Identification of a new regulatory pathway for the K-State in bacillus subtilis

Bacillus subtilis, une bactérie Gram-positive présente dans le sol, peut lorsque les nutriments sont en concentrations limitantes, sporuler, former des biofilms ou devenir compétente. La compétence est, chez B. subtilis, caractérisée par un arrêt de la division cellulaire, une tolérance aux antibiotiques et l'expression de plus d'une centaine de gènes. L'expression de la compétence, aussi désignée sous le nom de " K-state ", est dépendante de la synthèse du facteur de transcription ComK et se fait de façon stochastique résultant en la formation de deux sous-populations bactériennes, non-compétentes et compétentes. L'émergence, à partir de cellules génétiquement identiques, de deux sous-populations distinctes est une stratégie de survie très répandue chez les procaryotes, connue sous le nom de " bet-hedging ". Bien que les mécanismes de régulation du développement de la compétence ont, chez B. subtilis, largement été étudiés au cours des dernières années, la raison pour laquelle les souches non-domestiques sont très peu transformables (1-2% de la population) comparé aux souches domestiques (~15%) reste méconnue. Nous démontrons ici que c'est essentiellement dû à une mutation de transition dans le promoteur du gène degQ. Cette mutation diminue la synthèse de DegQ, protéine impliquée dans la régulation de la formation de biofilms, de la synthèse d'exoprotéases et de la transformation génétique. DegQ est une protéine impliquée dans le transfert d'un groupe phosphoryl entre la kinase DegS et son substrat DegU. Une faible quantité de DegQ diminue la concentration en DegU~P ce qui a pour conséquence la désinhibition de l'opéron srfA entrainant une accumulation de ComK et l'expression de la compétence. C'est ainsi que, dans les souches domestiques de B. subtilis, un plus grand nombre de bactéries atteignent le niveau nécessaire en ComK pour activer une boucle d'auto-activation transcriptionnelle de comK. Nous démontrons aussi que l'activation transcriptionnelle de srfA est, dans les souches non-domestiques, transitoire alors que la population bactérienne entre en phase stationnaire de croissance. Ces données indiquent que le développement de la compétence est moins fréquent et plus transitoire dans les souches non-domestiques de B. subtilis. De plus, cette limitation du K-state dans les souches non-domestiques est plus importante que précédemment " pensé " probablement dû à la domestication de B. subtilis au cours de ces 50 dernières années. Ce travail reflète non seulement, l'importance de l'utilisation de souches non-domestiques dans la caractérisation des voies de régulation de la compétence chez B. subtilis, mais aussi la portée du choix de modèle biologique dans l'étude de phénomènes biologiques complexes.Bacillus subtilis, a Gram-positive soil bacterium, can enter into several developmental pathways such as sporulation, biofilm formation and competence development for DNA transformation when it becomes limited for essential nutrients. During competence, cells do not divide, are tolerant to antibiotics and competent cells express more than a 100 genes. The competent state has been named the K-state after its master regulator ComK. In B. subtilis, the entry into the K-state is stochastically determined by the activation of the transcription factor ComK and occurs, in the domesticated strains of B. subtilis, in approximately 15% of the population. The emergence from genetically identical cells of two distinct subpopulations (competent cells and non-competent cells) is known to be a classic survival strategy for bacteria, known as bet-hedging. Regulation of entry into the K-state has been intensively studied and is well understood; however, the reasons why undomesticated isolates of B. subtilis are poorly transformable compared to the domesticated strains remained unexplained. We show here that fewer cells enter the K-state, suggesting that some regulatory pathway limiting its expression has been lost in the domesticated backgrounds. We demonstrate that this is largely due to an inactivating point mutation in the degQ promoter region resulting in a decrease of the amount of DegQ. DegQ is known to stimulate phosphate transfer from the DegS autokinase to its cognate response regulator DegU. A low level of DegQ thus decreases the concentration of the phosphorylated form of DegU, leading to the de-repression of the srfA operon, which increases the amount of ComS leading to the stabilization of ComK. Thus, in domesticated strains of B. subtilis, more cells reach the concentration threshold of ComK needed to activate the positive auto-regulatory loop of ComK acting on its own promoter. We also show that the activation of srfA transcription in undomesticated strains is transient, as it is turned off when cells enter the stationary phase. Taken together, these data indicate that the K-state and transformability are less frequent and more transient in the undomesticated strains of B. subtilis. Consideration of the regulatory mechanisms and the fitness advantages and costs of the K-state must from now on take these features into consideration. These results underscore that our understanding of real-life biology requires the use of wild isolates

