The toxin-antitoxin εζ system: Role of ζ toxin in regulating ATP, GTP, (p)ppGpp and uridine diphosphate N-acetylglucosamine pool to cope with stress

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-10-2015The toxin-antitoxin (TA) systems are compact modules, usually comprising a pair of genes coding for a toxin and its cognate antitoxin. These systems are present in the chromosomes of Bacteria, Archaea, in phages and in the large majority of low copy number plasmids. Basically, toxins are proteins whose activity usually leads to the inhibition of cell proliferation by interfering with cellular processes such as DNA replication, translation, cell division, membrane biosynthesis or ATP synthesis. Toxins of the ζ family (ζ, PezT, etc.) are one the most ubiquitous in nature. In B. subtilis the expression of the ζ toxin at or near physiological concentrations induces a state of dormancy, which can be reversed by de novo synthesis of the homodimeric ε antitoxin, lyses a small fraction of cells and leaves a subpopulation (as little as 10-4) refractory or tolerant to the toxin action. Here we studied the type of dormancy induced by the toxin and by different antimicrobial agents. We show that the combined action of ζ and different antimicrobial agents (Amp, Van, Fos, Tri, Cip, Ery) does not increase the number of tolerant cells. Indeed the toxin ζ enhances the efficacy of the antimicrobial agents used and thus potentiates cell killing, suggesting that there are more than one way to induce dormancy. In E. coli deletion of monofunctional RelA is necessary to reduce the proportion of tolerant cells. Conversely in B. subtilis the deletion of the bifunctional RelA, and the presence of low uncontrolled (p)ppGpp levels leads to hypertolerance of toxin and/or of antimicrobials. Physiological or low (p)ppGpp levels (as in wild type, sasA-, sasB- and relA- sasA-) show a normal toxin and antimicrobial tolerance and lower (p)ppGpp levels (relA- sasB-) or absence of (p)ppGpp (relA- sasA- sasB-) in concert with elevated GTP levels, potentiate the efficacy of both the toxin and the antimicrobial action, rendering tolerance vulnerable to eradicate. Reduction of the GTP levels overcomes this phenotype. In vitro experiments reveal that the ζ toxin phosphorylates the Uracil-N-acetylglucosamine (UNAG), leading to unreactive UNAG- 3P. In silico analysis suggest that the phosphotransfer reaction is uncoupled to the ATP hydrolysis. Biochemical analysis reveals that in vitro ζ toxin is a strong UNAGdependent ATPase and phosphorylates only a fraction of UNAG. Indeed, in vivo experiments show that the ζ toxin does not deplete the (UNAG) pool, because expression of the ζ toxin enhances the efficacy of genuine cell wall inhibitors, which act in the following step of the peptidoglycan biosynthesises (Fos, Amp and Van). Mutagenesis of several amino acids of the ζ toxin indicated that the residues D67, E100, E116, R158 and R171 are important for the binding of the protein with the UNAG, ATP and Mg++ and for its mechanism of action. The ζ toxin reduces the nucleotide pool (ATP and GTP) and indirectly “macromolecule synthesis” rendering cells “metabolically inactive” .Los sistemas toxina-antitoxina están formados por dos componentes: una toxina de larga vida media y una antitoxina, con una vida media mucho más corta. Estos sistemas se encuentran en los cromosomas de las Bacterias, Archaea, en fagos y en la gran mayoría de los plásmidos de bajo número de copia. Las toxinas son proteínas que inhiben el crecimiento celular, interfieren en la replicación del ADN, la traducción, la división celular, la biosíntesis de la membrana y la síntesis de ATP. Las toxinas de la familia ζ (ζ, PezT, etc.) son una de las toxinas más extendidas en la naturaleza. En B. Subtilis concentraciones fisiológicas de la toxina ζ inducen un estado de inactividad, que puede ser revertido mediante la síntesis de novo de homodimeros de la antitoxina ε2. También lisa una pequeña fracción de las células y deja una subpoblación (del orden de 10-4) tolerante a la acción de la toxina. En esta tesis doctoral se estudió el tipo de tolerancia inducida por la toxina y la tolerancia inducida por distintos agentes antimicrobianos. Se ha demostrado que la acción combinada de la toxina ζ junto con la acción de diferentes agentes antimicrobianos (Amp, Van, Fos, Tri, Cip, Ery) no incrementa el número de células tolerantes sino que aumenta la eficacia de los agentes antimicrobianos utilizados, potenciando la muerte celular. Estos datos sugieren que hay más de una vía para inducir el estado de tolerancia. En E. coli la supresión de la enzima monofuncional RelA es necesaria para reducir la proporción de células tolerantes. Sin embargo en B. Subtilis la inactivación de la enzima bifuncional RelA, y la presencia de niveles bajos y “desregulados” de (p)ppGpp conduce a la hipertolerancia hacia la toxina y/o de los antimicrobianos. Niveles bajos o fisiológicos de (p)ppGpp (cepa silvestre, sasA-, sasB- y relA -sasA-) muestran una tolerancia normal tanto a la toxina cuanto a los agentes antimicrobianos. Niveles muy bajos (relA-sasB-) o ausentes (relA -sasB- sasA-) juntos con altos niveles de GTP potencian el efecto tanto de la toxina como de los agentes antimicrobianos, lo que hace que la tolerancia sea vulnerables a la erradicación. La reducción de los niveles de GTP revierte esto fenotipo. En experimentos in vitro se ha visto que la toxina ζ fosforila el Uracil N-acetilglucosamina (UNAG), conduciendo a la formación de un substrato no reactivo (UNAG-3P). El análisis in silico sugiere que la reacción de fosfotransferencia está desacoplada con la hidrólisis del ATP. El análisis bioquímico reveló que in vitro la toxina ζ es una ATPasa dependiente del (UNAG) y que fosforila solo una fracción de UNAG. Además, en experimentos in vivo se ha visto que la toxina ζ no agota las reservas de UNAG, porque su expresión mejora la eficacia de los inhibidores de la pared celular (Fos, Amp y Van) que actúan en las siguientes etapas de la biosíntesis del peptidoglicano. La mutagénesis de varios aminoácidos de la toxina ζ indicó que los residuos D67, E100, E116, R158 y R171 son importantes para la unión de la proteína con el UNAG, ATP y Mg++ y para su mecanismo de acción. La toxina ζ reduce la reservas de ATP y GTP, e indirectamente la síntesis de macromoléculas dejando las células metabólicamente inactiva

