapid responses to environmental stresses and are thought to facilitate growth arrest, persistence, and tolerance to antibiotics. The vapBC-1 locus of NTHi encodes a type II TA system, comprising the ribonuclease toxin VapC1 and its cognate antitoxin VapB1. The activity of VapC1 has been linked to the survival of NTHi during antibiotic treatment both in vivo and ex vivo. Therefore, inhibitors of VapC1 might serve as adjuvants to antibiotics, preventing NTHi from entering growth arrest and surviving; however, none have been reported to date. A truncated VapB1 peptide from a crystal structure of the VapBC-1 complex was used to generate pharmacophore queries to facilitate a scaffold hopping approach for the identification of small-molecule VapC1 inhibitors. The National Center for Advancing Translational Sciences small-molecule library was virtually screened using the shape-based method rapid overlay of chemical structures (ROCS), and the top-ranking hits were docked into the VapB1 binding pocket of VapC1. Two hundred virtual screening hits with the best docking scores were selected and tested in a biochemical VapC1 activity assay, which confirmed eight compounds as VapC1 inhibitors. An additional 60 compounds were selected with structural similarities to the confirmed VapC1 inhibitors, of which 20 inhibited VapC1 activity. Intracellular target engagement of five inhibitors was indicated by the destabilization of VapC1 within bacterial cells from a cellular thermal shift assay; however, no impact on bacterial growth was observed. Thus, this virtual screening and scaffold hopping approach enabled the discovery of VapC1 ribonuclease inhibitors that might serve as starting points for preclinical development.


Assuntos
Antitoxinas , Toxinas Bacterianas , Antitoxinas/química , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Haemophilus influenzae/química , Haemophilus influenzae/metabolismo , Humanos , Ribonucleases/metabolismo
7.
J Microbiol ; 60(2): 192-206, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35102526

RESUMO

Toxin-antitoxin (TA) systems are growth-controlling genetic elements consisting of an intracellular toxin protein and its cognate antitoxin. TA systems have been spread among microbial genomes through horizontal gene transfer and are now prevalent in most bacterial and archaeal genomes. Under normal growth conditions, antitoxins tightly counteract the activity of the toxins. Upon stresses, antitoxins are inactivated, releasing activated toxins, which induce growth arrest or cell death. In this study, among nine functional TA modules in Bosea sp. PAMC 26642 living in Arctic lichen, we investigated the functionality of BoHigBA2. BohigBA2 is located close to a genomic island and adjacent to flagellar gene clusters. The expression of BohigB2 induced the inhibition of E. coli growth at 37°C, which was more manifest at 18°C, and this growth defect was reversed when BohigA2 was co-expressed, suggesting that this BoHigBA2 module might be an active TA module in Bosea sp. PAMC 26642. Live/dead staining and viable count analyses revealed that the BoHigB2 toxin had a bactericidal effect, causing cell death. Furthermore, we demonstrated that BoHigB2 possessed mRNA-specific ribonuclease activity on various mRNAs and cleaved only mRNAs being translated, which might impede overall translation and consequently lead to cell death. Our study provides the insight to understand the cold adaptation of Bosea sp. PAMC 26642 living in the Arctic.


Assuntos
Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Bradyrhizobiaceae/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Sistemas Toxina-Antitoxina , Antitoxinas/genética , Regiões Árticas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Família Multigênica , RNA Mensageiro/metabolismo
8.
Nat Commun ; 13(1): 836, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35149704

RESUMO

Microbial composition and functions in the rhizosphere-an important microbial hotspot-are among the most fascinating yet elusive topics in microbial ecology. We used 557 pairs of published 16S rDNA amplicon sequences from the bulk soils and rhizosphere in different ecosystems around the world to generalize bacterial characteristics with respect to community diversity, composition, and functions. The rhizosphere selects microorganisms from bulk soil to function as a seed bank, reducing microbial diversity. The rhizosphere is enriched in Bacteroidetes, Proteobacteria, and other copiotrophs. Highly modular but unstable bacterial networks in the rhizosphere (common for r-strategists) reflect the interactions and adaptations of microorganisms to dynamic conditions. Dormancy strategies in the rhizosphere are dominated by toxin-antitoxin systems, while sporulation is common in bulk soils. Functional predictions showed that genes involved in organic compound conversion, nitrogen fixation, and denitrification were strongly enriched in the rhizosphere (11-182%), while genes involved in nitrification were strongly depleted.


