Pyruvate Kinase regulates the Pentose-Phosphate pathway in Response to Hypoxia in Mycobacterium tuberculosis

In response to the stress of infection, Mycobacterium tuberculosis (Mtb) reprograms its metabolism to accommodate nutrient and energetic demands in a changing environment. Pyruvate kinase (PYK) is an essential glycolytic enzyme in the phosphoenolpyruvate–pyruvate–oxaloacetate node that is a central switch point for carbon flux distribution. Here we show that the competitive binding of pentose monophosphate inhibitors or the activator glucose 6-phosphate (G6P) to MtbPYK tightly regulates the metabolic flux. Intriguingly, pentose monophosphates were found to share the same binding site with G6P. The determination of a crystal structure of MtbPYK with bound ribose 5-phosphate (R5P), combined with biochemical analyses and molecular dynamic simulations, revealed that the allosteric inhibitor pentose monophosphate increases PYK structural dynamics, weakens the structural network communication, and impairs substrate binding. G6P, on the other hand, primes and activates the tetramer by decreasing protein flexibility and strengthening allosteric coupling. Therefore, we propose that MtbPYK uses these differences in conformational dynamics to up- and down-regulate enzymic activity. Importantly, metabolome profiling in mycobacteria reveals a significant increase in the levels of pentose monophosphate during hypoxia, which provides insights into how PYK uses dynamics of the tetramer as a competitive allosteric mechanism to retard glycolysis and facilitate metabolic reprogramming toward the pentose-phosphate pathway for achieving redox balance and an anticipatory metabolic response in Mtb

Caractérisation structurale et fonctionnelle de l’opéron acc chez Agrobacterium tumefaciensAgrobacterium\ tumefaciens C58

