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The toxin from a ParDE toxin-antitoxin system found in Pseudomonas aeruginosa offers protection to cells challenged with anti-gyrase antibiotics.

ABSTRACT: Toxin-antitoxin systems are mediators of diverse activities in bacterial physiology. For the ParE-type toxins, their reported role of gyrase inhibition utilized during plasmid-segregation killing indicates they are toxic. However, their location throughout chromosomes leads to questions about function, including potential non-toxic outcomes. The current study has characterized a ParDE system from the opportunistic human pathogen Pseudomonas aeruginosa (Pa). We identified a protective function for this ParE toxin, PaParE, against effects of quinolone and other antibiotics. However, higher concentrations of PaParE are themselves toxic to cells, indicating the phenotypic outcome can vary based on its concentration. Our assays confirmed PaParE inhibition of gyrase-mediated supercoiling of DNA with an IC₅₀ value in the low micromolar range, a species-specificity that resulted in more efficacious inhibition of Escherichia coli derived gyrase versus Pa gyrase, and overexpression in the absence of antitoxin yielded an expected filamentous morphology with multi-foci nucleic acid material. Additional data revealed that the PaParE toxin is monomeric and interacts with dimeric PaParD antitoxin with a K_D in the lower picomolar range, yielding a heterotetramer. This work provides novel insights into chromosome-encoded ParE function, whereby its expression can impart partial protection to cultures from selected antibiotics.

SUBMITTER: Muthuramalingam M

PROVIDER: S-EPMC6368863 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A ParDE-family toxin antitoxin system in major resistance plasmids of Enterobacteriaceae confers antibiotic and heat tolerance.

Project description:Toxin-antitoxin (TA) systems were initially discovered as plasmid addiction systems on low-copy-number plasmids. Thousands of TA loci have since been identified on chromosomes, plasmids and mobile elements in bacteria and archaea with diverse roles in bacterial physiology and in maintenance of genetic elements. Here, we identified and characterised a plasmid mediated type II TA system in Enterobacteriaceae as a member of the ParDE super family. This system (hereafter, ParDEI) is distributed among IncI and IncF-type antibiotic resistance and virulence plasmids found in avian and human-source Escherichia coli and Salmonella. It is found that ParDEI is a plasmid stability and stress response module that increases tolerance of aminoglycoside, quinolone and β-lactam antibiotics in E. coli by ~100-1,000-fold, and thus to levels beyond those achievable in the course of antibiotic therapy for human infections. ParDEI also confers a clear survival advantage at 42 °C and expression of the ParEI toxin in trans induces the SOS response, inhibits cell division and promotes biofilm formation. This transmissible high-level antibiotic tolerance is likely to be an important factor in the success of the IncI and IncF plasmids which carry it and the important pathogens in which these are resident.

| S-EPMC6614396 | biostudies-literature

The solution structure of ParD, the antidote of the ParDE toxin antitoxin module, provides the structural basis for DNA and toxin binding.

Project description:ParD is the antidote of the plasmid-encoded toxin-antitoxin (TA) system ParD-ParE. These modules rely on differential stabilities of a highly expressed but labile antidote and a stable toxin expressed from one operon. Consequently, loss of the coding plasmid results in loss of the protective antidote and poisoning of the cell. The antidote protein usually also exhibits an autoregulatory function of the operon. In this paper, we present the solution structure of ParD. The repressor activity of ParD is mediated by the N-terminal half of the protein, which adopts a ribbon-helix-helix (RHH) fold. The C-terminal half of the protein is unstructured in the absence of its cognate binding partner ParE. Based on homology with other RHH proteins, we present a model of the ParD-DNA interaction, with the antiparallel beta-strand being inserted into the major groove of DNA. The fusion of the N-terminal DNA-binding RHH motif to the toxin-binding unstructured C-terminal domain is discussed in its evolutionary context.

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ClpAP protease is a universal factor that activates the parDE toxin-antitoxin system from a broad host range RK2 plasmid.

Project description:The activity of type II toxin-antitoxin systems (TA), which are responsible for many important features of bacterial cells, is based on the differences between toxin and antitoxin stabilities. The antitoxin lability results from bacterial protease activity. Here, we investigated how particular Escherichia coli cytosolic proteases, namely, Lon, ClpAP, ClpXP, and ClpYQ, affect the stability of both the toxin and antitoxin components of the parDE system from the broad host range plasmid RK2. The results of our in vivo and in vitro experiments show that the ParD antitoxin is degraded by the ClpAP protease, and dsDNA stimulates this process. The ParE toxin is not degraded by any of these proteases and can therefore cause growth inhibition of plasmid-free cells after an unequal plasmid distribution during cell division. We also demonstrate that the ParE toxin interaction with ParD prevents antitoxin proteolysis by ClpAP; however, this interaction does not prevent the ClpAP interaction with ParD. We show that ClpAP protease homologs affect plasmid stability in other bacterial species, indicating that ClpAP is a universal activator of the parDE system and that ParD is a universal substrate for ClpAP.

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Structural Insights Into the Transcriptional Regulation of HigBA Toxin-Antitoxin System by Antitoxin HigA in Pseudomonas aeruginosa.