Semaglutide and cardiovascular outcomes in patients with obesity and prevalent heart failure: a prespecified analysis of the SELECT trial

Background: Semaglutide, a GLP-1 receptor agonist, reduces the risk of major adverse cardiovascular events (MACE) in people with overweight or obesity, but the effects of this drug on outcomes in patients with atherosclerotic cardiovascular disease and heart failure are unknown. We report a prespecified analysis of the effect of once-weekly subcutaneous semaglutide 2·4 mg on ischaemic and heart failure cardiovascular outcomes. We aimed to investigate if semaglutide was beneficial in patients with atherosclerotic cardiovascular disease with a history of heart failure compared with placebo; if there was a difference in outcome in patients designated as having heart failure with preserved ejection fraction compared with heart failure with reduced ejection fraction; and if the efficacy and safety of semaglutide in patients with heart failure was related to baseline characteristics or subtype of heart failure. Methods: The SELECT trial was a randomised, double-blind, multicentre, placebo-controlled, event-driven phase 3 trial in 41 countries. Adults aged 45 years and older, with a BMI of 27 kg/m2 or greater and established cardiovascular disease were eligible for the study. Patients were randomly assigned (1:1) with a block size of four using an interactive web response system in a double-blind manner to escalating doses of once-weekly subcutaneous semaglutide over 16 weeks to a target dose of 2·4 mg, or placebo. In a prespecified analysis, we examined the effect of semaglutide compared with placebo in patients with and without a history of heart failure at enrolment, subclassified as heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, or unclassified heart failure. Endpoints comprised MACE (a composite of non-fatal myocardial infarction, non-fatal stroke, and cardiovascular death); a composite heart failure outcome (cardiovascular death or hospitalisation or urgent hospital visit for heart failure); cardiovascular death; and all-cause death. The study is registered with ClinicalTrials.gov, NCT03574597. Findings: Between Oct 31, 2018, and March 31, 2021, 17 604 patients with a mean age of 61·6 years (SD 8·9) and a mean BMI of 33·4 kg/m2 (5·0) were randomly assigned to receive semaglutide (8803 [50·0%] patients) or placebo (8801 [50·0%] patients). 4286 (24·3%) of 17 604 patients had a history of investigator-defined heart failure at enrolment: 2273 (53·0%) of 4286 patients had heart failure with preserved ejection fraction, 1347 (31·4%) had heart failure with reduced ejection fraction, and 666 (15·5%) had unclassified heart failure. Baseline characteristics were similar between patients with and without heart failure. Patients with heart failure had a higher incidence of clinical events. Semaglutide improved all outcome measures in patients with heart failure at random assignment compared with those without heart failure (hazard ratio [HR] 0·72, 95% CI 0·60-0·87 for MACE; 0·79, 0·64-0·98 for the heart failure composite endpoint; 0·76, 0·59-0·97 for cardiovascular death; and 0·81, 0·66-1·00 for all-cause death; all pinteraction>0·19). Treatment with semaglutide resulted in improved outcomes in both the heart failure with reduced ejection fraction (HR 0·65, 95% CI 0·49-0·87 for MACE; 0·79, 0·58-1·08 for the composite heart failure endpoint) and heart failure with preserved ejection fraction groups (0·69, 0·51-0·91 for MACE; 0·75, 0·52-1·07 for the composite heart failure endpoint), although patients with heart failure with reduced ejection fraction had higher absolute event rates than those with heart failure with preserved ejection fraction. For MACE and the heart failure composite, there were no significant differences in benefits across baseline age, sex, BMI, New York Heart Association status, and diuretic use. Serious adverse events were less frequent with semaglutide versus placebo, regardless of heart failure subtype. Interpretation: In patients with atherosclerotic cardiovascular diease and overweight or obesity, treatment with semaglutide 2·4 mg reduced MACE and composite heart failure endpoints compared with placebo in those with and without clinical heart failure, regardless of heart failure subtype. Our findings could facilitate prescribing and result in improved clinical outcomes for this patient group. Funding: Novo Nordisk

Identification d’une nouvelle voie de regulation du K-state chez Bacillus subtilis

