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Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex at 3.1 Å resolution. Unexpectedly, the BoNT/X complex is stable and protease resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency bothin vitroandin vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents weak ganglioside binding and exposed hydrophobic surfaces.

Resistance to β-lactam antibacterials, importantly via production of β-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) β-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C β-lactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied β-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D β-lactamases, our data support the proposal that bicyclic boronates are broad-spectrum β-lactamase inhibitors that work by mimicking a high energy ‘tetrahedral’ intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful β-lactamase inhibition.

OBJECTIFS : Évaluer les résultats de la cimentoplastie vertébrale percutanée en tant que traitement de première intention des fractures thoraco-lombaires traumatiques chez les patients présentant un rachis rigide. MÉTHODE : Trente-sept patients souffrant de fractures thoraco-lombaires dans un segment rachidien ankylosé, sans atteinte neurologique ont été évalués rétrospectivement. Trente etun patients ont bénéficié d'une cimentoplastie vertébrale percutanée et six patients d'un traitement conservateur. Tous les patients ont été suivis à six semaines et un an après le traitement. Les pathologies ankylosantes, les sites et types de fractures, la consolidation radiologique, les complications rachidiennes et la douleur ont été évalués. Des mesures de la densité osseuse du corps vertébral ont également été effectuées. Les rapports de hauteur corporéale antéro-postérieure ont été mesurés avant et après la procédure. RÉSULTATS : Il y avait 39 fractures traitées avec une cimentoplastie vertébrale percutanée et huit fractures traitées de manière conservatrice. Le taux de réussite primaire de la procédure était de 71,2%, cinq patients présentaient de nouvelles fractures. Le taux de réussite secondaire était de 89,7%. Les fractures de 50% des patients traités de manière conservatrice ne se sont pas consolidées au dernier contrôle avec un recul d'un an. Le score de douleur était nul dans 87,1% un an après la cimentoplastie, alors que tous les patients traités de manière conservatrice présentaient une douleur résiduelle. Les rapports de hauteur vertébrale sont restés stables avant et après la procédure (0,62 à 0,60) et ont diminué chez les témoins (0,66 à 0,58). La densité osseuse vertébrale était de 75,9 ± 43,7 UH dans le groupe de réussite primaire, de 64,5 ± 23,8 UH dans le groupe de patients présentant une nouvelle fracture et de 60,0 ± 16,4 UH dans le groupe en échec thérapeutique. CONCLUSION : La cimentoplastie percutanée a présenté un taux de réussite global élevé et a été efficace dans le contrôle de la douleur chez les patients souffrant de fractures vertébrales impliquant des segments ankylosés de la colonne vertébrale.

The SOERE-PRO (Observatory for monitoring and experimentation on agroecosystems fertilized with organic residues) is a long term field experiments network dedicated to the study of risks and benefits associated with organic residues usage. This network includes 4 main experimental sites in France equipped with sensors and monitoring material (La Mare, Reunion Island ; QualiAgro, Ile de France ; Colmar, Alsace ; EFELE, Bretagne). These sites are used to monitor closely the effects of organic residues application on the different compartments (soil, vegetation, water, air) of the agroecosystems. For some sites, this monitoring has been ongoing for more than 15 years. At SOERE-PRO Réunion, the following organic residues are applied to different plots of the sugarcane crop field: 1/ sludge pellets from Grand Prado waste water treatment plant, each year (BA); 2/ sludge compost from Grand Prado waste water treatment plant, every 4 years (CB); 3/ pig manure, each year (LP); 4/ poultry manure, every 4 years (LV). The effect of these organic residues is then compared to mineral-based fertilization (N, P, K). The field experiment was installed at " La Mare ", a location in Reunion Island, in November 2013. Sugarcane was planted on a total area of 1.2 hectares, which includes 30 plots. Each plot has an area of 294 m2, with dimensions 28m x 10m, and is organized in a randomized block design (5 blocks, 6 plots per block). At SOERE-PRO Reunion, instrumentation includes mainly: -TDR probes and tensiometers along the soil profile for soil water status monitoring -Lysimeters and soil water samplers for soil circulating water collection -Automated gas chambers, which close at regular intervals to measure gas fluxes in order to assess soil nitrous oxide (N2O) and carbon dioxide (CO2) emissions. -NH3 collectors (alpha badges) to estimate NH3 volatilisation

