Subtle selectivity in a pheromone sensor triumvirate desynchronizes competence and predation in a human gut commensal

Constantly surrounded by kin or alien organisms in nature, eukaryotes and prokaryotes developed various communication systems to coordinate adaptive multi-entity behavior. In complex and overcrowded environments, they require to discriminate relevant signals in a myriad of pheromones to execute appropriate responses. In the human gut commensal Streptococcus salivarius, the cytoplasmic Rgg/RNPP regulator ComR couples competence to bacteriocin-mediated predation. Here, we describe a paralogous sensor duo, ScuR and SarF, which circumvents ComR in order to disconnect these two physiological processes. We highlighted the recurring role of Rgg/RNPP in the production of antimicrobials and designed a robust genetic screen to unveil potent/optimized peptide pheromones. Further mutational and biochemical analyses dissected the modifiable selectivity toward their pheromone and operating sequences at the subtle molecular level. Additionally, our results highlight how we might mobilize antimicrobial molecules while silencing competence in endogenous populations of human microflora and temper gut disorders provoked by bacterial pathogens

The Structure of the SlrP-hTrx1 Complex Sheds Light on the Autoinhibition Mechanism of the Type-III Secretion System Effectors of the NEL Family

Salmonella infections are a leading cause of bacterial foodborne illness in the United States and the European Union. Antimicrobial therapy is often administered to treat the infection but increasing isolates are being detected that demonstrate resistance to multiple antibiotics. Salmonella enterica contains two virulence related type-III secretion systems (T3SS): one promotes invasion of the intestine and the other one mediates systemic disease. Both of them secrete the SlrP protein acting as E3 ubiquitin ligase in human host cells where it targets thioredoxin-1 (Trx1). SlrP belongs to the NEL family of bacterial E3 ubiquitin ligases that have been observed in two distinct autoinhibitory conformations. We solved the 3D structure of the SlrP/Trx1 complex and determined the Trx1 ubiquitination site. The description of the substrate-binding mode sheds light on the first step of the activation mechanism of SlrP. Comparison with the available structural data of other NEL effectors allowed us to gain new insights into their autoinhibitory mechanism. We propose a molecular mechanism for the regulation of SlrP in which structural constraints sequestrating the NEL domain would be sequentially released. This work thus constitutes a new milestone in the understanding of how these T3SS effectors influence pathogen virulence. It also provides the fundamental basis for future development of new antimicrobials.Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, Spain. Grant P08-CVI-03487Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund. SAF2010-15015 and SAF2013-46229-RSpanish Ministry of Science and Innovation. Grant FR2009-0103Programme Picasso-2010 from the Partenariat Hubert Curie

Structural Basis for the Regulation Mechanism of the Tyrosine Kinase CapB from Staphylococcus aureus

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function

Etudes structurales et fonctionnelles de l'HPr Kinase/Phosphorylase, de sa voie de signalisation et de sa nouvelle famille de protéine-kinases à P-loop

Mon travail de thèse s'articule autour de l'étude d'une protéine-kinase bactérienne d'un nouveau genre, l'HPr Kinase/Phosphorylase (HPrK/P). Cette enzyme bifonctionnelle régule chez les bactéries à Gram positif la répression cataabolique du carbone, qui permet aux bactéries de s'adapter rapidement à la disponibilité en source de carbone. HPrK/P ne présente aucune similarité structurale avec les protéine-kinases eucaryotes. Elle phosphoryle la serine 46 des protéines HPr et Crh, qui activent alors le régulateur transcriptionnel CcpA. La conformation phosphorylase de l'enzyme est connue depuis 2001. Lors de ma thèse, l'étude cristallographique du mutant V267F du domaine catalytique de l'HPrK/P de L. casei a permis de résoudre sa conformation kinase. L'analyse structurale des interactions entre CcpA et les formes phosphorylées de HPr et Crh a également été réalisée. Enfin, j'ai étudié les protéines Fap7 et YFH7 de S. cerevisiae dans le cadre d'un projet d'analyse systématique des protéines à P-loop de fonction inconnue chez la levure.My PhD work concerns the study of a new type of bacterial protein-kinase, the HPr Kinase/Phosphorylase (HPrK/P). This bifunctional enzyme is a key sensor of the Carbon catabolite regulation mechanism in low G+C Gram positive bacteria. HPrK/P exhibits no similiarity with eukaryotic protein kinases. HPrK/P phosphorylates residue serine 46 of the HPr and Crh proteins, allowing them to activate the transcriptional regulator CcpA, and to regulate the expression of carbon catabolism genes. This mechanism enables bacteria to adapt to their environment. The structure of the phosphorylase conformation of HPrK/P was known. My work on mutant V267F of L. casei HPrK/P catalytic domain allowed me to gain insights into the kinase conformation. I also performed structural analysis of the interactions between CcpA and the phosphorylated forms of HPr and Crh. Finally, I analysed the proteins Fap7 and YFH7 of S. cerevisiae in a project of systematic study of yeast P-loop containing proteins of unknown function.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Etude structurale d'un switch moléculaire impliqué dans le quorum sensing chez Bacillus cereus