Bacillus subtilis, a Gram-positive soil bacterium, can enter into several developmental pathways such as sporulation, biofilm formation and competence development for DNA transformation when it becomes limited for essential nutrients. During competence, cells do not divide, are tolerant to antibiotics and competent cells express more than a 100 genes. The competent state has been named the K-state after its master regulator ComK. In B. subtilis, the entry into the K-state is stochastically determined by the activation of the transcription factor ComK and occurs, in the domesticated strains of B. subtilis, in approximately 15% of the population. The emergence from genetically identical cells of two distinct subpopulations (competent cells and non-competent cells) is known to be a classic survival strategy for bacteria, known as bet-hedging. Regulation of entry into the K-state has been intensively studied and is well understood; however, the reasons why undomesticated isolates of B. subtilis are poorly transformable compared to the domesticated strains remained unexplained. We show here that fewer cells enter the K-state, suggesting that some regulatory pathway limiting its expression has been lost in the domesticated backgrounds. We demonstrate that this is largely due to an inactivating point mutation in the degQ promoter region resulting in a decrease of the amount of DegQ. DegQ is known to stimulate phosphate transfer from the DegS autokinase to its cognate response regulator DegU. A low level of DegQ thus decreases the concentration of the phosphorylated form of DegU, leading to the de-repression of the srfA operon, which increases the amount of ComS leading to the stabilization of ComK. Thus, in domesticated strains of B. subtilis, more cells reach the concentration threshold of ComK needed to activate the positive auto-regulatory loop of ComK acting on its own promoter. We also show that the activation of srfA transcription in undomesticated strains is transient, as it is turned off when cells enter the stationary phase. Taken together, these data indicate that the K-state and transformability are less frequent and more transient in the undomesticated strains of B. subtilis. Consideration of the regulatory mechanisms and the fitness advantages and costs of the K-state must from now on take these features into consideration. These results underscore that our understanding of real-life biology requires the use of wild isolates.Bacillus subtilis, une bactérie Gram-positive présente dans le sol, peut lorsque les nutriments sont en concentrations limitantes, sporuler, former des biofilms ou devenir compétente. La compétence est, chez B. subtilis, caractérisée par un arrêt de la division cellulaire, une tolérance aux antibiotiques et l'expression de plus d'une centaine de gènes. L'expression de la compétence, aussi désignée sous le nom de " K-state ", est dépendante de la synthèse du facteur de transcription ComK et se fait de façon stochastique résultant en la formation de deux sous-populations bactériennes, non-compétentes et compétentes. L'émergence, à partir de cellules génétiquement identiques, de deux sous-populations distinctes est une stratégie de survie très répandue chez les procaryotes, connue sous le nom de " bet-hedging ". Bien que les mécanismes de régulation du développement de la compétence ont, chez B. subtilis, largement été étudiés au cours des dernières années, la raison pour laquelle les souches non-domestiques sont très peu transformables (1-2% de la population) comparé aux souches domestiques (~15%) reste méconnue. Nous démontrons ici que c'est essentiellement dû à une mutation de transition dans le promoteur du gène degQ. Cette mutation diminue la synthèse de DegQ, protéine impliquée dans la régulation de la formation de biofilms, de la synthèse d'exoprotéases et de la transformation génétique. DegQ est une protéine impliquée dans le transfert d'un groupe phosphoryl entre la kinase DegS et son substrat DegU. Une faible quantité de DegQ diminue la concentration en DegU~P ce qui a pour conséquence la désinhibition de l'opéron srfA entrainant une accumulation de ComK et l'expression de la compétence. C'est ainsi que, dans les souches domestiques de B. subtilis, un plus grand nombre de bactéries atteignent le niveau nécessaire en ComK pour activer une boucle d'auto-activation transcriptionnelle de comK. Nous démontrons aussi que l'activation transcriptionnelle de srfA est, dans les souches non-domestiques, transitoire alors que la population bactérienne entre en phase stationnaire de croissance. Ces données indiquent que le développement de la compétence est moins fréquent et plus transitoire dans les souches non-domestiques de B. subtilis. De plus, cette limitation du K-state dans les souches non-domestiques est plus importante que précédemment " pensé " probablement dû à la domestication de B. subtilis au cours de ces 50 dernières années. Ce travail reflète non seulement, l'importance de l'utilisation de souches non-domestiques dans la caractérisation des voies de régulation de la compétence chez B. subtilis, mais aussi la portée du choix de modèle biologique dans l'étude de phénomènes biologiques complexes

Identification d’une nouvelle voie de regulation du K-state chez Bacillus subtilis