Project description:HigB-HigA is a bacterial toxin-antitoxin (TA) system in which the antitoxin HigA can mask the endoribonuclease activity of toxin HigB and repress the transcription of the TA operon by binding to its own promoter region. The opportunistic pathogen Pseudomonas aeruginosa HigBA (PaHigBA) is closely associated with the pathogenicity by reducing the production of multiple virulence factors and biofilm formation. However, the molecular mechanism underlying HigBA TA operon transcription by PaHigA remains elusive. Here, we report the crystal structure of PaHigA binding to the promoter region of higBA operon containing two identical palindromic sequences at 3.14 Å resolution. The promoter DNA is bound by two cooperative dimers to essentially encircle the intact palindrome region. The helix-turn-helix (HTH) motifs from the two dimers insert into the major grooves of the DNA at the opposite sides. The DNA adopts a canonical B-DNA conformation and all the hydrogen bonds between protein and DNA are mediated by the DNA phosphate backbone. A higher resolution structure of PaHigA-DNA complex at 2.50 Å further revealed three water molecules bridged the DNA-binding interface and mediated the interactions between the bases of palindromic sequences and PaHigA (Thr40, Asp43, and Arg49). Structure-based mutagenesis confirmed these residues are essential for the specific DNA-binding ability of PaHigA. Our structure-function studies therefore elucidated the cooperative dimer-dimer transcription repression mechanism, and may help to understand the regulation of multiple virulence factors by PaHigA in P. aeruginosa.

| S-EPMC6987408 | biostudies-literature

E.coli toxin-antitoxin systems

Project description:This SuperSeries is composed of the SubSeries listed below.

2020-06-11 | GSE144030 | GEO

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

Project description:Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf4 inhibition toxin and Rorf0727 be named PfiA for PfiT antitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5'-AATTCN5 GTTAA-3') overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.

| S-EPMC7264888 | biostudies-literature

Antitoxin CrlA of CrlTA Toxin-Antitoxin System in a Clinical Isolate <i>Pseudomonas aeruginosa</i> Inhibits Lytic Phage Infection.

Project description:Pseudomonas aeruginosa is an important opportunistic pathogen in cystic fibrosis patients and immunocompromised individuals, and the toxin-antitoxin (TA) system is involved in bacterial virulence and phage resistance. However, the roles of TA systems in P. aeruginosa are relatively less studied and no phage Cro-like regulators were identified as TA components. Here, we identified and characterized a chromosome-encoded prophage Cro-like antitoxin (CrlA) in the clinical isolate P. aeruginosa WK172. CrlA neutralized the toxicity of the toxin CrlA (CrlT) which cleaves mRNA, and they formed a type II TA system. Specifically, crlA and crlT are co-transcribed and their protein products interact with each other directly. The autorepression of CrlA is abolished by CrlT through the formation of the CrlTA complex. Furthermore, crlTA is induced in the stationary phase, and crlA is expressed at higher levels than crlT. The excess CrlA inhibits the infection of lytic Pseudomonas phages. CrlA is widely distributed among Pseudomonas and in other bacterial strains and may provide antiphage activities.

| S-EPMC9127804 | biostudies-literature

Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against Clostridium difficile infection.

Project description:The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile. Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab')2 fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens.

| S-EPMC4288887 | biostudies-literature

Predicting toxins found in toxin-antitoxin systems with a role in host-induced Burkholderia pseudomallei persistence.

Project description:Burkholderia pseudomallei (Bpm) is a bacterial pathogen that causes Melioidosis, a disease with up to 40% mortality and an infection relapse of 15-23% despite antibiotic treatment. Ineffective clearance of Bpm by antibiotics is believed to be due to persistence, a hibernation-like survival mechanism modulated, in part, by toxin-antitoxin systems (TAS). Several organisms possess a repertoire of TASs but defining environmental cues eliciting their activity is hindered by laborious in vitro experiments, especially when there are many toxins with redundant function. Here, we identified which of 103 proteins in Bpm that share features found in toxins of the TAS and repurposed transcriptional data to identify which ones play a role in surviving intracellular host defenses. Putative toxins with the strongest transcriptional response were found to have low conservation between Bpm strains, while toxins that were constitutively expressed were highly conserved. Further examination of highly conserved toxins BPSS0899, BPSS1321, and BPSL1494 showed that they were functional, and their mutation led to reduce survival within macrophages and reduced in vivo persistence-associated pathology (abscesses) during treatment, but did not affect macrophages persistence. These findings highlight the utility of a data-driven approach to select putative toxins and suggests a selective role for some TAS in host survival.

| S-EPMC7547725 | biostudies-literature

Targeting bacterial gyrase with cystobactamid, fluoroquinolone and aminocoumarin antibiotics induces distinct molecular signatures in Pseudomonas aeruginosa

Project description:The design of novel antibiotics that break antimicrobial resistance relies on a profound understanding of their mechanism of action. While it has been shown that the cellular effects of antibiotics cluster according to their molecular targets, we investigated whether compounds binding to different sites of the same target can be differentiated by their transcriptome or metabolome signatures. The effects of three fluoroquinolones, two aminocoumarins and two cystobactamids, all inhibiting bacterial gyrase, on Pseudomonas aeruginosa at sub-inhibitory concentrations were captured by RNA sequencing.

2021-05-03 | GSE166602 | GEO

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