Les bactéries utilisent un mode de communication appelé quorum sensing pour régulerl expression des gènes en fonction de la densité de population et contrôler ainsi de façonmulticellulaire des processus tels que la sporulation, la compétence ou la virulence. Chez les bactériesà Gram-positif, le quorum sensing repose principalement sur la production, la sécrétion et la détectionde petits peptides de signalisation.Le projet porte sur l étude du système quorum sensing: NprR/NprX chez Bacillus cereus, oùNprR est l effecteur qui reconnait spécifiquement le peptide de signalisation NprX. NprR est uneprotéine bi-fonctionnelle. Seule, elle agit en tant qu inhibiteur de la sporulation, en complexe avecNprX, elle perd sa fonction initiale au profit d une activité facteur de transcription impliquée dans lavirulence. NprR appartient à une famille d effecteurs de quorum sensing appelée RNPP (Rap, NprR,PlcR et PrgX) encore mal caractérisée au niveau structural. Mon projet de thèse a consisté en l analysestructure-fonction du système NprR/NprX.Pour comprendre la régulation fonctionnelle de NprR par NprX, des études en solution (SECMALSet DLS) ont permis de mettre en évidence un switch moléculaire qui repose sur un changementd oligomérisation. Ainsi NprX fait basculer NprR d une conformation Apo dimérique à uneconformation compléxée tétramérique.L étude structurale par cristallographie a aboutit à la résolution de la structure du complexeNprR/NprX. L analyse de ce tétramère suggère la reconnaissance de 2 sites distincts sur l ADN.L étude structurale par SAXS, a quant à elle, permis de proposer une conformation dimérique de laforme Apo NprR, modèle conforté grâce à une étude par mutagénèse dirigée des résidus d interface. Ils agit d un mode de dimérisation semblable à celui des protéines Rap (membres de la famille RNPP).La caractérisation par ITC de l interaction NprR/NprX avec différentes formes du peptide,ainsi que l analyse de la poche de fixation du complexe, ont permis de mieux comprendre la spécificitéd interaction et de mettre en évidence deux résidus clés de l effecteur : l Asn275 essentielle à lafixation du peptide et l Arg 126 essentielle à l activation de la fonction facteur de transcription.Ces travaux ont contribué à une meilleure compréhension du système quorum sensingNprR/NprX grâce à l élucidation du switch moléculaire contrôlé par NprX mais aussi à une meilleureconnaissance de la famille d effecteurs RNPP.Bacteria use a communication mode named quorum sensing to regulate gene expression depending on the population density and thus to control processes such as sporulation, competence or virulence in a multicellular manner. In Gram-positive bacteria, the quorum sensing relies mostly on the production, the secretion and the detection of small signaling peptides. The project focuses on the study of the quorum sensing system NprR/NprX in Bacillus cereus, where NprR is the effector, which recognizes specifically the signaling peptide NprX. NprR is a bi-functional protein. In the absence of peptide, it acts as a sporulation inhibitor while in complex with NprX, it acts as transcription factor implicated in virulence. NprR belongs to a family of quorum sensing effectors named RNPP (for the first identified members: Rap, NprR, PlcR and PrgX) still not well characterized at a structural level. My PhD project consisted to perform the structure/function analysis of the NprR/NprX system. To understand the functional regulation of NprR by NprX, I carried out different studies in solution (SEC-MALS and DLS). These results allowed me to highlight a molecular switch based on a changing of the oligomerisation state of the protein. NprX binding switches NprR from an Apo dimeric conformation to a tetrameric complex. The structural study by crystallography led to the resolution of the tetrameric NprR/NprX complex structure. The analysis of this tetramer suggests the recognition of 2 DNA binding sites. The structural study of the dimeric conformation of Apo NprR by SAXS, allowed me to propose a model similar to that of the Rap dimers (members of RNPP family). This model is supported by a directed mutagenesis study of interface residues. The characterization by ITC of the NprR/NprX interaction with different forms of the peptide, as well as the analysis of the binding pocket in the complex, led to a better understanding of the specificity of the interaction. Two key residues of the effector were highlighted: Asn275, essential to peptide binding and Arg126, essential to the activation of the transcription factor function. These results have contributed to a better understanding of the NprR/NprX quorum sensing system thanks to the elucidation of the molecular switch controlled by NprX but also in a better knowledge of the RNPP effectors family.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Analyse structurale des tyrosine-kinases bactériennes (BY-kinases) et leurs substrats