Bacillus subtilis, une bactérie Gram-positive présente dans le sol, peut lorsque les nutriments sont en concentrations limitantes, sporuler, former des biofilms ou devenir compétente. La compétence est, chez B. subtilis, caractérisée par un arrêt de la division cellulaire, une tolérance aux antibiotiques et l'expression de plus d'une centaine de gènes. L'expression de la compétence, aussi désignée sous le nom de " K-state ", est dépendante de la synthèse du facteur de transcription ComK et se fait de façon stochastique résultant en la formation de deux sous-populations bactériennes, non-compétentes et compétentes. L'émergence, à partir de cellules génétiquement identiques, de deux sous-populations distinctes est une stratégie de survie très répandue chez les procaryotes, connue sous le nom de " bet-hedging ". Bien que les mécanismes de régulation du développement de la compétence ont, chez B. subtilis, largement été étudiés au cours des dernières années, la raison pour laquelle les souches non-domestiques sont très peu transformables (1-2% de la population) comparé aux souches domestiques (~15%) reste méconnue. Nous démontrons ici que c'est essentiellement dû à une mutation de transition dans le promoteur du gène degQ. Cette mutation diminue la synthèse de DegQ, protéine impliquée dans la régulation de la formation de biofilms, de la synthèse d'exoprotéases et de la transformation génétique. DegQ est une protéine impliquée dans le transfert d'un groupe phosphoryl entre la kinase DegS et son substrat DegU. Une faible quantité de DegQ diminue la concentration en DegU~P ce qui a pour conséquence la désinhibition de l'opéron srfA entrainant une accumulation de ComK et l'expression de la compétence. C'est ainsi que, dans les souches domestiques de B. subtilis, un plus grand nombre de bactéries atteignent le niveau nécessaire en ComK pour activer une boucle d'auto-activation transcriptionnelle de comK. Nous démontrons aussi que l'activation transcriptionnelle de srfA est, dans les souches non-domestiques, transitoire alors que la population bactérienne entre en phase stationnaire de croissance. Ces données indiquent que le développement de la compétence est moins fréquent et plus transitoire dans les souches non-domestiques de B. subtilis. De plus, cette limitation du K-state dans les souches non-domestiques est plus importante que précédemment " pensé " probablement dû à la domestication de B. subtilis au cours de ces 50 dernières années. Ce travail reflète non seulement, l'importance de l'utilisation de souches non-domestiques dans la caractérisation des voies de régulation de la compétence chez B. subtilis, mais aussi la portée du choix de modèle biologique dans l'étude de phénomènes biologiques complexes.Bacillus subtilis, a Gram-positive soil bacterium, can enter into several developmental pathways such as sporulation, biofilm formation and competence development for DNA transformation when it becomes limited for essential nutrients. During competence, cells do not divide, are tolerant to antibiotics and competent cells express more than a 100 genes. The competent state has been named the K-state after its master regulator ComK. In B. subtilis, the entry into the K-state is stochastically determined by the activation of the transcription factor ComK and occurs, in the domesticated strains of B. subtilis, in approximately 15% of the population. The emergence from genetically identical cells of two distinct subpopulations (competent cells and non-competent cells) is known to be a classic survival strategy for bacteria, known as bet-hedging. Regulation of entry into the K-state has been intensively studied and is well understood; however, the reasons why undomesticated isolates of B. subtilis are poorly transformable compared to the domesticated strains remained unexplained. We show here that fewer cells enter the K-state, suggesting that some regulatory pathway limiting its expression has been lost in the domesticated backgrounds. We demonstrate that this is largely due to an inactivating point mutation in the degQ promoter region resulting in a decrease of the amount of DegQ. DegQ is known to stimulate phosphate transfer from the DegS autokinase to its cognate response regulator DegU. A low level of DegQ thus decreases the concentration of the phosphorylated form of DegU, leading to the de-repression of the srfA operon, which increases the amount of ComS leading to the stabilization of ComK. Thus, in domesticated strains of B. subtilis, more cells reach the concentration threshold of ComK needed to activate the positive auto-regulatory loop of ComK acting on its own promoter. We also show that the activation of srfA transcription in undomesticated strains is transient, as it is turned off when cells enter the stationary phase. Taken together, these data indicate that the K-state and transformability are less frequent and more transient in the undomesticated strains of B. subtilis. Consideration of the regulatory mechanisms and the fitness advantages and costs of the K-state must from now on take these features into consideration. These results underscore that our understanding of real-life biology requires the use of wild isolates

Structural characterization of a novel subfamily of leucine-rich repeat proteins from the human pathogen Leptospira interrogans.