Assuntos
Bactérias/classificação , Fenômenos Fisiológicos Bacterianos , Rizosfera , Microbiologia do Solo , Antitoxinas , Bactérias/genética , Bacteroidetes , Biodiversidade , Microbiota/genética , Proteobactérias , RNA Ribossômico 16S/genética , Solo
9.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35121656

RESUMO

Toxin-antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription and translation as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain.


Assuntos
Antitoxinas/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Domínios Proteicos/genética , Sistemas Toxina-Antitoxina/genética , Proteínas de Bactérias/genética , Burkholderia/genética , Regulação Bacteriana da Expressão Gênica/genética , Guanosina Pentafosfato/genética , Óperon/genética , Prófagos/genética
10.
Front Immunol ; 13: 831536, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35185923

RESUMO

Abrin, a type-II ribosome inactivating protein from the seed of Abrus precatorius, is classified as a Category B bioterrorism warfare agent. Due to its high toxicity, ingestion by animals or humans will lead to death from multiple organ failure. Currently, no effective agents have been reported to treat abrin poisoning. In this study, a novel anti-abrin neutralizing antibody (S008) was humanized using computer-aided design, which possessed lower immunogenicity. Similar to the parent antibody, a mouse anti-abrin monoclonal antibody, S008 possessed high affinity and showed a protective effect against abrin both in vitro and in vivo, and protected mice that S008 was administered 6 hours after abrin. S008 was found that it did not inhibit entry of abrin into cells, suggesting an intracellular blockade capacity against the toxin. In conclusion, this work demonstrates that S008 is a high affinity anti-abrin antibody with both a neutralizing and protective effect and may be an excellent candidate for clinical treatment of abrin poisoning.


Assuntos
Abrina/imunologia , Abrina/toxicidade , Anticorpos Monoclonais Humanizados/imunologia , Antitoxinas/imunologia , Envenenamento/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Antitoxinas/administração & dosagem , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Taxa de Sobrevida
11.
Nucleic Acids Res ; 50(4): 2319-2333, 2022 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-35141752

RESUMO

Staphylococcus aureus is a notorious and globally distributed pathogenic bacterium. New strategies to develop novel antibiotics based on intrinsic bacterial toxin-antitoxin (TA) systems have been recently reported. Because TA systems are present only in bacteria and not in humans, these distinctive systems are attractive targets for developing antibiotics with new modes of action. S. aureus PemIK is a type II TA system, comprising the toxin protein PemK and the labile antitoxin protein PemI. Here, we determined the crystal structures of both PemK and the PemIK complex, in which PemK is neutralized by PemI. Our biochemical approaches, including fluorescence quenching and polarization assays, identified Glu20, Arg25, Thr48, Thr49, and Arg84 of PemK as being important for RNase function. Our study indicates that the active site and RNA-binding residues of PemK are covered by PemI, leading to unique conformational changes in PemK accompanied by repositioning of the loop between ß1 and ß2. These changes can interfere with RNA binding by PemK. Overall, PemK adopts particular open and closed forms for precise neutralization by PemI. This structural and functional information on PemIK will contribute to the discovery and development of novel antibiotics in the form of peptides or small molecules inhibiting direct binding between PemI and PemK.


Assuntos
Antitoxinas , Staphylococcus aureus , Antibacterianos/metabolismo , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
12.
Microb Pathog ; 164: 105423, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35092834

RESUMO

The increase in antibiotic non-responsive bacteria is the leading concern in current research oriented to eliminate pathogens. Nowadays, the excess use of antibiotics without specifically understanding the potentiality of killing pathogens and bacterial survival patterns has helped bacteria emerge indefatigably. Bacteria use various mechanisms such as resistance, persistence, and tolerance to ensure survival. Among these, persistence is a mechanism by which bacteria reside in their dormant state, bypassing the effects of treatments, making it crucial for bacterial survival. Persistent bacterial cells arise from the normal bacterial population as a slow-growing subset of bacteria with no metabolic flux. This behavior renders it to survive for a longer duration and at higher concentrations of antibiotics. They are one of the underlying causes of recurrence of bacterial infections. The present article explains the detailed molecular mechanisms and strategies of bacterial persistence, including the toxin-antitoxin modules, DNA damage, the formation of inactive ribosomal complexes, (p)ppGpp network, antibiotic-induced persistence, which are triggered by drug-induced stress. The article also comprehensively covers the epigenetic memory of persistence in bacteria, and anti-persistent therapeutics like antimicrobial molecules, synthetic peptides, acyldepsipeptide antibiotics, and endolysin therapy to reduce persister cell formation and control their frequency. These strategies could be utilized in combating the pathogenic bacteria undergoing persistence.


