Toxin Induction or Inhibition of Transcription or Translation Posttreatment Increases Persistence to Fluoroquinolones.

Toxin-antitoxin modules are widespread in prokaryotes, and the capacity of toxin accumulation to increase the tolerances of bacteria to antibiotics has been well documented. The conventional model for this functionality implies that an overabundance of toxin arrests bacterial growth, which inhibits processes targeted by antibiotics and thereby limits their corruption and the lethal damage that would ensue. Implicit in this model is that toxins exert their influence on antibiotic lethality before and/or during treatment, even though they are also present and functional after treatment concludes. Given recent evidence establishing that the period following antibiotic treatment (recovery) is important for the survival of nongrowing bacterial populations treated with fluoroquinolones (FQs), we assayed to what extent toxins influence bacterial survival during the recovery period. With both LdrD and MazF, toxins of type I and II systems, respectively, controlling accumulation to occur only after FQ treatment of nongrowing cultures resulted in significant increases in persisters. Further genetic investigation revealed important roles for homologous recombination and nucleotide excision repair machinery. Focusing on the wild type, we did not observe any SOS-induced toxin functioning in this manner; however, an analogous phenomenon was observed for wild-type Escherichia coli as well as uropathogenic E. coli (UPEC) when transcription or translation was inhibited during the post-FQ recovery period. Collectively, these data reveal the capacity of toxins to thwart FQ killing even after the treatment has concluded and show that FQ treatment of nongrowing bacteria can be rendered largely ineffective if bacteria cannot readily resume translation and growth at the conclusion of treatment. IMPORTANCE Overabundances of toxins have been shown to increase the antibiotic tolerances of bacteria. Largely, these effects have been attributed to the abilities of toxins to inhibit bacterial growth before and during antibiotic exposure. In this study, we assessed to what extent toxins can influence bacterial survival following antibiotic treatment, rather than before or during. Using two mechanistically distinct toxins, we show that their accumulations after antibiotic exposure have the capacity to increase the abundances of fluoroquinolone persisters from nongrowing populations. Further, we show with wild-type and uropathogenic E. coli that chemical inhibition of growth, not just that induced by toxins, produces analogous results. These observations reveal another dimension of how toxins influence antibiotic tolerance and highlight the importance of postantibiotic physiology on bacterial survival.

The higBA-Type Toxin-Antitoxin System in IncC Plasmids Is a Mobilizable Ciprofloxacin-Inducible System.

A putative type II toxin-antitoxin (TA) module almost exclusively associated with conjugative IncC plasmids is homologous to the higBA family of TA systems found in chromosomes and plasmids of several species of bacteria. Despite the clinical significance and strong association with high-profile antimicrobial resistance (AMR) genes, the TA system of IncC plasmids remains largely uncharacterized. In this study, we present evidence that IncC plasmids encode a bona fide HigB-like toxin that strongly inhibits bacterial growth and results in cell elongation in Escherichia coli. IncC HigB toxin acts as a ribosome-dependent endoribonuclease that significantly reduces the transcript abundance of a subset of adenine-rich mRNA transcripts. A glycine residue at amino acid position 64 is highly conserved in HigB toxins from different bacterial species, and its replacement with valine (G64V) abolishes the toxicity and the mRNA cleavage activity of the IncC HigB toxin. The IncC plasmid higBA TA system functions as an effective addiction module that maintains plasmid stability in an antibiotic-free environment. This higBA addiction module is the only TA system that we identified in the IncC backbone and appears essential for the stable maintenance of IncC plasmids. We also observed that exposure to subinhibitory concentrations of ciprofloxacin, a DNA-damaging fluoroquinolone antibiotic, results in elevated higBA expression, which raises interesting questions about its regulatory mechanisms. A better understanding of this higBA-type TA module potentially allows for its subversion as part of an AMR eradication strategy. IMPORTANCE Toxin-antitoxin (TA) systems play vital roles in maintaining plasmids in bacteria. Plasmids with incompatibility group C are large plasmids that disseminate via conjugation and carry high-profile antibiotic resistance genes. We present experimental evidence that IncC plasmids carry a TA system that functions as an effective addiction module and maintains plasmid stability in an antibiotic-free environment. The toxin of IncC plasmids acts as an endoribonuclease that targets a subset of mRNA transcripts. Overexpressing the IncC toxin gene strongly inhibits bacterial growth and results in cell elongation in Escherichia coli hosts. We also identify a conserved amino acid residue in the toxin protein that is essential for its toxicity and show that the expression of this TA system is activated by a DNA-damaging antibiotic, ciprofloxacin. This mobile TA system may contribute to managing bacterial stress associated with DNA-damaging antibiotics.

