Serine/Threonine Protein Phosphatase-Mediated Control of the Peptidoglycan Cross-Linking L,D-Transpeptidase Pathway in Enterococcus faecium

International audienceThe last step of peptidoglycan polymerization involves two families of unrelated transpeptidases that are the essential targets of ␤-lactam antibiotics. D,D-transpeptidases of the penicillin-binding protein (PBP) family are active-site serine enzymes that use pentapeptide precursors and are the main or exclusive cross-linking enzymes in nearly all bacteria. However, pepti-doglycan cross-linking is performed mainly by active-site cysteine L,D-transpeptidases that use tetrapeptides in Mycobacterium tuberculosis, Clostridium difficile, and ␤-lactam-resistant mutants of Enterococcus faecium. We have investigated reprogramming of the E. faecium peptidoglycan assembly pathway by a switch from pentapeptide to tetrapeptide precursors and bypass of PBPs by L,D-transpeptidase Ldt fm. Mutational alterations of two signal transduction systems were necessary and sufficient for activation of the L,D-transpeptidation pathway, which is essentially cryptic in wild-type strains. The first one is a classical two-component regulatory system, DdcRS, that controls the activity of Ldt fm at the substrate level. As previously described, loss of DdcS phosphatase activity leads to production of the D,D-carboxypeptidase DdcY and conversion of the pentapeptide into the tetrapeptide substrate of Ldt fm. Here we show that full bypass of PBPs by Ldt fm also requires increased Ser/Thr protein phos-phorylation resulting from impaired activity of phosphoprotein phosphatase StpA. This enzyme negatively controlled the level of protein phosphorylation both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase Stk. In combination with production of DdcY, increased protein phosphorylation by this eukaryotic-enzyme-like Ser/Thr protein kinase was sufficient for activation of the L,D-transpeptidation pathway in the absence of mutational alteration of pepti-doglycan synthesis enzymes

Multiframe Scene Flow with Piecewise Rigid Motion

We introduce a novel multiframe scene flow approach that jointly optimizes the consistency of the patch appearances and their local rigid motions from RGB-D image sequences. In contrast to the competing methods, we take advantage of an oversegmentation of the reference frame and robust optimization techniques. We formulate scene flow recovery as a global non-linear least squares problem which is iteratively solved by a damped Gauss-Newton approach. As a result, we obtain a qualitatively new level of accuracy in RGB-D based scene flow estimation which can potentially run in real-time. Our method can handle challenging cases with rigid, piecewise rigid, articulated and moderate non-rigid motion, and does not rely on prior knowledge about the types of motions and deformations. Extensive experiments on synthetic and real data show that our method outperforms state-of-the-art.Comment: International Conference on 3D Vision (3DV), Qingdao, China, October 201

Comparative Molecular and Microbiologic Diagnosis of Bacterial Endocarditis

Sequencing of 16S rDNA, and of sodAint and rpoBint in some cases, was applied to DNA from heart valves of 46 patients (36 with definite and 10 with possible endocarditis). Sequence-based identifications were compared with those obtained with conventional methods. Among the 36 definite cases, 30 had positive blood cultures and 6 had negative cultures. Among the 30 positive cases, sequencing of 16S rDNA permitted identification of species (18), genus (8), or neither (4); sodAint and rpoBint sequencing was necessary for species identification in 8 cases. Species identifications were identical in only 61.5%, when conventional techniques and DNA sequencing were used. In five of the six blood culture–negative endocarditis cases, sequencing identified Bartonella quintana (3), B. henselae (1), and Streptococcus gallolyticus (1). Our results demonstrate a clear benefit of molecular identification, particularly in cases of blood culture–negative endocarditis and of possible endocarditis, to confirm or invalidate the diagnosis. Moreover, in 19.4% of the definite cases, the improvement in species identification by sequencing led to improved patient management