The ζ Toxin Induces a Set of Protective Responses and Dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε2) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20–30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1–5×10−5). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K+. We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε2 antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε2 antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity

Toxin ζ reversible induces dormancy and reduces the UDP-N-acetylglucosamine pool as one of the protective responses to cope with stress

Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.The research was partially financed by the Ministerio de Economía y Competividad (MINECO) BFU2012-39879-C02-01 to J.C.A., by the MINECO (BFU2012-39879-C02-02) and by the Comunidad de Madrid (CM-BIO0260-2006) to S.A. MT is a PhD fellow of the International Fellowship Programme of La Caixa Foundation (La Caixa/CNB).We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

The interaction of ω2 with the RNA polymerase β′ subunit functions as an activation to repression switch

The ω gene is encoded in broad-host range and low-copy plasmids. It is genetically linked to antibiotic resistance genes of the major human pathogens of phylum Firmicutes. The homodimeric forms of ω (ω) coordinate the plasmid copy number control, faithful partition (ω and δ) and better-than-random segregation (ζεζ) systems. The promoter (P) of the ωεζ operon (P) transiently interacts with ω. Adding δ facilitates the formation of stable ω·P complexes. Here we show that limiting ω interacts with the N-terminal domain of the β′ subunit of the Bacillus subtilis RNA polymerase (RNAP-σ) vegetative holoenzyme. In this way ω recruits RNAP-σ onto P DNA. Partial P occupancy by ω increases the rate at which RNAP-σ complex shifts from its closed (RP) to open (RP) form. This shift increases transcription activation. Adding δ further increases the rate of P transcription initiation, perhaps by stabilizing the ω·P complex. In contrast, full operator occupancy by ω facilitates RP formation, but it blocks RP isomerization and represses P utilization. The stimulation and inhibition of RP formation is the mechanism whereby ω mediates copy number fluctuation and stable plasmid segregation. By this mechanism, ω also indirectly influences the acquisition of antibiotic resistance genes.Funding for open access charge: Dirección General de Investigación-Ministerio de Economía y Competitivida

Toxin ζ Reversible Induces Dormancy and Reduces the UDP-N-Acetylglucosamine Pool as One of the Protective Responses to Cope with Stress

Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes

The effect of acute moderate‑intensity exercise on the serum and fecal metabolomes and the gut microbiota of cross‑country endurance athletes