Toxin-Antitoxin Systems in Pathogenic Bacteria.

Toxin-antitoxin (TA) systems, which are ubiquitously present in plasmids, bacterial and archaeal genomes, are classified as types I to VI, according to the nature of the antitoxin and to the mode of toxin inhibition [...].

Combination of Antibiotics-Nisin Reduces the Formation of Persister Cell in Listeria monocytogenes.

Persister cells are a subpopulation of bacteria with the ability of survival when exposed to lethal doses of antibiotics, and are responsible for antibiotic therapy failure and infection recurrences. In this study, we investigated persister cell formation and the role of nisin in combination with antibiotics in reducing persistence in Listeria monocytogenes. We also examined the expression of toxin-antitoxin (TA) systems in persister cells of L. monocytogenes to gain a better understanding of the effect of TA systems on persister cell formation. To induce persistence, L. monocytogenes were exposed to high doses of different antibiotics over a period of 24 hr, and the expression levels of TA system was genes were measured 5 hr after the addition of antibiotics by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method. To investigate the effect of nisin, L. monocytogenes was exposed to a combination of nisin and antibiotics. According to our results, L. monocytogenes was highly capable of persister cell formation, and the combination of nisin and antibiotics resulted in reduced persistence. qRT-PCR results showed a significant increase in GNAT/RHH expression among the studied systems. Overall, our results demonstrated the potential of the combination of nisin and antibiotics in reducing persister cell formation, and emphasized the role of the GNAT/RHH system in bacterial persistence.

Structure-based design of peptides that trigger Streptococcus pneumoniae cell death.

Toxin-antitoxin (TA) systems regulate key cellular functions in bacteria. Here, we report a unique structure of the Streptococcus pneumoniae HigBA system and a novel antimicrobial agent that activates HigB toxin, which results in mRNA degradation as an antibacterial strategy. In this study, protein structure-based peptides were designed and successfully penetrated the S. pneumoniae cell membrane and exerted bactericidal activity. This result represents the time during which inhibitors triggered S. pneumoniae cell death via the TA system. This discovery is a remarkable milestone in the treatment of antibiotic-resistant S. pneumoniae, and the mechanism of bactericidal activity is completely different from those of current antibiotics. Furthermore, we found that the HigBA complex shows a crossed-scissor interface with two intermolecular ß-sheets at both the N and C termini of the HigA antitoxin. Our biochemical and structural studies provided valuable information regarding the transcriptional regulation mechanisms associated with the structural variability of HigAs. Our in vivo study also revealed the potential catalytic residues of HigB and their functional relationships. An inhibition study with peptides additionally proved that peptide binding may allosterically inhibit HigB activity. Overall, our results provide insights into the molecular basis of HigBA TA systems in S. pneumoniae, which can be applied for the development of new antibacterial strategies. DATABASES: Structural data are available in the PDB database under the accession number 6AF4.

Structural and Functional Study of the Klebsiella pneumoniae VapBC Toxin-Antitoxin System, Including the Development of an Inhibitor That Activates VapC.

