Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis

As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase

Penicillin Binding Proteins as Danger Signals: Meningococcal Penicillin Binding Protein 2 Activates Dendritic Cells through Toll-Like Receptor 4

Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml±0.1), CD80 (LOGEC50 = 4.88 µg/ml±0.15) and CD86 (LOGEC50 = 5.36 µg/ml±0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7±5.1% cells versus 12±2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed tha

Variabilité de la température dans le bassin hydraulique d'Oum Er Rbia (Maroc), durant la période (1990-2020) : Étude des anomalies et des tendances

Le cinquième rapport du GIEC a fait la constatation, qu'il est certain que les températures mondiales ont augmenté au cours du 20ème siècle. Au Maroc, plusieurs études ont révélé que le pays, suite à sa situation géographique, subit les impacts du réchauffement climatique depuis plusieurs décennies. Les études ont principalement porté sur l'évolution des précipitations et la récurrence des sécheresses. Néanmoins, les variations de la température restent moins abordées, surtout au niveau des grandes échelles spatiales. Le présent article vise à étudier la variabilité des températures moyennes annuelles dans le grand bassin hydraulique d'Oum Er Rbia, en se penchant exactement sur leurs tendances et leurs anomalies, ainsi que sur leurs impacts. A partir des données de température moyenne mensuelle de dix stations dispersées sur l'ensemble du bassin, cette étude, basée sur une analyse statistique, utilisant le coefficient de variation, l'indice d'anomalie de la température et le test de tendance de Mann Kendall, a révélé que la variabilité marque l’évolution des moyennes annuelles de la température durant la période (1990-2020), avec une tendance générale à la hausse. Les résultats obtenus ont également montré que cette augmentation de la température a engendré des impacts négatifs sur les ressources en eau et l'agriculture

Évaluation de la Qualité du Sol face à la Dégradation : Cas du Bassin Versant d’Oum Er-rbia Amont Ouled Sidi Driss - Maroc

Le sol est une ressource cruciale pour la vie humaine, car il fournit 95 % de ce que nous consommons. Sa formation nécessite un laps de temps considérable, cependant, il est extrêmement vulnérable à la dégradation, ce qui rend sa protection absolument essentielle.  Le bassin versant de l'Oum Er-rbia (amont Ouled Sidi Driss) illustre l'impact des activités humaines et du changement climatique sur la qualité du sol. Ce bassin couvre une superficie de 11 152 km² et englobe divers types de sols tels que le Rendziniforme, les Régosols et les Lithosols. Malheureusement, ces sols sont devenus moins résistants face aux processus d'érosion et de dégradation résultant des activités humaines et du changement climatique.  Dans cet article, nous visons à étudier la résistance du sol à la dégradation en évaluant sa qualité grâce à une analyse de données relatives à la lithologie, à la profondeur du sol, à la texture du sol, à la pente du terrain et à la densité du drainage. Le traitement de ces données à l'aide d'un système d'information géographique nous a permis de cartographier la qualité du sol dans le bassin versant. Nous avons constaté que les sols de qualité moyenne à élevée couvrent une superficie de 6 837 km², soit 61 % de la superficie totale du bassin versant

Characterization of YbjG, a pyrophosphate phosphatase from E. coli involved in the lipid carrier undecaprenyl phosphate metabolism

•Background Undecaprenyl phosphate (C55-P) is an essential lipid carrier involved in the biosynthesis of cell surface carbohydrate polymers such as the peptidoglycan. C55-P is the result of the dephosphorylation of the undecaprenyl pyrophosphate (C55-PP) by specific phosphatases. In Escherichia coli this dephosphorylation can be performed by four integral membrane proteins, BacA, and three members of the type 2 phosphatidic acid phosphatase family (PAP2), PgpB, YbjG, and LpxT. •Objectives The aim of this project is to characterize YbjG and contributes to the understanding of the physiological role and mechanism of action of this enzyme in the C55-P metabolism. The C55-PP phosphatases could become an interesting target in the search for new molecules with antibacterial activity. •Methods In parallel the stability of YbjG and its activity against C15-PP were assessed in 94 different detergents. Moreover the enzymatic activity of YbjG was studied: substrate specificity, optimum pH and temperature, effect of detergent concentration. •Conclusions For the first time, YbjG has been purified and we show its ability to dephosphorylate C15-PP, DGPP and C55-PP in vitro with respectively decreasing efficiency. No activity has been detected on five other potential substrates (PPi, PA, C5-PP, G6P & PNPP). The phosphatase activity on C15-PP is maximum at pH 6,5 and 25 °C. Moreover Cymal6, LMNG, & ωUDM are good detergent both for the stability and the C15-PP phosphatase activity of YbjG, but approximately half of the 94 tested detergents show C15-PP phosphatase activity on the qualitative enzymatic test.Structural Study of C55-PP phosphatase

Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.

Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C55-PP molecules on the same (outer) side of the plasma membrane

Catalytic mechanism of MraY and WecA, two paralogues of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily.

International audienceThe MraY transferase catalyzes the first membrane step of bacterial cell wall peptidoglycan biosynthesis, namely the transfer of the N-acetylmuramoyl-pentapeptide moiety of the cytoplasmic precursor UDP-MurNAc-pentapeptide to the membrane transporter undecaprenyl phosphate (C55P), yielding C55-PP-MurNAc-pentapeptide (lipid I). A paralogue of MraY, WecA, catalyzes the transfer of the phospho-GlcNAc moiety of UDP-N-acetylglucosamine onto the same lipid carrier, leading to the formation of C55-PP-GlcNAc that is essential for the synthesis of various bacterial cell envelope components. These two enzymes are members of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, which are essential for bacterial envelope biogenesis. Despite the availability of detailed biochemical information on the MraY enzyme, and the recently published crystal structure of MraY of Aquifex aeolicus, the molecular basis for its catalysis remains poorly understood. This knowledge can contribute to the design of potential inhibitors. Here, we report a detailed catalytic study of the Bacillus subtilis MraY and Thermotoga maritima WecA transferases. Both forward and reverse exchange reactions required the presence of the second substrate, C55P and uridine monophosphate (UMP), respectively. Both enzymes did not display any pyrophosphatase activity on the nucleotide substrate. Moreover, we showed that the nucleotide substrate UDP-MurNAc-pentapeptide, as well as the nucleotide product UMP, can bind to MraY in the absence of lipid ligands. Therefore, our data are in favour of a single displacement mechanism. During this "one-step" mechanism, the oxyanion of the polyprenyl-phosphate attacks the β-phosphate of the nucleotide substrate, leading to the formation of lipid product and the liberation of UMP. The involvement of an invariant aspartyl residue in the deprotonation of the lipid substrate is discussed

Molecular architecture of the PBP2–MreC core bacterial cell wall synthesis complex

Bacterial wall biosynthesis is a complex process that requires the coordination of multiple enzymes. Here, the authors structurally characterize the PBP2:MreC complex involved in peptidoglycan elongation and cross-linking, and demonstrate that its disruption leads to loss of H. pylori shape and inability to sustain growth