Agrobacterium tumefaciens is a soil bacterium responsible of the crown gall in plants when it possesses the Tumor inducing plasmid (pTi) which is also the virulence plasmid. The bacterium transfers a piece of DNA from the pTi into the plant genome. The transferred DNA codes for plant hormone synthesis, leading to the formation of tumors which are colonized by bacteria, on one hand, and on the other hand, for the synthesis of small molecules (opines) that are used as nutrients by A. tumefaciens. The opine agrocinopine A induces the production of quorum sensing signals responsible for the spread of the virulence plasmid from pathogenic to nonpathogenic bacterium. Agrobacterium radiobacter K84, a nonpathogenic bacterium, produces the agrocin 84, an antibiotic that kills A. tumefaciens.Import and catabolism of agrocinopine A are operated by acc operon, present on the pTi. The periplasmic binding protein AccA (PBP AccA) associated with the ABC transporter imports the opine into the periplasm where it is degraded by AccF and AccG. AccR regulates the expression of the acc operon and that of the transcription factor TraR, central in quorum sensing signaling. AccA also imports agrocin 84, which is activated by AccF. My PhD work focused on AccA and AccF specificity through structure-function studies and I initiated the study of the transcription factor AccR. The structural study of AccA in complex with agrocinopine A, agrocin 84 and derivatives from these molecules revealed that only the pyranose-2-phosphate motif, common in these two molecules, was recognized. Microcalorimetry and autofluorescence measurements confirmed this conclusion. The pyranose-2-phosphate motif would allow any compound possessing this motif at one end to be transported. The structure of the enzyme AccF showed that again only the pyranose-2-phosphate group is recognized. From the structure and molecular modelling of the substrate in the active site, an original mechanism of the phosphodiester bond cleavage is proposed. Microcalorimetry affinity measures showed that only the arabinose-2-phosphate and glucose-2-phosphate are capable of interacting with AccR. In cellulo experiments confirm that both compounds regulate the expression of quorum sensing.My work sheds light on import and use of agrocinopine in A. tumefaciens. Recognition specificity of the PBP AccA for a part of the imported molecule is observed in other PBPs and opens new ways for rational design of antibiotic compounds that, similarly to agrocin 84, would use the “Trojan horse” strategy.Agrobacterium tumefaciens est une bactérie du sol responsable de la galle du collet chez les plantes lorsqu'elle possède le plasmide Ti (Tumor inducing) dit de virulence (pTi). La bactérie transfère un morceau d’ADN du pTi dans le génome de la plante qui code d'une part la production d’hormones de plantes, à l’origine de la formation de tumeurs colonisées par les bactéries et d'autre part la production de petites molécules (opines) qui servent de nutriment à A. tumefaciens. L'opine, agrocinopine A induit la production de signaux quorum sensing à l’origine de la dissémination du plasmide de virulence vers des bactéries non pathogènes. Agrobacterium radiobacter K84, une bactérie non pathogène, produit de l’agrocine 84, un antibiotique qui tue A. tumefaciens.L’import et le catabolisme de l’agrocinopine A sont réalisés par l’opéron acc présent sur le pTi. La protéine périplasmique (PBP) AccA associée à un transporteur ABC importe l’opine dans le cytoplasme qui est ensuite dégradée par AccF et AccG. AccR régule l’expression de l’opéron acc et celle du facteur de transcription TraR, central dans la signalisation quorum sensing. AccA importe l’agrocine 84 qui est activée par AccF. Mon travail de doctorat a permis par des études structure-fonction de caractériser la spécificité d'AccA et d’AccF et d’initier l’étude du facteur de transcription AccR. L’étude structurale de la PBP en complexe avec l’agrocinopine A, l’agrocine 84 et des dérivés de ces molécules a révélé que seul le motif pyranose-2-phosphate commun aux 2 molécules était reconnu par AccA. Cela a été confirmé par microcalorimétrie et autofluorescence. Le motif pyranose-2-phosphate permettrait donc l’entrée de toute molécule qui le possède à une extrémité. La structure de l’enzyme AccF a montré que là encore seul le groupement pyranose-2-phosphate est reconnu. A partir de la structure obtenue et de modélisation du substrat dans le site actif, un mécanisme enzymatique original pour l’hydrolyse de la liaison phosphodiester est proposé. Les mesures d’affinité par microcalorimétrie montrent que seuls l’arabinose-2-phosphate et le glucose-2-phosphate sont capables de fixer AccR. Des expériences in cellulo ont confirmé qu'ils régulent bien l'expression du QS.Mes travaux apportent un éclairage nouveau sur l’import et l'utilisation de l’agrocinopine chez A. tumefaciens. La spécificité de reconnaissance de la PBP pour une partie de la molécule importée est observée chez d’autres PBP, et ouvre la voie à la conception de molécules antibiotiques qui, à l’image de l’agrocine 84, utilisent une stratégie de type « cheval de Troie »

Structural and functionnal characterization of acc operon from Agrobacterium tumefaciens