Assuntos
Antitoxinas , Infecções Bacterianas , Antibacterianos/metabolismo , Bactérias , Infecções Bacterianas/microbiologia , Tolerância a Medicamentos , Humanos
13.
J Biol Chem ; 298(2): 101557, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34974059

RESUMO

It is well established that the antitoxins of toxin-antitoxin (TA) systems are selectively degraded by bacterial proteases in response to stress. However, how distinct stressors result in the selective degradation of specific antitoxins remain unanswered. MqsRA is a TA system activated by various stresses, including oxidation. Here, we reconstituted the Escherichia coli ClpXP proteolytic machinery in vitro to monitor degradation of MqsRA TA components. We show that the MqsA antitoxin is a ClpXP proteolysis substrate, and that its degradation is regulated by both zinc occupancy in MqsA and MqsR toxin binding. Using NMR chemical shift perturbation mapping, we show that MqsA is targeted directly to ClpXP via the ClpX substrate targeting N-domain, and ClpX mutations that disrupt N-domain binding inhibit ClpXP-mediated degradation in vitro. Finally, we discovered that MqsA contains a cryptic N-domain recognition sequence that is accessible only in the absence of zinc and MqsR toxin, both of which stabilize the MqsA fold. This recognition sequence is transplantable and sufficient to target a fusion protein for degradation in vitro and in vivo. Based on these results, we propose a model in which stress selectively targets nascent and zinc-free MqsA, resulting in exposure of the ClpX recognition motif for ClpXP-mediated degradation.


Assuntos
Antitoxinas , Proteínas de Ligação a DNA , Endopeptidase Clp , Proteínas de Escherichia coli , Escherichia coli , Zinco , Antitoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Oxirredução , Peptídeo Hidrolases/metabolismo , Proteólise , Zinco/metabolismo
14.
J Bacteriol ; 204(3): e0051921, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-34978459

RESUMO

Shigella sonnei is a major cause of bacillary dysentery and an increasing concern due to the spread of multidrug resistance. S. sonnei harbors pINV, an ∼210 kb plasmid that encodes a type III secretion system (T3SS), which is essential for virulence. During growth in the laboratory, avirulence arises spontaneously in S. sonnei at high frequency, hampering studies on and vaccine development against this important pathogen. Here, we investigated the molecular basis for the emergence of avirulence in S. sonnei and showed that avirulence mainly results from pINV loss, which is consistent with previous findings. Ancestral deletions have led to the loss from S. sonnei pINV of two toxin-antitoxin (TA) systems involved in plasmid maintenance, CcdAB and GmvAT, which are found on pINV in Shigella flexneri. We showed that the introduction of these TA systems into S. sonnei pINV reduced but did not eliminate pINV loss, while the single amino acid polymorphisms found in the S. sonnei VapBC TA system compared with S. flexneri VapBC also contributed to pINV loss. Avirulence also resulted from deletions of T3SS-associated genes in pINV through recombination between insertion sequences (ISs) on the plasmid. These events differed from those observed in S. flexneri due to the different distribution and repertoire of ISs. Our findings demonstrated that TA systems and ISs influenced plasmid dynamics and loss in S. sonnei and could be exploited for the design and evaluation of vaccines. IMPORTANCE Shigella sonnei is the major cause of shigellosis in high-income and industrializing countries and is an emerging, multidrug-resistant pathogen. A significant challenge when studying this bacterium is that it spontaneously becomes avirulent during growth in the laboratory through loss of its virulence plasmid (pINV). Here, we deciphered the mechanisms leading to avirulence in S. sonnei and how the limited repertoire and amino acid sequences of plasmid-encoded toxin-antitoxin (TA) systems make the maintenance of pINV in this bacterium less efficient compared with Shigella flexneri. Our findings highlighted how subtle differences in plasmids in closely related species have marked effects and could be exploited to reduce plasmid loss in S. sonnei. This should facilitate research on this bacterium and vaccine development.