International audiencePathogenic Leptospira spp. are the agents of leptospirosis, an emerging zoonotic disease. Analyses of Leptospira genomes have shown that the pathogenic leptospires (but not the saprophytes) possess a large number of genes encoding proteins containing leucine-rich repeat (LRR) domains. In other pathogenic bacteria, proteins with LRR domains have been shown to be involved in mediating host-cell attachment and invasion, but their functions remain unknown in Leptospira. To gain insight into the potential function of leptospiral LRR proteins, the crystal structures of four LRR proteins that represent a novel subfamily with consecutive stretches of a 23-amino-acid LRR repeat motif have been solved. The four proteins analyzed adopt the characteristic α/β-solenoid horseshoe fold. The exposed residues of the inner concave surfaces of the solenoid, which constitute a putative functional binding site, are not conserved. The various leptospiral LRR proteins could therefore recognize distinct structural motifs of different host proteins and thus serve separate and complementary functions in the physiology of these bacteria

An extracellular Leptospira interrogans leucine‐rich repeat protein binds human E‐ and VE‐cadherins

International audiencePathogenic Leptospira bacteria are the causative agents of leptospirosis, a zoonotic disease affecting animals and humans worldwide. These pathogenic species have the ability to rapidly cross host tissue barriers by a yet unknown mechanism. A comparative analysis of pathogens and saprophytes revealed a higher abundance of genes encoding proteins with leucine-rich repeat (LRR) domains in the genomes of pathogens. In other bacterial pathogens, proteins with LRR domains have been shown to be involved in mediating host cell attachment and invasion. One protein from the pathogenic species Leptospira interrogans, LIC10831, has been previously analysed via X-ray crystallography, with findings suggesting it may be an important bacterial adhesin. Herein we show that LIC10831 elicits an antibody response in infected animals, is actively secreted by the bacterium, and binds human E- and VE-cadherins. These results provide biochemical and cellular evidences of LRR protein-mediated host-pathogen interactions and identify a new multireceptor binding protein from this infectious Leptospira species

Structural and Functional Characterization of an Orphan ATP-Binding Cassette ATPase Involved in Manganese Utilization and Tolerance in Leptospira spp

International audienceABSTRACT Pathogenic Leptospira species are the etiological agents of the widespread zoonotic disease leptospirosis. Most organisms, including Leptospira , require divalent cations for proper growth, but because of their high reactivity, these metals are toxic at high concentrations. Therefore, bacteria have acquired strategies to maintain metal homeostasis, such as metal import and efflux. By screening Leptospira biflexa transposon mutants for their ability to use Mn 2+ , we have identified a gene encoding a putative orphan ATP-binding cassette (ABC) ATPase of unknown function. Inactivation of this gene in both L. biflexa and L. interrogans strains led to mutants unable to grow in medium in which iron was replaced by Mn 2+ , suggesting an involvement of this ABC ATPase in divalent cation uptake. A mutation in this ATPase-coding gene increased susceptibility to Mn 2+ toxicity. Recombinant ABC ATPase of the pathogen L. interrogans exhibited Mg 2+ -dependent ATPase activity involving a P-loop motif. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. Each monomer adopted a canonical two-subdomain organization of the ABC ATPase fold with an α/β subdomain containing the Walker motifs and an α subdomain containing the ABC signature motif (LSSGE). The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. These results provide the first structural and functional characterization of a leptospiral ABC ATPase

Quaternary Structure of Fur Proteins, a New Subfamily of Tetrameric Proteins

International audienceThe ferric uptake regulator (Fur) belongs to the family of the DNA-binding metal-responsive transcriptional regulators. Fur is a global regulator found in all proteobacteria. It controls the transcription of a wide variety of genes involved in iron metabolism but also in oxidative stress or virulence factor synthesis. When bound to ferrous iron, Fur can bind to specific DNA sequences, called Fur boxes. This binding triggers the repression or the activation of gene expression, depending on the regulated genes. As a general view, Fur proteins are considered to be dimeric proteins both in solution and when bound to DNA. In this study, we have purified Fur from four pathogenic strains (Pseudomonas aeruginosa, Francisella tularensis, Yersinia pestis, and Legionella pneumophila) and compared them to Fur from Escherichia coli (EcFur), the best characterized of this family. By using a series of “in solution” techniques, including multiangle laser light scattering and small-angle X-ray scattering, as well as cross-linking experiments, we have shown that the Fur proteins can be classified into two groups, according to their quaternary structure. The group of dimers is represented by EcFur and YpFur and the group of very stable tetramers by PaFur, FtFur, and LpFur. Using PaFur as a case study, we also showed that the dissociation of the tetramers into dimers is necessary for binding of Fur to DNA, and that this dissociation requires the combined effect of metal ion binding and DNA proximity