A case of phage therapy against pandrug-resistant Achromobacter xylosoxidans in a 12-year-old lung-transplanted cystic fibrosis patient

Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains

Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis

Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which l-Phe is shown to be transferred from Phe-tRNAPhe to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides

Inhibition of the β-lactamase BlaMab by avibactam improves the in vitro and in vivo efficacy of imipenem against mycobacterium abscessus

Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a β-lactam (cefoxitin or imipenem). The triple combination is used without any β-lactamase inhibitor, even though M. abscessus produces the broad-spectrum β-lactamase BlaMab. We determine whether inhibition of BlaMab by avibactam improves the activity of imipenem against M. abscessus. The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of BlaMab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 μg/ml), amikacin (32 μg/ml), and avibactam (4 μg/ml) was bactericidal in broth (<0.1% survival), with 3.2- and 4.3-log10 reductions in the number of CFU being achieved at 72 h when imipenem was used at 8 and 32 μg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 μg/ml) and high (32 μg/ml) dosages of imipenem, respectively. In vivo inhibition of BlaMab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding BlaMab was induced (20-fold) in the infected macrophages. Inhibition of BlaMab by avibactam improved the efficacy of imipenem against M. abscessus in vitro, in macrophages, and in zebrafish embryos, indicating that this β-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of BlaMab, since the production of the β-lactamase is inducible in macrophages

Rôle de la L,D-transpeptidation dans biosynthèse du peptidoglycane et la résistance aux bêta-lactamines chez les Actinomycetales

Les PLP qui assurent la polymérisation finale du peptidoglycane sont les cibles des b-lactamines. Un nouveau mécanisme de résistance à ces antibiotiques par activation d'une autre voie de synthèse (L,D-transpeptidation) a été récemment caractérisé. L'existence et l'impact de la voie de L,D-transpeptidation ont été étudiés chez C. jeikeium et M. tuberculosis. Leur L,D-transpeptidases utilisent un substrat tétrapeptidique et sont inhibées par les carbapénèmes. Chez C. jeikeium la voie des PLP est prédominante. La disponibilité du substrat tétrapeptidique généré par la Pbp4 est le facteur limitant la L,D-transpeptidation. La Pbp2C a été identifiée comme responsable de la résistance aux b-lactamines. Chez M. tuberculosis, le peptidoglycane des formes non réplicatives comporte très majoritairement des ponts générés par L,D-transpeptidation suggérant que les L,D-transpeptidases et les carbapénèmes représentent une cible et une famille d'antibiotiques pour le traitement de la tuberculose.The PBP that catalyze the last cross-linking step of peptidoglycan synthesis are the targets of b-lactams. A novel mechanism of b-lactam resistance due to activation of another pathway (L,D-transpeptidation) has been recently characterized. The aim of the study was to investigate the impact of the L,D-transpeptidation pathway in C. jeikeium and M. tuberculosis. Their L,D-transpeptidases use substrates containing a tetrapeptide stem and are inhibited by carbapenems. In C. jeikeium, the PBP pathway is predominant. The supply in tetrapeptide substrate generated by Pbp4 is the limiting factor for L,D-transpeptidation pathway. Pbp2C was identified as the cross-linking enzyme responsible for b-lactam resistance in C. jeikeium. In M. tuberculosis, the peptidoglycan of non-replicating cells predominantly contains crosslinks generated by L,D-transpeptidation. L,D-transpeptidases and carbapenems may represent a target and a drug family relevant to the eradication of persistent M. tuberculosis.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Rôle d'une D,D-carboxypeptidase dans une nouvelle voie de synthèse du peptidoglycane chez Enterococcus faecium résistant à l'ampicilline