Assuntos
Antitoxinas , Disenteria Bacilar , Sistemas Toxina-Antitoxina , Sequência de Aminoácidos , Antitoxinas/genética , Elementos de DNA Transponíveis , Disenteria Bacilar/microbiologia , Disenteria Bacilar/prevenção & controle , Humanos , Plasmídeos/genética , Shigella flexneri/genética , Shigella sonnei/genética , Sistemas Toxina-Antitoxina/genética , Virulência/genética
15.
Nucleic Acids Res ; 50(3): 1687-1700, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35018473

RESUMO

Toxin-antitoxin (TA) systems are proposed to play crucial roles in bacterial growth under stress conditions such as phage infection. The type III TA systems consist of a protein toxin whose activity is inhibited by a noncoding RNA antitoxin. The toxin is an endoribonuclease, while the antitoxin consists of multiple repeats of RNA. The toxin assembles with the individual antitoxin repeats into a cyclic complex in which the antitoxin forms a pseudoknot structure. While structure and functions of some type III TA systems are characterized, the complex assembly process is not well understood. Using bioinformatics analysis, we have identified type III TA systems belonging to the ToxIN family across different Escherichia coli strains and found them to be clustered into at least five distinct clusters. Furthermore, we report a 2.097 Å resolution crystal structure of the first E. coli ToxIN complex that revealed the overall assembly of the protein-RNA complex. Isothermal titration calorimetry experiments showed that toxin forms a high-affinity complex with antitoxin RNA resulting from two independent (5' and 3' sides of RNA) RNA binding sites on the protein. These results further our understanding of the assembly of type III TA complexes in bacteria.


Assuntos
Antitoxinas , Toxinas Bacterianas , Escherichia coli/química , Sistemas Toxina-Antitoxina , Antitoxinas/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Escherichia coli/metabolismo , RNA/metabolismo
16.
Interdiscip Sci ; 14(1): 80-88, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34664198

RESUMO

Levilactobacillus brevis are present in various environments, such as beer, fermented foods, silage, and animal host. Like other lactic acid bacteria, L. brevis might adopt the viable but nonculturable (VBNC) state under unfavorable conditions. The toxin-antitoxin (TA) system, known to regulate cell growth in response to environmental stresses, is found to control the dynamic of the VBNC state. Here, we investigate the type II TA locus prevalence and compare the TA diversity in L. brevis genomes. Using the TAfinder software, we identified a total of 273 putative type II TA loci in 110 replicons of 21 completely sequenced genomes. Genome size does not appear to correlate with the amount of putative type II TA in L. brevis. Besides, type II TA loci are distributed differently among the chromosomes and plasmids. The most prevalent toxin domain is MazF-like in the chromosomes, and RelE/RelE-like in the plasmids; while for antitoxin, Xre-like and Phd-like domains are the most common in the chromosomes and plasmids, respectively. We also observed a unique GNAT-like/ArsR-like TA pair that presents only in the L. brevis chromosome. Detection of 273 putative type II TA loci in 21 complete genomes of Levilactobacillus brevis.


Assuntos
Antitoxinas , Sistemas Toxina-Antitoxina , Animais , Antitoxinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plasmídeos/genética , Sistemas Toxina-Antitoxina/genética
17.
Microb Pathog ; 162: 105309, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34839000

RESUMO

Xenorhabdus nematophila is an entomopathogenic bacterium that synthesizes numerous toxins and kills its larval insect host. Apart from such toxins, its genome also has a plethora of toxin-antitoxin (TA) systems. The role of TA systems in bacterial physiology is debatable; however, they are associated with maintaining bacterial genomic stability and their survival under adverse environmental conditions. Here, we explored the functionality and transcriptional regulation of the type II hipBAXn2 TA system. This TA system was identified in the genome of X. nematophila ATCC 19061, which consists of the hipAXn2 toxin gene encoding 278 amino acid residues and hipBXn2 encoding antitoxin of 135 amino acid residues. We showed that overexpression of HipAXn2 toxin reduced the growth of Escherichia coli cells in a bacteriostatic manner, and amino-acids G8, H164, N167, and S169 were key residues for this growth reduction. Promoter activity and expression profiling of the hipBAXn2 TA system was showed that transcription was induced in both E. coli as well as X. nematophila upon exposure to different stress conditions. Further, we have exhibited the binding features of HipAXn2 toxin and HipBXn2 antitoxin to their promoter. This study provides evidence for the presence of a functional and well-regulated hipBAXn2 TA system in X. nematophila.