Klebsiella pneumoniae is one of the most critical opportunistic pathogens. TA systems are promising drug targets because they are related to the survival of bacterial pathogens. However, structural information on TA systems in K. pneumoniae remains lacking; therefore, it is necessary to explore this information for the development of antibacterial agents. Here, we present the first crystal structure of the VapBC complex from K. pneumoniae at a resolution of 2.00 Å. We determined the toxin inhibitory mechanism of the VapB antitoxin through an Mg2+ switch, in which Mg2+ is displaced by R79 of VapB. This inhibitory mechanism of the active site is a novel finding and the first to be identified in a bacterial TA system. Furthermore, inhibitors, including peptides and small molecules, that activate the VapC toxin were discovered and investigated. These inhibitors can act as antimicrobial agents by disrupting the VapBC complex and activating VapC. Our comprehensive investigation of the K. pneumoniae VapBC system will help elucidate an unsolved conundrum in VapBC systems and develop potential antimicrobial agents.

MazEF-rifampicin interaction suggests a mechanism for rifampicin induced inhibition of persisters.

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.

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

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.

Targeting Type II Toxin-Antitoxin Systems as Antibacterial Strategies.

The identification of novel targets for antimicrobial agents is crucial for combating infectious diseases caused by evolving bacterial pathogens. Components of bacterial toxin-antitoxin (TA) systems have been recognized as promising therapeutic targets. These widespread genetic modules are usually composed of two genes that encode a toxic protein targeting an essential cellular process and an antitoxin that counteracts the activity of the toxin. Uncontrolled toxin expression may elicit a bactericidal effect, so they may be considered "intracellular molecular bombs" that can lead to elimination of their host cells. Based on the molecular nature of antitoxins and their mode of interaction with toxins, TA systems have been classified into six groups. The most prevalent are type II TA systems. Due to their ubiquity among clinical isolates of pathogenic bacteria and the essential processes targeted, they are promising candidates for the development of novel antimicrobial strategies. In this review, we describe the distribution of type II TA systems in clinically relevant human pathogens, examine how these systems could be developed as the targets for novel antibacterials, and discuss possible undesirable effects of such therapeutic intervention, such as the induction of persister cells, biofilm formation and toxicity to eukaryotic cells.

Chromosomal toxin-antitoxin systems in Pseudomonas putida are rather selfish than beneficial.

Chromosomal toxin-antitoxin (TA) systems are widespread genetic elements among bacteria, yet, despite extensive studies in the last decade, their biological importance remains ambivalent. The ability of TA-encoded toxins to affect stress tolerance when overexpressed supports the hypothesis of TA systems being associated with stress adaptation. However, the deletion of TA genes has usually no effects on stress tolerance, supporting the selfish elements hypothesis. Here, we aimed to evaluate the cost and benefits of chromosomal TA systems to Pseudomonas putida. We show that multiple TA systems do not confer fitness benefits to this bacterium as deletion of 13 TA loci does not influence stress tolerance, persistence or biofilm formation. Our results instead show that TA loci are costly and decrease the competitive fitness of P. putida. Still, the cost of multiple TA systems is low and detectable in certain conditions only. Construction of antitoxin deletion strains showed that only five TA systems code for toxic proteins, while other TA loci have evolved towards reduced toxicity and encode non-toxic or moderately potent proteins. Analysis of P. putida TA systems' homologs among fully sequenced Pseudomonads suggests that the TA loci have been subjected to purifying selection and that TA systems spread among bacteria by horizontal gene transfer.

Multi-Stress Induction of the Mycobacterium tuberculosis MbcTA Bactericidal Toxin-Antitoxin System.

MbcTA is a type II toxin/antitoxin (TA) system of Mycobacterium tuberculosis. The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD+ and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the mbcAT operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in M. tuberculosis and in the fast growing model species Mycobacterium smegmatis to: (i) assess the autoregulation of mbcAT; (ii) perform a genetic dissection of the mbcA promoter/operator region; and (iii) explore the regulation of mbcAT transcription from the mbcA promoter (PmbcA) in a variety of stress conditions, including in vivo in mice and in macrophages.

