Analyse des effets de souches probiotiques anti-inflammatoires

Inflammatory bowel diseases are characterized by abnormal inflammation of digestive tract. Several studies have shown positive effects of anti-inflammatory probiotic (native or recombinant) or commensal bacterial strains. The first part of this PhD thesis describes different optimization strategies of the use of lactic acid bacteria as proteins delivery vector. We have shown that the modification of peptidoglycan of Lactococcus lactis influencing lysis rate does not confer any advantage in both persistence in gastrointestinal tract and proteins delivery vector abilities. We showed that nature of bacterial vector is an essential parameter to deliver protein of health interest: Bifidobacterium infantis could increase higher immune response against E7 antigen than the ones obtained with L. lactis and Lactobacillus plantarum. In the second part of this PhD thesis, we analized anti-inflammatory effects of recombinant probiotic or commensal bacteria. We showed that Lb. casei BL23 producing superoxide dismutase could significantly protect mice from dextran sodium sulfate induced colitis damages. Finally, we observed anti-inflammatory properties on cellulars and animals models of Faecalibacterium prausnitzii, the first commensal anti-inflammatory bacterium identified by analysis of human clinical data.Les maladies inflammatoires chroniques de l'intestin sont caractérisées par une inflammation anormale et récurrente du tractus digestif. De nombreuses études ont démontré des effets bénéfiques de souches probiotiques anti-inflammatoires recombinantes ou non. La première partie de cette thèse décrit différentes stratégies d'optimisation de souches de bactéries lactiques en tant que vecteurs de protéines d'intérêt santé. Nous avons ainsi démontré qu'une modification du peptidoglycane de la paroie de Lactococcus lactis influençant la lyse bactérienne ne permettait pas de moduler l'immunogénicité de l'antigène E7 délivré par L. lactis. Nous avons également démontré que la nature du vecteur bactérien était un paramètre essentiel dans la vectorisation de la protéine délivrée : ainsi l'espèce Bifidobacterium infantis induit une réponse immunitaire spécifique à l'antigène E7 supérieure à celle obtenue avec les vecteurs L. lactis et Lactobacillus plantarum. La deuxième partie de cette thèse porte sur l'étude des effets anti-inflammatoires de bactéries recombinantes ou non. Nous avons ainsi démontré que la souche Lb. casei BL23 produisant une superoxyde dismutase à manganèse permettait de diminuer significativement des colites murines induites par administration de dextran sodium sulfate. Enfin, nous avons mis en évidence des propriétés anti-inflammatoires sur divers modèles d'inflammation in vitro / in vivo de Faecalibacterium prausnitzii, première bactérie commensale anti-inflammatoire identifiée sur la base de données cliniques humaines

A single point mutation alters the transglycosylation/hydrolysis partition, significantly enhancing the synthetic capability of an endo-glycoceramidase

The mutation of D311 to tyrosine in endo-glycoceramidase II from Rhodococcus sp. and the use of a poorly recognized substrate, 2-chloro-4-nitrophenyl beta-cellobioside, have provided appropriate conditions for the efficient synthesis of alkyl beta-cellobioside derivatives. The mutant D311Y was characterized by a lowered KM value for the hydrolysis of 2-chloro-4-nitrophenyl beta-cellobioside and increased transglycosylation when using aliphatic 1,3-diols or alcohols bearing a delta-hydroxy ketone function as acceptors. Closer analysis revealed that the transglycosylation/hydrolysis ratio in reactions catalyzed by the mutant was completely inversed and weak secondary hydrolysis was postponed, thus providing the basis for high transglycosylation yields (between 68 and 93%). Overall, results confirm that the enhancement of transglycosylation in glycoside hydrolases can be achieved by a combination of destabilized transition states and increased recognition for acceptor molecules

Use of superoxide dismutase and catalase producing lactic acid bacteria in TNBS induced Crohn's disease in mice