Assuntos
Antitoxinas , Proteínas de Escherichia coli , Sistemas Toxina-Antitoxina , Xenorhabdus , Antitoxinas/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA , Escherichia coli/genética , Sistemas Toxina-Antitoxina/genética , Xenorhabdus/genética
18.
Nat Rev Microbiol ; 20(5): 285-298, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-34837014

RESUMO

Clostridioides difficile is a Gram-positive anaerobe that can cause a spectrum of disorders that range in severity from mild diarrhoea to fulminant colitis and/or death. The bacterium produces up to three toxins, which are considered the major virulence factors in C. difficile infection. These toxins promote inflammation, tissue damage and diarrhoea. In this Review, we highlight recent biochemical and structural advances in our understanding of the mechanisms that govern host-toxin interactions. Understanding how C. difficile toxins affect the host forms a foundation for developing novel strategies for treatment and prevention of C. difficile infection.


Assuntos
Antitoxinas , Toxinas Bacterianas , Clostridioides difficile , Antitoxinas/uso terapêutico , Proteínas de Bactérias , Diarreia/tratamento farmacológico , Humanos
19.
J Biol Chem ; 298(1): 101457, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34861238

RESUMO

Toxin-antitoxin (TA) systems are ubiquitous regulatory modules for bacterial growth and cell survival following stress. YefM-YoeB, the most prevalent type II TA system, is present in a variety of bacterial species. In Staphylococcus aureus, the YefM-YoeB system exists as two independent paralogous copies. Our previous research resolved crystal structures of the two oligomeric states (heterotetramer and heterohexamer-DNA ternary complex) of the first paralog as well as the molecular mechanism of transcriptional autoregulation of this module. However, structural details reflecting molecular diversity in both paralogs have been relatively unexplored. To understand the molecular mechanism of how Sa2YoeB and Sa2YefM regulate their own transcription and how each paralog functions independently, we solved a series of crystal structures of the Sa2YoeB-Sa2YefM. Our structural and biochemical data demonstrated that both paralogous copies adopt similar mechanisms of transcriptional autoregulation. In addition, structural analysis suggested that molecular diversity between the two paralogs might be reflected in the interaction profile of YefM and YoeB and the recognition pattern of promoter DNA by YefM. Interaction analysis revealed unique conformational and activating force effected by the interface between Sa2YoeB and Sa2YefM. In addition, the recognition pattern analysis demonstrated that residues Thr7 and Tyr14 of Sa2YefM specifically recognizes the flanking sequences (G and C) of the promoter DNA. Together, these results provide the structural insights into the molecular diversity and independent function of the paralogous copies of the YoeB-YefM TA system.


Assuntos
Antitoxinas , Toxinas Bacterianas , Proteínas de Escherichia coli , Sistemas Toxina-Antitoxina , Antitoxinas/química , Toxinas Bacterianas/química , Proteínas de Escherichia coli/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Sistemas Toxina-Antitoxina/genética
20.
Life Sci ; 288: 120149, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34843738

RESUMO

AIM: An enormous presence and their identified role as stress managers, antibiotic resistance, persistence, and biofilm formation is the reason why the research on Toxin-Antitoxin (TA) loci is getting more and more emphasis. These set of genes consist of poison (Toxin) and its antidote (Antitoxin) expressing in an operon where the toxin inhibits the cellular process and antitoxin which can be a protein or non-coding RNA, rescues the toxin. Most recent progress in genomics and in silico studies have revealed new TA families, and types of TA on bacterial chromosome. However, there is almost no or very little is known about the TA in H. pylori. Therefore, this study aims to identify the TA genes in human pathogen using a comprehensive in silico approach. METHODOLOGY: Here, we have collected the genome-wide data of TA in H. pylori 26695 using TASmania, a new TA database. Further, entire TA dataset was validated with several other databases available for TA, operon analysis and experimental data available. KEY FINDINGS: The study revealed the presence of 80 putative TA genes in H. pylori and highlighted their similarity as well as uniqueness in comparison to other three known TA carrying human pathogens. SIGNIFICANCE: The present study indicates the presence of a large number of TA genes in H. pylori which make biofilm and goes into persistence. Hence, our innovative approach unlocks the prospect for characterizing these putative TA genes and their role as stress managers.


Assuntos
Antitoxinas/genética , Toxinas Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Helicobacter pylori/genética , Óperon , Sistemas Toxina-Antitoxina/genética , Genômica/métodos , Humanos
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