Agrobacterium tumefaciens est une bactérie du sol responsable de la galle du collet chez les plantes lorsqu'elle possède le plasmide Ti (Tumor inducing) dit de virulence (pTi). La bactérie transfère un morceau d’ADN du pTi dans le génome de la plante qui code d'une part la production d’hormones de plantes, à l’origine de la formation de tumeurs colonisées par les bactéries et d'autre part la production de petites molécules (opines) qui servent de nutriment à A. tumefaciens. L'opine, agrocinopine A induit la production de signaux quorum sensing à l’origine de la dissémination du plasmide de virulence vers des bactéries non pathogènes. Agrobacterium radiobacter K84, une bactérie non pathogène, produit de l’agrocine 84, un antibiotique qui tue A. tumefaciens.L’import et le catabolisme de l’agrocinopine A sont réalisés par l’opéron acc présent sur le pTi. La protéine périplasmique (PBP) AccA associée à un transporteur ABC importe l’opine dans le cytoplasme qui est ensuite dégradée par AccF et AccG. AccR régule l’expression de l’opéron acc et celle du facteur de transcription TraR, central dans la signalisation quorum sensing. AccA importe l’agrocine 84 qui est activée par AccF. Mon travail de doctorat a permis par des études structure-fonction de caractériser la spécificité d'AccA et d’AccF et d’initier l’étude du facteur de transcription AccR. L’étude structurale de la PBP en complexe avec l’agrocinopine A, l’agrocine 84 et des dérivés de ces molécules a révélé que seul le motif pyranose-2-phosphate commun aux 2 molécules était reconnu par AccA. Cela a été confirmé par microcalorimétrie et autofluorescence. Le motif pyranose-2-phosphate permettrait donc l’entrée de toute molécule qui le possède à une extrémité. La structure de l’enzyme AccF a montré que là encore seul le groupement pyranose-2-phosphate est reconnu. A partir de la structure obtenue et de modélisation du substrat dans le site actif, un mécanisme enzymatique original pour l’hydrolyse de la liaison phosphodiester est proposé. Les mesures d’affinité par microcalorimétrie montrent que seuls l’arabinose-2-phosphate et le glucose-2-phosphate sont capables de fixer AccR. Des expériences in cellulo ont confirmé qu'ils régulent bien l'expression du QS.Mes travaux apportent un éclairage nouveau sur l’import et l'utilisation de l’agrocinopine chez A. tumefaciens. La spécificité de reconnaissance de la PBP pour une partie de la molécule importée est observée chez d’autres PBP, et ouvre la voie à la conception de molécules antibiotiques qui, à l’image de l’agrocine 84, utilisent une stratégie de type « cheval de Troie ».Agrobacterium tumefaciens is a soil bacterium responsible of the crown gall in plants when it possesses the Tumor inducing plasmid (pTi) which is also the virulence plasmid. The bacterium transfers a piece of DNA from the pTi into the plant genome. The transferred DNA codes for plant hormone synthesis, leading to the formation of tumors which are colonized by bacteria, on one hand, and on the other hand, for the synthesis of small molecules (opines) that are used as nutrients by A. tumefaciens. The opine agrocinopine A induces the production of quorum sensing signals responsible for the spread of the virulence plasmid from pathogenic to nonpathogenic bacterium. Agrobacterium radiobacter K84, a nonpathogenic bacterium, produces the agrocin 84, an antibiotic that kills A. tumefaciens.Import and catabolism of agrocinopine A are operated by acc operon, present on the pTi. The periplasmic binding protein AccA (PBP AccA) associated with the ABC transporter imports the opine into the periplasm where it is degraded by AccF and AccG. AccR regulates the expression of the acc operon and that of the transcription factor TraR, central in quorum sensing signaling. AccA also imports agrocin 84, which is activated by AccF. My PhD work focused on AccA and AccF specificity through structure-function studies and I initiated the study of the transcription factor AccR. The structural study of AccA in complex with agrocinopine A, agrocin 84 and derivatives from these molecules revealed that only the pyranose-2-phosphate motif, common in these two molecules, was recognized. Microcalorimetry and autofluorescence measurements confirmed this conclusion. The pyranose-2-phosphate motif would allow any compound possessing this motif at one end to be transported. The structure of the enzyme AccF showed that again only the pyranose-2-phosphate group is recognized. From the structure and molecular modelling of the substrate in the active site, an original mechanism of the phosphodiester bond cleavage is proposed. Microcalorimetry affinity measures showed that only the arabinose-2-phosphate and glucose-2-phosphate are capable of interacting with AccR. In cellulo experiments confirm that both compounds regulate the expression of quorum sensing.My work sheds light on import and use of agrocinopine in A. tumefaciens. Recognition specificity of the PBP AccA for a part of the imported molecule is observed in other PBPs and opens new ways for rational design of antibiotic compounds that, similarly to agrocin 84, would use the “Trojan horse” strategy