The human pathogen Streptococcus pneumoniae has been treated for decades with β-lactam antibiotics. Its resistance is now widespread, mediated by the expression of mosaic variants of the target enzymes, the penicillin-binding proteins (PBPs). Understanding the mode of action of β-lactams, not only in molecular detail but also in their physiological consequences, will be crucial to improving these drugs and any counterresistances. In this work, we investigate the piperacillin paradox, by which this β-lactam selects primarily variants of PBP2b, whereas its most reactive target is PBP2x. These PBPs are both essential monofunctional transpeptidases involved in peptidoglycan assembly. PBP2x participates in septal synthesis, while PBP2b functions in peripheral elongation. The formation of the “lemon”-shaped cells induced by piperacillin treatment is consistent with the inhibition of PBP2x. Following the examination of treated and untreated cells by electron microscopy, the localization of the PBPs by epifluorescence microscopy, and the determination of the inhibition time course of the different PBPs, we propose a model of peptidoglycan assembly that accounts for the piperacillin paradox.

International audience; The morphogenesis of ovococci has been reviewed extensively. Recent results have provided new insights concerning the mechanisms of elongation in ovoid bacteria. We present here the proteins involved in the elongation (firmly established and more or less hypothetical) and discuss the relationship between elongation and division of ovococci

Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan glycosyltrasferase-transpeptidase PBP1A interacts with the cell elongation-specific transpeptidase PBP2 in vitro and in the cell. Cells lacking PBP1A are thinner and initiate cell division later in the cell cycle. PBP1A localizes mainly to the cylindrical wall of the cell, supporting its role in cell elongation. Our in vitro peptidoglycan synthesis assays provide novel insights into the cooperativity of peptidoglycan synthases with different activities. PBP2 stimulates the glycosyltransferase activity of PBP1A, and PBP1A and PBP2 cooperate to attach newly synthesized peptidoglycan to sacculi. PBP2 has peptidoglycan transpeptidase activity in the presence of active PBP1A. Our data also provide a possible explanation for the depletion of lipid II precursors in penicillin-treated cells.

International audience; The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.

Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan glycosyltrasferase-transpeptidase PBP1A interacts with the cell elongation-specific transpeptidase PBP2 in vitro and in the cell. Cells lacking PBP1A are thinner and initiate cell division later in the cell cycle. PBP1A localizes mainly to the cylindrical wall of the cell, supporting its role in cell elongation. Our in vitro peptidoglycan synthesis assays provide novel insights into the cooperativity of peptidoglycan synthases with different activities. PBP2 stimulates the glycosyltransferase activity of PBP1A, and PBP1A and PBP2 cooperate to attach newly synthesized peptidoglycan to sacculi. PBP2 has peptidoglycan transpeptidase activity in the presence of active PBP1A. Our data also provide a possible explanation for the depletion of lipid II precursors in penicillin-treated cells.

A total of 221 isolates of M. tuberculosis were sampled from hospitals and the general population in the northern plain of Vietnam, one of the most populated region of the country. Genotypic composition and diversity were characterized, and we investigated how they are affected by sampling (hospital vs. general population), correcting for potential confounding effects (location, age and gender of the patients). Spoligotyping and 12 MIRU-VNTR typing were used as first line. Then 15 MIRU-VNTR standard set was used, making 21 MIRU-VNTR typing for the clustered isolates. Result showed that 8 lineages and 13 sub-lineages were circulating in the region. The most predominant lineages were Beijing (38.5%) and EAI (38.5%). Others appeared with small proportions H (1.4%), LAM (1.8%), T (8.1%), X (0.9%), MANU (2.3%), and Zero (0.4%). Higher clustering rate was found in the hospital samples (17.9% in urban and 19.2% in rural areas) compared to the population ones (0%). The typical Vietnamese EAI4-VNM sub-lineage of EAI lineage accounted for 67% of EAI strains and was associated with older ages. Beijing genotypes were associated with younger, urban population and were characterized by high clustering rates. These characteristics strongly suggest that Beijing strains are invading the population, replacing the local EAI-VNM4, thus predicting a more serious tuberculosis situation in the future in the absence of more effective control strategies.