Des tyrosine-kinases bactériennes atypiques (appelées BY-kinases) ont été identifiées comme constituant d'un complexe multiprotéique transmembranaire responsable de la synthèse et l'export des polysaccharides de la capsule bactérienne. Les BY-kinases s'autophosphorylent sur un cluster de tyrosine C-terminal et phosphorylent des protéines endogènes de la bactérie comme des UDP-sucres déshydrogénases impliquées dans la synthèse des précurseurs des exopolysaccharides. Les données structurales et fonctionnelles disponibles posaient la question de la conservation du degré d'oligomérisation et du mécanisme d'autophosphorylation entre BY-kinases de firmicutes et de protéobactéries. J'ai donc résolu la structure cristalline du domaine cytoplasmique de la BY-kinase Wzc de la protéobactérie E. coli. Cette nouvelle structure montre que, comme la BY-kinase CapAB du firmicute S. aureus, Wzc forme un anneau octamérique expliquant le mécanisme d'autophosphorylation intermoléculaire. Des mesures d'affinité par fluorimétrie m'ont également permis de montrer qu'une tyrosine interne Y569, initialement supposée réguler la trans-autophosphorylation du tyrosine-cluster, est directement impliquée dans la fixation du nucléotide. Nous montrons également qu'une boucle flexible riche en résidus basiques joue un rôle essentiel dans la synthèse de la capsule, probablement en interagissant avec d'autres protéines impliquées dans ce processus. De plus, j'ai résolu la structure cristalline de l'UDP-N-acétylmannosamine déshydrogenase CapO, substrat de la BY-kinase CapAB de S. aureus. Cette structure révèle la formation d'un pont disulfure entre la cystéine catalytique C258 et le résidu C92 et la présence de potentiels sites de phosphorylation, Y89 et Y264, à proximité de ces deux cystéines. L'analyse de mutants est en cours afin d'élucider le mécanisme de régulation de cette enzyme. La comparaison avec les structures d'autres membres de cette famille de déshydrogénases permet également de mieux comprendre leur spécificité de substrat.Atypical bacterial tyrosine kinases (BY-kinases) have been identified as part of a multiprotein transmembrane complex responsible of the biosynthesis and export of capsular polysaccharides. BY-kinases autophosphorylate on a C-terminal tyrosine cluster and phosphorylate endogenous bacterial proteins like UDP-sugar dehydrogenases involved in the synthesis of exopolysaccharide precursors. Available structural and functional data raised the question of the conservation of the oligomerization state and of the autophosphorylation mechanism between BY-kinases from proteobacteria and firmicutes. I thus solved the crystal structure of the cytoplasmic domain of the BY-kinase Wzc from E. coli. This new structure shows that, like the BY-kinase CapAB from the firmicute S. aureus, Wzc forms an octameric ring explaining the intermolecular autophosphorylation mechanism. Fluorimetric affinity measurements further allowed me to show that the internal tyrosine Y569, initially supposed to regulate tyrosine-cluster trans-autophosphorylation, is directly involved in nucleotide binding. We also show that a flexible loop rich in basic residues plays an essential role in capsule synthesis, most probably through interaction with other proteins involved in this process. Moreover, I solved the crystal structure of UDP-N-acetylmannosamine dehydrogenase CapO, the substrate of the BY-kinases CapAB from S. aureus. The structure reveals the formation of a disulfide bridge between the catalytic cysteine C258 and residue C92 and the presence of potential phosphorylation sites, Y89 and Y264, close to these two cysteines. Mutational analysis is underway in order to elucidate the regulatory mechanism of this enzyme. Comparison with the structures of other members of this family of dehydrogenases also allows us to shed light on their specific substrate recognition.ORSAY-PARIS 11-BU Sciences (914712101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Structural basis for the activation mechanism of the PlcR virulence regulator by the quorum-sensing signal peptide PapR

The quorum-sensing regulator PlcR is the master regulator of most known virulence factors in Bacillus cereus. It is a helix-turn-helix (HTH)-type transcription factor activated upon binding of its cognate signaling peptide PapR on a tetratricopeptide repeat-type regulatory domain. The structural and functional properties of PlcR have defined a new family of sensor regulators, called the RNPP family (for Rap, NprR, PrgX, and PlcR), in Gram-positive bacteria. To fully understand the activation mechanism of PlcR, we took a closer look at the conformation changes induced upon binding of PapR and of its target DNA, known as PlcR-box. For that purpose we have determined the structures of the apoform of PlcR (Apo PlcR) and of the ternary complex of PlcR with PapR and the PlcR-box from the plcA promoter. Comparison of the apoform of PlcR with the previously published structure of the PlcR-PapR binary complex shows how a small conformational change induced in the C-terminal region of the tetratricopeptide repeat (TPR) domain upon peptide binding propagates via the linker helix to the N-terminal HTH DNA-binding domain. Further comparison with the PlcR-PapR-DNA ternary complex shows how the activation of the PlcR dimer allows the linker helix to undergo a drastic conformational change and subsequent proper positioning of the HTH domains in the major groove of the two half sites of the pseudopalindromic PlcR-box. Together with random mutagenesis experiments and interaction measurements using peptides from distinct pherogroups, this structural analysis allows us to propose a molecular mechanism for this functional switch

HPr Kinase/Phosphorylase, the Sensor Enzyme of Catabolite Repression in Gram-Positive Bacteria: Structural Aspects of the Enzyme and the Complex with Its Protein Substrate

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