Effects of sub-inhibitory concentrations of antibiotics and oxidative stress on the expression of type II toxin-antitoxin system genes in Klebsiella pneumoniae.

OBJECTIVES: Sub-inhibitory concentrations (sub-MICs) of antibiotics reflect the conditions that bacteria encounter in tissues and the natural environment. Sub-MICs of antibiotics can induce stress and alter the expression of different bacterial genes. Bacteria react to stress conditions using different mechanisms, one of which is the toxin-antitoxin (TA) system. This study investigated the expression of the TA system genes under oxidative and antibiotic stresses in Klebsiella pneumoniae (K. pneumoniae). METHODS: To determine the effects of sub-MICs of gentamicin, nalidixic acid, ceftazidime, and certain concentrations of H2O2 on bacterial survival and growth, colony forming units were quantitated and turbidity was assessed following the treatment of K. pneumoniae with ½ MICs of antibiotics and 5 mM H2O2 at different time intervals. The expression of TA system genes in K. pneumoniae was evaluated 1 h after treatment using the quantitative real-time PCR (qRT-PCR) method. RESULTS: The results revealed reduced K. pneumoniae growth in the presence of sub-MICs of antibiotics and 5 mM H2O2 compared to the control. Furthermore, according to the results of the qRT-PCR assay, only the presence of gentamicin could increase the expression of TA system genes. CONCLUSION: Although the exact role of the TA systems in response to stress is still unclear, this study provided information on the effect of the type II TA systems under oxidative and antibiotic stress conditions.

Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells.

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicillin. Moreover, microfluidic experiments performed with a strain reporting the induction of the yefM-yoeB TA system allowed the observation of a small number of type II persister cells that resume growth after removal of ampicillin. However, we were unable to establish a correlation between high fluorescence and persistence, since the fluorescence of persister cells was comparable to that of the bulk of the population and none of the cells showing high fluorescence were able to resume growth upon removal of the antibiotic. Altogether, these data show that there is no direct link between induction of TA systems and persistence to antibiotics.IMPORTANCE Within a growing bacterial population, a small subpopulation of cells is able to survive antibiotic treatment by entering a transient state of dormancy referred to as persistence. Persistence is thought to be the cause of relapsing bacterial infections and is a major public health concern. Type II toxin-antitoxin systems are small modules composed of a toxic protein and an antitoxin protein counteracting the toxin activity. These systems were thought to be pivotal players in persistence until recent developments in the field. Our results demonstrate that previous influential reports had technical flaws and that there is no direct link between induction of TA systems and persistence to antibiotics.

Characterization and Transcriptome Analysis of Acinetobacter baumannii Persister Cells.

Acinetobacter baumannii is a nonfermenting Gram-negative bacillus. A. baumannii resistance is a significant obstacle to clinical infection treatment. The existence of persister cells (persisters) might represent the reason for therapy failure and relapse, and such cells may be the driving force behind rising resistance rates. In this study, A. baumannii ATCC 19606 was used as a target to explore the essential features of A. baumannii persisters. Antibiotic treatment of A. baumannii cultures at 50-fold the minimum inhibitory concentration resulted in a distinct plateau of surviving drug-tolerant persisters. The sensitive bacteria were lysed with ceftazidime, and the nonreplicating bacteria were isolated for transcriptome analysis using RNA sequencing. We analyzed the transcriptome of A. baumannii persisters and identified significantly differentially expressed genes, as well as their enriched pathways. The results showed that both the GP49 (HigB)/Cro (HigA) and DUF1044/RelB toxin/antitoxin systems were significantly increased during the persister incubation period. In addition, the activities of certain metabolic pathways (such as electron transport, adenosine triphosphate [ATP], and the citrate cycle) decreased sharply after antibiotic treatment and remained low during the persister period, while aromatic compound degradation genes were only upregulated in persisters. These results suggest the involvement of aromatic compound degradation genes in persister formation and maintenance. They further provide the first insight into the mechanism of persister formation in A. baumannii.

