CARACTERISATION EXHAUSTIVE DES SUBSTITUTIONS<br />DE PENICILLIN-BINDING PROTEINS INTERVENANT<br />DANS LA RESISTANCE AUX β-LACTAMINES CHEZ<br />STREPTOCOCCUS PNEUMONIAE

Date de rédaction: avril 2006Penicillin-Binding Proteins (PBP) are enzymes catalyzing the final steps of the bacterial cell-wall synthesis and are the targets of the β-lactam antibiotics. There are many amino acid substitutions in PBPs of clinical isolates of Streptococcus pneumoniae resistant to β-lactams, leading to a decrease of the affinity of these enzymes for antibiotics. There are about 40 substitutions in the transpeptidase domain of the two major resistantdeterminants PBP2x and PBP1a.Former studies have described the role of four mutations of PBP2x and three of PBP1a. But these mutations explain only a part of the resistance phenomenon. Only a few mutations may be involved in the loss of affinity of PBPs for β-lactam antibiotics leading to an increase of the resistance level.To identify all the relevant mutations, a set of automated protocols allowing to do site-directed mutagenesis, expression, purification and functional characterization of enzymes using automated liquid-handling systems was developed. An exhaustive characterization of more than 40 mutants of PBP2x of the highly resistant clinical isolate 5204 was performed using this method. All the relevant substitutions were identified and a new molecularresistance mechanism to β-lactams was elucidated. Moreover, a functional and phenotypic study of the resistance involving PBP1a was performed.This work provides a global view on the molecular mechanisms of the PBP-mediated resistance of S.pneumoniae to β-lactams using an original exhaustive method.Les Penicillin-Binding Proteins (PBP) sont des enzymes intervenant dans les étapes finales de la synthèse de la paroi bactérienne et sont les cibles des antibiotiques de la famille des β-lactamines. Dans les souches cliniques de Streptococcus pneumoniae résistantes aux β-lactamines, les PBPs ont de nombreuses mutations qui ont pour effet une diminution d'affinité de ces enzymes pour les antibiotiques. Il y a en moyenne 40 substitutions dans le domaine transpeptidase des deux acteurs majeurs de la résistance PBP2x et PBP1a.Des études précédentes ont décrit le rôle de quatre mutations de PBP2x et de trois de PBP1a, mais celles-ci ne sont responsables que d'une partie de la résistance. Il n'y a très probablement qu'un nombre restreint de mutations responsables de la perte d'affinité des PBPs pour les β-lactamines ayant pour conséquence une augmentation du niveau de résistance.Pour identifier toutes les mutations impliquées, une série de protocoles automatisés permettant de faire de la mutagénèse dirigée, de l'expression, de la purification et de la caractérisation fonctionnelle d'enzymes en utilisant des robots de types manipulateurs de liquides ont été développés. L'application de cette méthode nous a permis de réaliser une caractérisation exhaustive de plus de 40 mutations de PBP2x de la souche cliniquerésistante 5204. Cette étude a abouti à l'identification de toutes les substitutions clés ainsi qu'à l'élucidation d'un nouveau mécanisme moléculaire de baisse d'affinité de PBP2x pour les β-lactamines. De plus, une étude fonctionnelle et phénotypique de la résistance impliquant PBP1a a été réalisée.Ce travail apporte une vue globale des mécanismes moléculaires de la résistance de S. pneumoniae aux β-lactamines impliquant les PBPs en utilisant une méthode exhaustive originale

Développement d’une approche multi-omique pour la recherche de biomarqueurs associés à la réponse au traitement dans les lymphomes

International audienc

The TRANSPLANTEX initiative

International audienceTRANSPLANTEX, a French "investment for the future" initiated consortium of leading transplant units and research laboratories across France and a number of European countries aims to unravel, through mainly high-throughput genomics (and other omics) analyses of donor and recipients, novel (a) non-HLA, histocompatibility antigens, whether inside, or outside the MHC; (b) pre/post transplantation biomarkers. This shall lead to our better understanding of the pathophysiology of (and eventually designing better therapeutics for) the graft-versus-host disease in hematopoietic cell transplants and that of chronic graft rejection after kidney transplant. Industrial developments as well as innovative teaching initiatives are also integral part of this program. The present issue of Human Immunology aims to present a first snapshot of some of the research performed by TRANPLANTEX partners

selectBoost : a general algorithm to enhance the performance of variable selection methods

Motivation: With the growth of big data, variable selection has become one of the critical challenges in statistics. Although many methods have been proposed in the literature, their performance in terms of recall (sensitivity) and precision (predictive positive value) is limited in a context where the number of variables by far exceeds the number of observations or in a highly correlated setting. Results: In this article, we propose a general algorithm, which improves the precision of any existing variable selection method. This algorithm is based on highly intensive simulations and takes into account the correlation structure of the data. Our algorithm can either produce a confidence index for variable selection or be used in an experimental design planning perspective. We demonstrate the performance of our algorithm on both simulated and real data. We then apply it in two different ways to improve biological network reverse-engineering

