De 1956 à 1986, l'utilisation des glycopeptides en thérapeutique infectieuse a été marquée par de francs succès dans le traitement des infections graves dues à des bactéries à Gram positif. La vancomycine et la téicoplanine sont des antibiotiques de grande importance thérapeutique utilisés à travers le monde pour traiter les infections graves dues aux staphylocoques, aux entérocoques, aux streptocoques et à Clostridium difficile à cause de la résistance de ces bactéries à d'autres antibiotiques. Malheureusement, depuis 1986, on a assisté à l'émergence et la dissémination de la résistance aux glycopeptides chez un grand nombre d'espèces pathogènes telles que les entérocoques et plus récemment Staphylococcus aureus résistant à la méticilline. Dans ce contexte de dissémination de la résistance aux glycopeptides, mon projet de doctorat fournit d'importantes données grâce à la mise en évidence d'un réservoir des gènes vanB, vanD et vanG dans la flore intestinale humaine. Nous avons caractérisé pour la première fois plusieurs allèles du gène vanG dans la flore humaine. Puis, nous avons isolé et caractérisé deux nouvelles espèces bactériennes anaérobies résistantes aux glycopeptides. Les analyses phylogénétiques ont montré que ces nouvelles espèces appartiennent au groupe phylogénétique XlVa des Clostridium. La première espèce a été nommée Clostridium lavalense sp. nov. CCRI-9842T . Cette souche est résistante à la vancomycine du fait de la présence du locus vanB2 porté par le transposon Tn5382. La deuxième espèce isolée a été nommée Ruminococcus gauvreaui sp. nov. CCRI-161101 . Cette souche est résistante à la vancomycine et à la téicoplanine et contient le locus vanD qui confère la résistance aux glycopeptides ainsi qu'un locus apparenté au locus vanG décrit chez les ERG. Il s'agit de la première description du locus vanD de résistance aux glycopeptides dans une espèce autre que les entérocoques habituellement connus comme réservoir de ce locus de résistance. De plus, un nouvel allèle du gène vanG a été caractérisé dans la souche R. gauvreaui CCRI-16110'. Ainsi les résultats de nos travaux ont permis de montrer que le tube digestif humain représente un important réservoir des gènes de résistance aux glycopeptides qui sont parfois présents dans des espèces bactériennes encore inconnues du tube digestif. Ces nouvelles données vont permettre de mieux adapter les outils de contrôle des infections dues à des bactéries résistantes aux glycopeptides. / From 1956 to 1986, glycopeptide use in infectious disease therapy was marked by astounding success for the treatment of serious Gram-positive bacterial infections. Vancomycin and teicoplanin are considered as the last resort glycopeptide antibiotics presently in use for the treatment of serious Staphylococcus spp., Enterococcus spp., Streptococcus spp. and Clostridium difficile drug-resistant infections. Unfortunately, the last twenty years have seen the emergence and dissemination of glycopeptide-resistant pathogenic strains such as methicillin-resistant Enterococcus spp. and, more recently, methicillin-resistant Staphylococcus aureus. In this context of glycopeptide resistance dissemination, my thesis serves as an important contribution through the identification of vanB, vanD and vanG reservoir genes in the human intestinal flora. We have, for the first time, characterized several alleles of the vanG gene in the human flora and have isolated and characterized two new anaerobic glycopeptide-resistant bacterial strains. Phylogenetic analyses have shown that these new strains belong to the Clostridium XlVa phylogenetic group. The first strain was named Clostridium lavalense sp. nov. CCRI-98421 . This strain is resistant to vancomycin, due to the presence of the vanB2 locus carried by Tn5382/1549 transposon. The second strain was named Ruminococcus gauvreaui sp. nov. CCRI-16110r and is resistant to vancomycin and teicoplanin. Ruminococcus gauvreaui CCRI-161101 contains the vanD locus, which confers glycopeptide resistance in enterococci, along with a vanG-like locus described for vancomycin-resistant Enterococcus. This is the first description of a glycopeptide resistance vanD locus in a non-Enterococcus strain, the usual vanD locus reservoir. In addition, a new allele of the vanG gene was characterized in the R. gauvreaui CCRI-16110T strain. Therefore, our results demonstrate that the human digestive tract represents an important reservoir of glycopeptide resistance genes, present in yet-to-be-described intestinal bacterial strains. These data will allow the fine tuning of infection control tools against glycopeptide-resistant bacteria.

No description available.