Glutamine-rich toxic proteins GrtA, GrtB and GrtC together with the antisense RNA AsgR constitute a toxin-antitoxin-like system in Corynebacterium glutamicum.

The Corynebacterium glutamicum R grtA (cgR_2936), grtB (cgR_2934) and grtC (cgR_2933) genes were identified as paralogs encoding glutamine-rich toxic proteins. We also identified a new antisense small RNA AsgR (antisense sRNA for grtA) that overlaps the 3' end of the grtA gene. Single over-expressions of grtA, grtB and grtC resulted in complete inhibition of Escherichia coli cell growth. This growth was rescued by co-expression of AsgR. Similar effects were observed in C. glutamicum, although the toxicities of these proteins were moderate. Inhibition of AsgR transcription resulted in increased levels and prolonged half-lives of grtA, grtB and grtC mRNAs. We also found that the expression levels of grtA, grtB and grtC were increased in an RNase III deletion mutant. Primer extension analysis revealed the RNase III cleavage site to be in the 3' untranslated region (3'-UTR) of the grtA mRNA. The expression levels of grtA, grtB and grtC were increased after exposure to several stresses, including heat shock, treatment with penicillin G, lysozyme or H2 O2 . The deletions of grtABC and asgR genes resulted in decreased survival rate under several stresses. These results indicate that GrtABC and AsgR constitute a type I toxin-antitoxin-like system in C. glutamicum.

mazE Antitoxin of Toxin Antitoxin System and fbpA as Reliable Targets to Eradication of Neisseria meningitidis.

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.

Co-expression network analysis of toxin-antitoxin loci in Mycobacterium tuberculosis reveals key modulators of cellular stress.

Research on toxin-antitoxin loci (TA loci) is gaining impetus due to their ubiquitous presence in bacterial genomes and their observed roles in stress survival, persistence and drug tolerance. The present study investigates the expression profile of all the seventy-nine TA loci found in Mycobacterium tuberculosis. The bacterium was subjected to multiple stress conditions to identify key players of cellular stress response and elucidate a TA-coexpression network. This study provides direct experimental evidence for transcriptional activation of each of the seventy-nine TA loci following mycobacterial exposure to growth-limiting environments clearly establishing TA loci as stress-responsive modules in M. tuberculosis. TA locus activation was found to be stress-specific with multiple loci activated in a duration-based response to a particular stress. Conditions resulting in arrest of cellular translation led to greater up-regulation of TA genes suggesting that TA loci have a primary role in arresting translation in the cell. Our study identifed higBA2 and vapBC46 as key loci that were activated in all the conditions tested. Besides, relBE1, higBA3, vapBC35, vapBC22 and higBA1 were also upregulated in multpile stresses. Certain TA modules exhibited co-activation across multiple conditions suggestive of a common regulatory mechanism.

Molecular mechanisms and clinical implications of bacterial persistence.

Any bacterial population harbors a small number of phenotypic variants that survive exposure to high concentrations of antibiotic. Importantly, these so-called 'persister cells' compromise successful antibiotic therapy of bacterial infections and are thought to contribute to the development of antibiotic resistance. Intriguingly, drug-tolerant persisters have also been identified as a factor underlying failure of chemotherapy in tumor cell populations. Recent studies have begun to unravel the complex molecular mechanisms underlying persister formation and revolve around stress responses and toxin-antitoxin modules. Additionally, in vitro evolution experiments are revealing insights into the evolutionary and adaptive aspects of this phenotype. Furthermore, ever-improving experimental techniques are stimulating efforts to investigate persisters in their natural, infection-associated, in vivo environment. This review summarizes recent insights into the molecular mechanisms of persister formation, explains how persisters complicate antibiotic treatment of infections, and outlines emerging strategies to combat these tolerant cells.