Reactive oxygen species are involved in various aspects of intestinal inflammation and tumor development. Decreasing their levels using antioxidant enzymes, such as catalase (CAT) or superoxide dismutase (SOD) could therefore be useful in the prevention of certain diseases. Lactic acid bacteria (LAB) are ideal candidates to deliver these enzymes in the gut. In this study, the anti-inflammatory effects of CAT or SOD producing Lwere evaluated using a trinitrobenzenesulfonic acid (TNBS) induced Crohn's disease murine model. Engineered Lactobacillus casei BL23 strains producing either CAT or SOD, or the native strain were given to mice before and after intrarectal administration of TNBS. Animal survival, live weight, intestinal morphology and histology, enzymatic activities, microbial translocation to the liver and cytokines released in the intestinal fluid were evaluated. The mice that received CAT or SOD-producing Lshowed a faster recovery of initial weight loss, increased enzymatic activities in the gut and lesser extent of intestinal inflammation compared to animals that received the wild-type strain or those that did not receive bacterial supplementation. Our findings suggest that genetically engineered Lthat produce antioxidant enzymes could be used to prevent or decrease the severity of certain intestinal pathologies. (C) 2010 Elsevier B.V. All rights reserved

A Single Point Mutation Alters the Transglycosylation/Hydrolysis Partition, Significantly Enhancing the Synthetic Capability of an <i>endo</i>-Glycoceramidase

The mutation of D311 to tyrosine in <i>endo</i>-glycoceramidase II from Rhodococcus sp. and the use of a poorly recognized substrate, 2-chloro-4-nitrophenyl β-cellobioside, have provided appropriate conditions for the efficient synthesis of alkyl β-cellobioside derivatives. The mutant D311Y was characterized by a lowered <i>K</i>_M value for the hydrolysis of 2-chloro-4-nitrophenyl β-cellobioside and increased transglycosylation when using aliphatic 1,3-diols or alcohols bearing a δ-hydroxy ketone function as acceptors. Closer analysis revealed that the transglycosylation/hydrolysis ratio in reactions catalyzed by the mutant was completely inversed and weak secondary hydrolysis was postponed, thus providing the basis for high transglycosylation yields (between 68 and 93%). Overall, results confirm that the enhancement of transglycosylation in glycoside hydrolases can be achieved by a combination of destabilized transition states and increased recognition for acceptor molecules

Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients

A decrease in the abundance and biodiversity of intestinal bacteria within the dominant phylum Firmicutes has been observed repeatedly in Crohn disease (CD) patients. In this study, we determined the composition of the mucosa-associated microbiota of CD patients at the time of surgical resection and 6 months later using FISH analysis. We found that a reduction of a major member of Firmicutes, Faecalibacterium prausnitzii, is associated with a higher risk of postoperative recurrence of ileal CD. A lower proportion of F. prausnitzii on resected ileal Crohn mucosa also was associated with endoscopic recurrence at 6 months. To evaluate the immunomodulatory properties of F. prausnitzii we analyzed the anti-inflammatory effects of F. prausnitzii in both in vitro (cellular models) and in vivo [2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced] colitis in mice. In Caco-2 cells transfected with a reporter gene for NF-κB activity, F. prausnitzii had no effect on IL-1β-induced NF-κB activity, whereas the supernatant abolished it. In vitro peripheral blood mononuclear cell stimulation by F. prausnitzii led to significantly lower IL-12 and IFN-γ production levels and higher secretion of IL-10. Oral administration of either live F. prausnitzii or its supernatant markedly reduced the severity of TNBS colitis and tended to correct the dysbiosis associated with TNBS colitis, as demonstrated by real-time quantitative PCR (qPCR) analysis. F. prausnitzii exhibits anti-inflammatory effects on cellular and TNBS colitis models, partly due to secreted metabolites able to block NF-κB activation and IL-8 production. These results suggest that counterbalancing dysbiosis using F. prausnitzii as a probiotic is a promising strategy in CD treatment