Physical exercise can produce changes in the microbiota, conferring health benefits through mechanisms that are not fully understood. We sought to determine the changes driven by exercise on the gut microbiota and on the serum and fecal metabolome using 16S rRNA gene analysis and untargeted metabolomics. A total of 85 serum and 12 fecal metabolites and six bacterial taxa (Romboutsia, Escherichia coli TOP498, Ruminococcaceae UCG-005, Blautia, Ruminiclostridium 9 and Clostridium phoceensis) were modified following a controlled acute exercise session. Among the bacterial taxa, Ruminiclostridium 9 was the most influenced by fecal and serum metabolites, as revealed by linear multivariate regression analysis. Exercise significantly increased the fecal ammonia content. Functional analysis revealed that alanine, aspartate and glutamate metabolism and the arginine and aminoacyl-tRNA biosynthesis pathways were the most relevant modified pathways in serum, whereas the phenylalanine, tyrosine and tryptophan biosynthesis pathway was the most relevant pathway modified in feces. Correlation analysis between fecal and serum metabolites suggested an exchange of metabolites between both compartments. Thus, the performance of a single exercise bout in cross-country non-professional athletes produces significant changes in the microbiota and in the serum and fecal metabolome, which may have health implications.AGL2016-77288-REMBO, STF 8131FPU grant 2016/011104.380 JCR (2020) Q1, 17/72 Multidisciplinary Sciences1.240 SJR (2020) Q1, 10/135 MultidisciplinaryNo data IDR 2020UE

Toxin zeta Triggers a Survival Response to Cope with Stress and Persistence

Bacteria have evolved complex regulatory controls in response to various environmental stresses. Protein toxins of the ζ superfamily, found in prominent human pathogens, are broadly distributed in nature. We show that ζ is a uridine diphosphate-N-acetylglucosamine (UNAG)-dependent ATPase whose activity is inhibited in vitro by stoichiometric concentrations of ε2 antitoxin. In vivo, transient ζ expression promotes a reversible multi-level response by altering the pool of signaling purine nucleotides, which leads to growth arrest (dormancy), although a small cell subpopulation persists rather than tolerating toxin action. High c-di-AMP levels (absence of phosphodiesterase GdpP) decrease, and low c-di-AMP levels (absence of diadenylate cyclase DisA) increase the rate of ζ persistence. The absence of CodY, a transition regulator from exponential to stationary phase, sensitizes cells to toxin action, and suppresses persisters formed in the ΔdisA context. These changes, which do not affect the levels of stochastic ampicillin (Amp) persistence, sensitize cells to toxin and Amp action. Our findings provide an explanation for the connection between ζ-mediated growth arrest (with alterations in the GTP and c-di-AMP pools) and persistence formation.Spanish Ministerio de Economía y Competividad and the European Union (MINECO-FEDER) BFU2015-67065-P4.019 JCR (2017) Q1, 31/125 MicrobiologyUE

Unraveling Gut Microbiota Signatures Associated with PPARD and PARGC1A Genetic Polymorphisms in a Healthy Population

Recent studies have revealed the importance of the gut microbiota in the regulation of metabolic phenotypes of highly prevalent metabolic diseases such as obesity, type II diabetes mellitus (T2DM) and cardiovascular disease. Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated nuclear receptors that interact with PPAR-γ co-activator-1α (PPARGC1A) to regulate lipid and glucose metabolism. Genetic polymorphisms in PPARD (rs 2267668; A/G) and PPARGC1A (rs 8192678; G/A) are linked to T2DM. We studied the association between the single-nucleotide polymorphisms (SNPs) rs 2267668 and rs 8192678 and microbiota signatures and their relation to predicted metagenome functions, with the aim of determining possible microbial markers in a healthy population. Body composition, physical exercise and diet were characterized as potential confounders. Microbiota analysis of subjects with PPARGC1A (rs 8192678) and PPARD (rs 2267668) SNPs revealed certain taxa associated with the development of insulin resistance and T2DM. Kyoto encyclopedia of gene and genomes analysis of metabolic pathways predicted from metagenomes highlighted an overrepresentation of ABC sugar transporters for the PPARGC1A (rs 8192678) SNP. Our findings suggest an association between sugar metabolism and the PPARGC1A rs 8192678 (G/A) genotype and support the notion of specific microbiota signatures as factors related to the onset of T2DM