<p>An efficient and versatile non-destructive method to analyze the progress of solid-phase glycoconjugate synthesis with gel-phase ¹⁹F NMR spectroscopy is described. The method relies on use of fluorinated linkers and building blocks carrying fluorinated protective groups. Commercially available fluorinated reagents have been utilized to attach the protective groups. </p><p>The influence of resin structures for seven commercial resins upon resolution of gel-phase ¹⁹F NMR spectra was investigated. Two different linkers for oligosaccharide synthesis were also developed and successfully employed in preparation of α-Gal trisaccharides and a n-pentenyl glycoside. Finally, reaction conditions for solid-phase peptide glycosylations were established.</p>

An efficient and versatile non-destructive method to analyze the progress of solid-phase glycoconjugate synthesis with gel-phase 19F NMR spectroscopy is described. The method relies on use of fluorinated linkers and building blocks carrying fluorinated protective groups. Commercially available fluorinated reagents have been utilized to attach the protective groups. The influence of resin structures for seven commercial resins upon resolution of gel-phase 19F NMR spectra was investigated. Two different linkers for oligosaccharide synthesis were also developed and successfully employed in preparation of α-Gal trisaccharides and a n-pentenyl glycoside. Finally, reaction conditions for solid-phase peptide glycosylations were established.

Mucin is a term used to describe a large family of heavily glycosylated proteins which are present on the surfaces of secretory epithelial cells and are overexpressed by many carcinomas. Membrane-bound mucin MUC1 is of special interest. Its backbone consists of repeating units of twenty amino acids with five potential glycosylation sites. These sites are expanded to structures like the T (Galβ(1-&gt;3)GalNAcα-Ser/Thr) and Tn (GalNAcα-Ser/Thr) antigens by the action of various glycosyltransferases. In different types of carcinomas these epitopes are being terminated by sialic acid residues to form among others: 2,3-sialyl-T and sialyl-Tn structures due to the elevated levels of different sialyltransferases. Solid-phase synthesis of the selected antigens derived from the mucin MUC1 has been developed and optimized. A chemoenzymatic approach has been used to effectively prepare 2,3-sialyl-T and 2,6-sialyl-Tn glycopeptides. The formation of intramolecular sialic acid lactones in presence of acetic acid was investigated. The stability of lactones formed from 2,3-sialyl-T towards water was studied using NMR spectroscopy and it appeared that 1''-&gt;2' lactone displayed remarkable strength to hydrolysis and it was suggested as a candidate for cancer vaccine. Gel-phase 19F NMR spectroscopy is known to be a very good tool to characterize resin-bound products using fluorinated protecting groups and linker molecules. The hydrophobic peptide LLLLTVLTV, which is a fragment from the MUC1 signal sequence, was prepared using solid-phase synthesis according to a modified Fmoc protocol with more active coupling reagent, stronger base, and the isopropylidene dipeptide Fmoc-Leu-Thr-(ΨMe,Mepro)-OH. Gel-phase 19F NMR spectroscopy was used to evaluate peptide chain aggregation and coupling and deprotection efficiency. A carbamate linker strategy proved to be effective in solid-phase synthesis of serine-based neoglycolipids with terminal amino functionality. Neoglycolipids were covalently bound to secondary amines in microtiter plates using squaric acid ester methodology. These arrays have potential to study the interactions between carbohydrates and e.g. proteins and microbes. The new fluorinated α-amino protective group [1-(4-(4-fluorophenyl)-2,6-dioxocyclohexylidene)ethyl] Fde was developed. This group is cleaved with hydrazine in DMF solution. By using amino acids protected with this group, it was possible to quantify the efficiency of peptide coupling using gel-phase 19F NMR spectroscopy.

La teicoplanine est utilisée par voie intraveineuse (IV) dans le traitement de la pneumonie acquise sous ventilation mécanique (PAVM) à Staphylocoque aureus méticilline résistant. Nous avons évalué la faisabilité et les bénéfices de la nébulisation de teicoplanine en ventilation mécanique (VM) par rapport à la voie IV. L’administration d’un aérosol de teicoplanine chez le rat a permis d’obtenir des concentrations pulmonaires 48 fois supérieures à celles après IV et des concentrations plasmatiques inférieures à celles après IV. Après avoir mesuré l’influence des nébuliseurs sur les paramètres ventilatoires du respirateur, nous avons sélectionné le nébuliseur Aeroneb Pro®. Les propriétés bactériologiques de l’antibiotique ne sont pas altérées par le nébuliseur et l’exposition d’un modèle d’épithélium pulmonaire in vitro à des doses 100 fois supérieures à celles utilisées en IV n’a pas montré de cytotoxicité. L’étude réalisée sur la nébulisation de teicoplanine chez le porc en VM a montré un dépôt pulmonaire moyen de 18,1%. En conclusion, la nébulisation de teicoplanine pourrait etre envisagée comme une nouvelle arme thérapeutique dans la PAVM. / Nebulization of teicoplanin should be an alternative way of administration in Ventilator-associated pneumonia (VAP) caused by Methicillin-resistant staphylococcus aureus. We assessed the faisibility and advantages of nebulization of teicoplanin in mechanical ventilation. After a nebulization of teicoplanin in a rat model, the lung concentrations were more than 48 times higher than the lung concentrations following intravenous administration. In the next study, we used an Aeroneb Pro® nebulizer selected according to its safe properties with ventilator which was evaluated in a bench study. Any cytotoxicity was observed with high doses of teicoplanin and nebulization did not affect its pharmacological properties. In mechanically ventilated piglets, teicoplanin was administered efficiently by nebulization wih an aerosol delivery of 18.1% [16.3-27.0]. Pharmacokinetic parameters are particularly consistent with this time-dependent antibiotic and emphasize the potential of airways delivery of teicoplanin. In conclusion, nebulization of teicoplanin adds a further challenge to the development of aerosol therapy of antibiotics in VAP.