Chez Enterococcus faecium, un mutant résisant à l'ampicliline a été obtenu après cinq étapes de sélection sur des concentrations croissantes d'ampiciline. Un nouverau type de pontage du peptidoglycane a été mis en évidence. La voie classique par D,D-transpeptidation (PLP), sensible à l'ampiciline, a été remplacé par une L,D-transpeptidation. Des activités D,D-carboxypeptidases et L,D-transpeptidase, insensibles aux b-lactalines, ont été détectées. L'activité de D,D-carboxypeptidase pourrait générer le substrat tétrapeptidique de la L,D-transpeptidase. Dans le génome de E. faecium, nous avons identifié deux gènes (Y1 et Y2) codant pour des protéines apparentées aux métallo-D,D-carboxypeptidases . Le gène Y2 appartient à un locus composé d'un système de régulation à deux composants (senseur et régulateur). Nous avons mis en evidence une mutation dans le senseur chez le mutant hautement résistant. Les D,D-carboxypeptidases, insensibles aux b-lactamines, apparaissent comme des enzymes clés dans l'expression de cette nouvelle voie de synthèse du peptidoglycane.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Analyse des recommandations américaines et européennes de 2015 pour la prise en charge des endocardites infectieuses

International audienceThe optimal management of infective endocarditis requires a broad range of expertise (infectious disease specialists, cardiologists, microbiologists, cardiac surgeons, and intensivists). Given the low level of evidence currently available to support the management of infective endocarditis, international guidelines have always been particularly awaited and rather well implemented. Their cautious analysis of the medical literature and the range of expertise combined within the groups in charge of these guidelines are usually broadly acknowledged and respected. The publications, a few weeks apart, of the 2015 updates of the American and European guidelines, was quite disturbing. Indeed, several discrepancies on major therapeutic propositions were observed, including empirical treatment (penicillin M + penicillin A + gentamicin for Europeans in acutely ill patients; penicillin A + beta-lactamase inhibitor + gentamicin for Americans), or first-line treatment for the most common pathogen responsible for endocarditis in 2016, Staphylococcus aureus (trimethoprim–sulfamethoxazole + clindamycin as an alternative in the European guidelines, while this regimen is not even mentioned in the American guidelines). Other discrepancies were observed, although less significant: the role of positron emission tomography labelled with 18F-fluorodeoxyglucose and administration modalities for aminoglycosides. We aimed to detail the main changes brought upon by these guidelines, their discrepancies, and the ‘pros’ and ‘cons’ that may help you select the best treatment regimen for your patients.La prise en charge optimale des endocardites infectieuses nécessite le recours à des expertises multiples (infectiologues, cardiologues, microbiologistes, chirurgiens cardiaques, réanimateurs). En l’absence de haut niveau de preuves pour la plupart des étapes de cette prise en charge, les recommandations internationales ont toujours été particulièrement attendues dans ce domaine, et dans l’ensemble plutôt bien respectées. L’analyse rigoureuse des données de la littérature, et les expertises qui composent les groupes multidisciplinaires chargés de ces recommandations, font généralement autorité. Dans ce contexte, la publication à quelques semaines d’écart, en 2015, des révisions des recommandations américaines et européennes, a semé le trouble. On y retrouve des discordances nettes sur des propositions thérapeutiques importantes comme le traitement empirique (pénicilline M + pénicilline A + gentamicine pour les Européens dans les endocardites aiguës sévères; pénicilline A + inhibiteur de bêta-lactamases + gentamicine pour les américains), ou le traitement de première intention des endocardites à Staphylococcus aureus, principal pathogène responsable d’endocardites en 2016 (triméthoprime–sulfaméthoxazole + clindamycine en alternative dans les recommandations européennes, tandis que ce régime n’est même pas mentionné dans les recommandations américaines). D’autres différences sont observées, moins radicales : la place de la tomographie par émission de positons marquée au 18F-fluorodésoxyglucose et les modalités d’administration des aminosides. Nous détaillerons les principaux changements apportés par ces recommandations, leurs désaccords et les arguments pour et contre qui peuvent vous aider à sélectionner le meilleur traitement pour vos patients