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Gram-positive bacteria of the species Lacticaseibacillus casei (formerly known as Lactobacillus casei) are fermentative bacilli found in dairy products that are known for their anti-inflammatory properties and beneficial effects against pathogens. Like all living organisms, L. casei produces nanostructures of 20 to 400 nm in diameter bounded by a lipid membrane called membrane vesicles (MVs). MVs have been shown to contain various cellular constituents and exert multiple functions. However, despite the growing interest in Gram-positive MVs over the past 10 years, the mechanisms of biogenesis and the roles played by these vesicles remain poorly characterized. In this context, the objectives of this thesis project are (i) to characterize the MVs released by the L. casei strain BL23, (ii) to identify the biogenesis mechanisms and finally (iii) to study the roles played by these MVs. In this work, we demonstrated the production of MVs by L. casei BL23 using electron microscopy, then the size and protein composition of the purified MVs were examined. Based on the characterization results, the contribution of L. casei BL23 prophages to the biogenesis of MVs was investigated by a site-directed mutagenesis approach. Our results suggest a contribution of the PLE2 prophage in the production of MVs by L. casei BL23 in the absence or presence of genotoxic stress. Furthermore, we also showed that the MVs of L. casei BL23 exhibited an antibiofilm activity against several pathogenic and spoilage bacteria. These results pave the way for the use of MVs of L. casei BL23 to fight against the formation of bacterial biofilms., Les bactéries à Gram positif de l’espèce Lacticaseibacillus casei (anciennement appelée Lactobacillus casei) sont des bacilles fermentaires présents dans les produits laitiers qui sont connues pour leurs propriétés anti-inflammatoires et leurs effets bénéfiques dans la lutte contre les pathogènes. Comme tous les organismes vivants, L. casei produit des nanostructures de 20 à 400 nm de diamètre délimitées par une membrane lipidique appelées vésicules membranaires (MVs). Les MVs ont été décrites comme pouvant contenir divers constituants cellulaires et exercer de multiples fonctions. Toutefois, malgré l’intérêt croissant accordé aux MVs de bactéries à Gram positif ces 10 dernières années, les mécanismes de biogenèse et les rôles exercés par ces vésicules restent encore peu caractérisés. Dans ce contexte, ce projet de thèse a pour objectifs (i) de caractériser les MVs libérées par la souche BL23 de L. casei, (ii) d’identifier les mécanismes de biogenèse et enfin (iii) d’étudier les rôles exercés par ces MVs. Dans ce travail, nous avons mis en évidence la production de MVs par L. casei BL23 grâce à des analyses de microscopie électronique, puis la taille et la composition protéique des MVs purifiées ont été étudiées. Sur la base des résultats de caractérisation, le rôle des prophages de L. casei BL23 dans la biogenèse des MVs a été examiné par une approche de mutagenèse dirigée. L’ensemble de nos résultats suggèrent une contribution du prophage PLE2 dans la production des MVs par L. casei BL23 en l’absence ou en présence de stress génotoxiques. Par ailleurs, nous avons mis en évidence une activité antibiofilm des MVs de L. casei BL23 sur un panel de bactéries pathogènes et de bactéries d’altération. Ainsi, ces résultats ouvrent la voie à l’utilisation de MVs de L. casei dans la lutte contre la formation de biofilms bactériens.

Objectives Rippling muscle disease (RMD) is characterized by muscle stiffness, muscle hypertrophy, and rippling muscle induced by stretching or percussion. Hereditary RMD is due to sequence variants in the CAV3 and PTRF/CAVIN1 genes encoding Caveolin-3 or Cavin-1, respectively; a few series of patients with acquired autoimmune forms of RMD (iRMD) associated with AChR antibody–positive myasthenia gravis and/or thymoma have also been described. Recently, MURC/caveolae-associated protein 4 (Cavin-4) autoantibody was identified in 8 of 10 patients without thymoma, highlighting its potential both as a biomarker and as a triggering agent of this pathology. Here, we report the case of a patient with iRMD-AchR antibody negative associated with thymoma. Methods We suspected a paraneoplastic origin and investigated the presence of specific autoantibodies targeting muscle antigens through a combination of Western blotting and affinity purification coupled with mass spectrometry–based proteomic approaches. Results We identified circulating MURC/Cavin-4 autoantibodies and found strong similarities between histologic features of the patient's muscle and those commonly reported in caveolinopathies. Strikingly, MURC/Cavin-4 autoantibody titer strongly decreased after tumor resection and immunotherapy correlating with complete disappearance of the rippling phenotype and full patient remission. Discussion MURC/Cavin-4 autoantibodies may play a pathogenic role in paraneoplastic iRMD associated with thymoma.

The last few decades have demonstrated that membrane vesicles (MVs) produced by microorganisms can have a wide variety of functions. This diversity places MVs at the crossroads of major research topics in current microbiology such as antibiotic resistance, horizontal gene transfer, cell communication, biofilm development, bacteriophage resistance, and pathogenesis.

International audience; Objective - Design - Human gastric epithelial cell lines were infected with 7.13, exposed or not to a DNA-damaging agent camptothecin (CPT), to mimic a genetic instability context. We quantified the expression of , and their target genes, we determined their subcellular localisation by immunofluorescence and examined USF1/p53 interaction. and INS-GAS mice were used to strengthen the findings in vivo and patient data examined for clinical relevance. Results - In vivo we revealed the dominant role of USF1 in protecting gastric cells against -induced carcinogenesis and its impact on p53 levels. In vitro, delocalises USF1 into foci close to cell membranes. prevents USF1/p53 nuclear built up and relocates these complexes in the cytoplasm, thereby impairing their transcriptional function. also inhibits CPT-induced USF1/p53 nuclear complexes, exacerbating CPT-dependent DNA damaging effects. Conclusion - Our data reveal that the depletion of USF1 and its de-localisation in the vicinity of cell membranes are essential events associated to the genotoxic activity of infection, thus promoting gastric carcinogenesis. These findings are also of clinical relevance, supporting USF1 expression as a potential marker of GC susceptibility.