Glycopeptides are the so-called last-resort antibiotics in clinical practice used to treat heavier, predominantly nosocomial infections caused by multi-resistant coagulase-negative staphylococci. The origin and genetic basis of resistance to glycopeptide antibiotics has not yet been elucidated within coagulase-negative staphylococci. Research on Staphylococcus aureus has shown, that intermediate resistance to glycopeptide antibiotics is associated with the presence of one or more mutations, rather than being conditioned by the support of a particular genetic element, such as in enterococci. By using various types of in vitro resistant mutant selection, we were able to obtain isogenic pairs of glycopeptide sensitive and resistant strains of Staphylococcus epidermidis and Staphylococcus haemolyticus. By sequencing the genomes of these pairs, one nucleotide polymorphisms were identified and predominantly found in metabolic and cell wall control systems. Phenotypic analysis did not reveal a direct association of glycopeptide resistance with increased biofilm formation. In clinical practice, the cross-resistance of glycopeptides and other antibiotics is problematic. For the non-glycopeptide antibiotics imipenem and rifampicin, the incidence of cross-resistance with glycopeptide antibiotics in S. aureus...

No description available.

A membrane protein VanZTei which is encoded by the gene vanZ from the vanA glycopeptide resistance gene cluster is a part of the large family of VanZ proteins. VanZTei confers resistance to teicoplanin in Enterococcus faecalis without the presence of other proteins encoded by the cluster. The aim of my work was to compare the ability of two orthologous proteins VanZTei and VanZg (from the genome of Enterococcus faecium) to confer resistance to glycopeptides in Staphylococcus aureus RN4220 and Enterococcus faecium. We have shown that VanZg increases resistance to teicoplanin (Tei) 8 to 16 times the and also to dalbavancin (Dalb) 8 times. VanZTei also confers resistance to Tei and Dalb, but the increase is only twofold. Conversely VanZTei confers resistance to newly synthetized glycopeptides more effectively than VanZg (fourfold increase of resistance confered by VanZTei and two to fourfold increase of resistance confered by VanZg). It suggests that both proteins have different specificity to antibiotics. In despite the mutants of S. aureus RN4220 VanZTei pRMC2 with increased resistance to teicoplanin (MICTei> 8 µg/ml) in which the resistance is dependent on vanZTei expression were selected. These resistant mutants do not carry mutation in a gene vanZTei or in its ribosomal binding site. Neither of the...

Deficiência na enzima lisossomal glucocerebrosidase (GCR) resulta na doença de Gaucher. O tratamento atual consiste na administração da enzima exógena, produzida em células CHO. Porém, o medicamento disponível no mercado é extremamente custoso. Neste trabalho, propusemos a clonagem e a expressão da GCR humana em células CHO, visando a obtenção de um clone celular produtor para viabilizar a produção futura da enzima, a um custo menor, no Instituto Butantan. A expressão estável da GCR recombinante foi obtida a partir da transfecção de células CHO-dhfr- com o plasmídeo pED de expressão em células de mamíferos contendo o cDNA da GCR, seguido de amplificação gênica por MTX. A GCR foi detectada no extrato celular (~ 64 kDa) e secretada para o sobrenadante (63-69 kDa) em ensaios de western blotting, usando o anticorpo policlonal anti-GCR gerado neste trabalho. A enzima secretada hidrolisou o substrato 4-MUG e a sua produtividade foi estimada em 5,14 pg/célula/dia para o melhor subclone produtor, selecionado para a produção futura da GCR em larga escala. / Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher\'s disease. Current treatment consists on enzyme replacement therapy by the administration of recombinant GCR produced in CHO cells. However, the medicine available in the market is extremely expensive. In this work, we proposed the cloning and expression of human GCR in CHO cells, in order to obtain a productive cellular clone for future production of GCR enzyme at a lower cost at the Butantan Institute. The stable expression of recombinant GCR was obtained after transfection of CHO-dhfr- cells with pED mammalian expression vector containing the GCR cDNA, followed by gene amplification with MTX. The GCR was detected by western blotting analysis, either as cell-associated (~ 64 kDa) or as secreted forms (63-69 kDa), using the anti-GCR polyclonal antibody produced in this work. The secreted enzyme was active on 4-MUG and was produced at a level of about 5,14 pg/cell/day for the best producer subclone, selected for subsequent steps of GCR production on large scale in next future.