Homozygosity for the V377I mutation in mevalonate kinase causes distinct clinical phenotypes in two sibs with hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS)

Mevalonate kinase (MVK) deficiency is a rare autosomal recessive auto-inflammatory disorder characterised by recurring episodes of fever associated with multiple non-specific inflammatory symptoms and caused by mutations in the MVK gene. The phenotypic spectrum is wide and depends mostly on the nature of the mutations. Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) is a relatively mild presentation and predominantly associated with a c.1129G>A (p.V377I) mutation in the MVK gene. We report cases of two sisters homozygous for this mutation but exhibiting distinct (symptomatic vs asymptomatic) phenotypes. Patient history was obtained; physical and clinical examination and laboratory tests were performed; lipopolysaccharide (LPS) response of peripheral blood mononuclear cells was quantified. Low MVK enzymatic activity is not necessarily associated with inflammatory symptoms. Increased inflammatory cytokine secretion in response to LPS is associated with symptomatic MVK deficiency. Individuals who are homozygous for the common p.V377I mutation in the MVK gene may not display the characteristic inflammatory episodes diagnostic of MKD and thus may be lost for correct and timely diagnosi

Exome sequencing identifies a novel missense variant in CTSC causing nonsyndromic aggressive periodontitis.

Cathepsin C (CatC) is a cysteine protease involved in a variety of immune and inflammatory pathways such as activation of cytotoxicity of various immune cells. Homozygous or compound heterozygous variants in the CatC coding gene CTSC cause different conditions that have in common severe periodontitis. Periodontitis may occur as part of Papillon-Lefèvre syndrome (PLS; OMIM#245000) or Haim-Munk syndrome (HMS; OMIM#245010), or may present as an isolated finding named aggressive periodontitis (AP1; OMIM#170650). AP1 generally affects young children and results in destruction of the periodontal support of the primary dentition. In the present study we report exome sequencing of a three generation consanguineous Turkish family with a recessive form of early-onset AP1. We identified a novel homozygous missense variant in exon 2 of CTSC (NM_148170, c.G302C, p.Trp101Ser) predicted to disrupt protein structure and to be disease causing. This is the first described CTSC variant specific to the nonsyndromic AP1 form. Given the broad phenotypic spectrum associated with CTSC variants, reporting this novel variant gives new insights on genotype/phenotype correlations and might improve diagnosis of patients with early-onset AP1

Multi-OMICS analyses unveil STAT1 as a potential modifier gene in mevalonate kinase deficiency

International audienceObjectives The objective of the present study was to explain why two siblings carrying both the same homozygous pathogenic mutation for the autoinflammatory disease hyper IgD syndrome, show opposite phenotypes, that is, the first being asymptomatic, the second presenting all classical characteristics of the disease.Methods Where single omics (mainly exome) analysis fails to identify culprit genes/mutations in human complex diseases, multiomics analyses may provide solutions, although this has been seldom used in a clinical setting. Here we combine exome, transcriptome and proteome analyses to decipher at a molecular level, the phenotypic differences between the two siblings.Results This multiomics approach led to the identification of a single gene—STAT1—which harboured a rare missense variant and showed a significant overexpression of both mRNA and protein in the symptomatic versus the asymptomatic sister. This variant was shown to be of gain of function nature, involved in an increased activation of the Janus kinase/signal transducer and activator of transcription signalling (JAK/STAT) pathway, known to play a critical role in inflammatory diseases and for which specific biotherapies presently exist. Pathway analyses based on information from differentially expressed transcripts and proteins confirmed the central role of STAT1 in the proposed regulatory network leading to an increased inflammatory phenotype in the symptomatic sibling.Conclusions This study demonstrates the power of a multiomics approach to uncover potential clinically actionable targets for a personalised therapy. In more general terms, we provide a proteogenomics analysis pipeline that takes advantage of subject-specific genomic and transcriptomic information to improve protein identification and hence advance individualised medicine.This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

Rab7-harboring vesicles are carriers of the transferrin receptor through the biosynthetic secretory pathway

International audienceThe biosynthetic secretory pathway is particularly challenging to investigate as it is underrepresented compared to the abundance of the other intracellular trafficking routes. Here, we combined the retention using selective hook (RUSH) to a CRISPR-Cas9 gene editing approach (eRUSH) and identified Rab7-harboring vesicles as an important intermediate compartment of the Golgi-to-plasma membrane transport of neosynthesized transferrin receptor (TfR). These vesicles did not exhibit degradative properties and were not associated to Rab6A-harboring vesicles. Rab7A was transiently associated to neosynthetic TfR-containing post-Golgi vesicles but dissociated before fusion with the plasma membrane. Together, our study reveals a role for Rab7 in the biosynthetic secretory pathway of the TfR, highlighting the diversity of the secretory vesicles' nature