Dengue Virus Non-Structural Protein 5

The World Health Organization estimates that the yearly number of dengue cases averages 390 million. This mosquito-borne virus disease is endemic in over 100 countries and will probably continue spreading, given the observed trend in global warming. So far, there is no antiviral drug available against dengue, but a vaccine has been recently marketed. Dengue virus also serves as a prototype for the study of other pathogenic flaviviruses that are emerging, like West Nile virus and Zika virus. Upon viral entry into the host cell and fusion of the viral lipid membrane with the endosomal membrane, the viral RNA is released and expressed as a polyprotein, that is then matured into three structural and seven non-structural (NS) proteins. The envelope, membrane and capsid proteins form the viral particle while NS1-NS2A-NS2B-NS3-NS4A-NS4B and NS5 assemble inside a cellular replication complex, which is embedded in endoplasmic reticulum (ER)-derived vesicles. In addition to their roles in RNA replication within the infected cell, NS proteins help the virus escape the host innate immunity and reshape the host-cell inner structure. This review focuses on recent progress in characterizing the structure and functions of NS5, a protein responsible for the replication and capping of viral RNA that represents a promising drug target

Structural basis for high specificity of octopine binding in the plant pathogen Agrobacterium tumefaciens

Agrobacterium pathogens of octopine-and nopaline-types force host plants to produce either octopine or nopaline compounds, which they use as nutrients. Two Agrobacterium ABC-transporters and their cognate periplasmic binding proteins (PBPs) OccJ and NocT import octopine and nopaline/octopine, respectively. Here, we show that both octopine transport and degradation confer a selective advantage to octopine-type A. tumefaciens when it colonizes plants. We report the X-ray structures of the unliganded PBP OccJ and its complex with octopine as well as a structural comparison with NocT and the related PBP LAO from Salmonella enterica, which binds amino acids (lysine, arginine and ornithine). We investigated the specificity of OccJ, NocT and LAO using several ligands such as amino acids, octopine, nopaline and octopine analogues. OccJ displays a high selectivity and nanomolar range affinity for octopine. Altogether, the structural and affinity data allowed to define an octopine binding signature in PBPs and to construct a OccJ mutant impaired in octopine binding, a selective octopine-binding NocT and a non-selective octopine-binding LAO by changing one single residue in these PBPs. We proposed the PBP OccJ as a major trait in the ecological specialization of octopine-type Agrobacterium pathogens when they colonize and exploit the plant host

On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation

Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell‐surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that covers the substrate binding site is proteolytically removed, exposing the active site to solvent and releasing a population of heterogenous active enzymes. The availability of a readily active ligase not requiring acid activation and subsequent purification of active forms would facilitate manufacturing and streamline applications. Here, we engineered the OaAEP1b‐C247A hyperactive ligase via serial truncations along the linker connecting the cap and core domain of the proenzyme. The recombinant expression of the truncated constructs was carried out in Escherichia coli. Following a solubilization/refolding protocol, one truncated construct termed ‘OaAEP1b‐C247A‐∆351’ could be overexpressed in the insoluble fraction, purified, and displayed a level of ligase activity comparable to the acid‐activated OaAEP1b‐C247A enzyme. This constitutively active protein can be stored for up to 2 years at −80 °C and readily used for peptide cyclization and protein conjugation. We were able to express and purify a stable constitutively active asparaginyl ligase that can be stored for months without significant activity loss. The removal of the low pH proenzyme activation step eliminates the heterogeneity introduced by this procedure. The yield of purified recombinant active ligase that can be routinely obtained per 100 mL of E. coli cell culture is about 0.9 mg. This recombinant active ligase can be used to carry out protein conjugation

Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases

Many eukaryotic organisms encode more than one RNA-dependent RNA polymerase (RdRP) that probably emerged as a result of gene duplication. Such RdRP paralogs often participate in distinct RNA silencing pathways and show characteristic repertoires of enzymatic activities in vitro. However, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. We show that orthologs of QDE-1, an RdRP component of the quelling pathway in Neurospora crassa, have rapidly diverged in evolution at the amino acid sequence level. Analyses of purified QDE-1 polymerases from N. crassa (QDE-1(Ncr)) and related fungi, Thielavia terrestris (QDE-1(Tte)) and Myceliophthora thermophila (QDE-1(Mth)), show that all three enzymes can synthesize RNA, but the precise modes of their action differ considerably. Unlike their QDE-1(Ncr) counterpart favoring processive RNA synthesis, QDE-1(Tte) and QDE-1(Mth) produce predominantly short RNA copies via primer-independent initiation. Surprisingly, a 3.19 Å resolution crystal structure of QDE-1(Tte) reveals a quasisymmetric dimer similar to QDE-1(Ncr). Further electron microscopy analyses confirm that QDE-1(Tte) occurs as a dimer in solution and retains this status upon interaction with a template. We conclude that divergence of orthologous RdRPs can result in functional innovation while retaining overall protein fold and quaternary structure

Structural basis for high specificity of octopine binding in the plant pathogen Agrobacterium tumefaciens

Abstract Agrobacterium pathogens of octopine- and nopaline-types force host plants to produce either octopine or nopaline compounds, which they use as nutrients. Two Agrobacterium ABC-transporters and their cognate periplasmic binding proteins (PBPs) OccJ and NocT import octopine and nopaline/octopine, respectively. Here, we show that both octopine transport and degradation confer a selective advantage to octopine-type A. tumefaciens when it colonizes plants. We report the X-ray structures of the unliganded PBP OccJ and its complex with octopine as well as a structural comparison with NocT and the related PBP LAO from Salmonella enterica, which binds amino acids (lysine, arginine and ornithine). We investigated the specificity of OccJ, NocT and LAO using several ligands such as amino acids, octopine, nopaline and octopine analogues. OccJ displays a high selectivity and nanomolar range affinity for octopine. Altogether, the structural and affinity data allowed to define an octopine binding signature in PBPs and to construct a OccJ mutant impaired in octopine binding, a selective octopine-binding NocT and a non-selective octopine-binding LAO by changing one single residue in these PBPs. We proposed the PBP OccJ as a major trait in the ecological specialization of octopine-type Agrobacterium pathogens when they colonize and exploit the plant host

Natural Guided Genome Engineering Reveals Transcriptional Regulators Controlling Quorum-Sensing Signal Degradation.

International audienceQuorum-quenching (QQ) are natural or engineered processes disrupting the quorum-sensing (QS) signalling which controls virulence and persistence (e.g. biofilm) in numerous bacteria. QQ involves different enzymes including lactonases, amidases, oxidases and reductases which degrade the QS molecules such as N-acylhomoserine lactones (NAHL). Rhodococcus erythropolis known to efficiently degrade NAHL is proposed as a biocontrol agent and a reservoir of QQ-enzymes for biotechnology. In R. erythropolis, regulation of QQ-enzymes remains unclear. In this work, we performed genome engineering on R. erythropolis, which is recalcitrant to reverse genetics, in order to investigate regulation of QQ-enzymes at a molecular and structural level with the aim to improve the QQ activity. Deep-sequencing of the R. erythropolis enhanced variants allowed identification of a punctual mutation in a key-transcriptional factor QsdR (Quorum sensing degradation Regulation) which regulates the sole QQ-lactonase QsdA identified so far. Using biophysical and structural studies on QsdR, we demonstrate that QQ activity can be improved by modifying the regulation of QQ-enzymes degrading QS signal. This modification requiring the change of only one amino-acid in a transcriptional factor leads to an enhanced R. erythropolis in which the QS-signal degradation pathway is strongly activated

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and alpha-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K-D of 0.6 mu M) greater than that for nopaline (KD of 3.7 mu M). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline/assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens