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, Le niveau d'expression des gènes codant pour les récepteurs (A) TLR9 et (B) TLR2. Les cellules HT-29 ont été stimulées par la souche sauvage Pf CIRM-BIA 129 (WT) ou la souche mutante Pf CIRM-BIA 129 KO SlpB (KO)

C. , En plus des effets stériques, l'acétylation modifie également la charge de la protéine, 2014.

C. , L'acylation est le processus par lequel une chaîne hydrocarbonée est ajoutée à la protéine, 2014.

. Beveridge, Cette structure joue un rôle important dans la croissance et la survie de ces microorganismes. Les nombreuses fonctions des Slps comprennent le maintien de l'intégrité de la cellule, la lutte contre des agents pathogènes, l'interaction avec l'hôte et son système immunitaire, Les protéines Slps (« S-Layer Proteins») constituent la couche S, une surface paracristalline structurée qui enveloppe la surface de certaines bactéries à Gram négatif et à Gram positif. Elle était initialement identifiée et bien caractérisée chez les archées, 1997.

, Cependant, il existe une différence en termes du taux de glycosylation observé entre les archées et les bactéries. En effet, les structures des N-glycosylations des Slps des archées présentent une grande diversité en termes de taille des glycanes, 2004.

. Anzengruber, Le nombre de résidus modifiés ainsi que la longueur des chaînes glycanes (50 unités identiques) sont moindres chez les protéines S-layer des bactéries. Les glycosylations des protéines S-layer, décrites essentiellement chez les espèces lactiques, peuvent être ancrées en N ou O sur le squelette peptidique et peuvent se composer d'hexoses, de pentoses, d'heptoses ou de désoxyhexose neutres et d'osamines, 2004.

. Cavallero, , 2010.

, Les modifications covalentes des protéines S-layer de P. freudenreichii Annexes

P. Chez and . Freudenreichii, SlpE et l'internaline like (Inl). L'analyse par spectrométrie de masse a permis de démontrer la présence de modifications covalentes (Figure 1), notamment des sites probables de glycosylation, à la fois des N-glycosylations et d'O-glycosylations, l'analyse des extraits de protéines Slps provenant de différentes souches a révélé la présence d'isoformes de point isoélectrique pour les protéines SlpA, SlpB, 2015.

, Figure 1. Les modifications covalentes ainsi que les sites modifiés identifiés par spectrométrie de masse. Les différentes isoformes des protéines SlpA, SlpB, SlpE et Internaline-like ont été prélevées sur les gels bidimensionnels. Les peptides issus de la protéolyse par trypsine des différentes isoformes ont été analysés par LC-MS/MS. Des sites potentiels de glycosylation ainsi que de phosphorylation ont été identifiés

L. Maréchal, Plusieurs cultures successives sur l'ultrafiltrat de lait ont été réalisées afin de constituer un stock de protéines Slps. Après centrifugation, les culots bactériens sont lavés deux fois par le tampon PBS (Phosphate Buffer saline). Les culots correspondants à une densité optique de 40 sont ensuite repris dans 2, Une extraction des protéines Slps a été réalisée en utilisant l'agent chaotrope, le chlorure de guanidine, tel que décrit précédemment, 2015.

, Les mélanges sont incubés pendant 15 minutes à 50°C. Les surnageants contenant les protéines Slps sont récupérés par centrifugation. Les extraits protéiques successifs obtenus sont dialysés contre de l'eau déminéralisée

, La concentration protéique des différents extraits protéiques obtenus a été déterminée en utilisant le kit 2-D Quant Kit et en suivant les instructions du fabricant

. Cao, La migration a été effectuée à un voltage constant jusqu'à la sortie du front de migration indiqué par le bleu de bromophénol. Les gels ont été ensuite transférés sur des membranes PVDF (Polyvinylidene difluoride) (GE Healthcare) en utilisant le système Trans-Blot® Electrophoretic Transfer Cell (250 mA, 30 V, 1h30). Les membranes ont été incubées à température ambiante dans une, Lectinblot Le lectinblot est une technique d'identification et de caractérisation de motifs glucidiques présents sur des protéines, et qui se repose sur les interactions lectine-glycane. Selon les caractéristiques des lectines, différents types de sucres peuvent être reconnus, 2013.

. Nacl, Les différentes lectines ont été diluées dans une solution de dilution (50 mM de Tris-HCl, 0.25% de gélatine, 0,05% de tween 20, 0,15 M de NaCl, 5 mM EDTA, pH=7,5) à une concentration de 10 µg/ml. Les membranes ont été ensuite rincées, puis incubées avec une solution de R.T.U (Horseradish Peroxidase Streptavidin, Vector Laboratories, Inc). Les membranes ont été révélées en utilisant le système ECL plus ® (GE Healthcare, Vélizy, France) et ensuite scannées en utilisant la GBox Syngene, pH=7,5), les membranes ont été incubées toute la nuit à une température ambiante avec les différentes lectines biotinylées (BIOTINYLATED LECTIN KIT I, Vector Laboratories, Inc)

, Invitrogen) a été utilisé en tant qu'un témoin positif. Les gels obtenus ont été fixés toute la nuit dans une solution contenant 50% méthanol et 10% d'acide acétique, afin de s'assurer que tout le SDS est éliminé du gel. Les gels sont ensuite rincés (3 rinçage de 10 minutes) à l'eau ultra-pure afin d'éliminer toute trace d'acide acétique ou de méthanol. Les gels sont ensuite incubés pendant 2 h dans une solution Pro-Q® Diamond, Les extraits protéiques de Slps, environ 25 µg ont été analysés par SDS-PAGE, tel que décrit précédemment. Un standard de poids moléculaire contenant deux phosphoprotéines

, Les gels ont été visualisés en utilisant la GBox Syngene (Ozyme, Saint-Quentin-en-Yvelines, France), avec une longueur d'excitation de 532 nm

, La quantité protéique en Slps nécessaire à la réalisation d'un gel bidimensionnels a été précipitée avec le kit 2D-Clean Up (GE Healthcare, Vélizy, France) Les protéines sont ensuite réhydratées dans 100 ?L de tampon Destreak, pp.4-7

, Ces bandelettes sont préalablement réhydratées une nuit dans 450 ?L de tampon Destreak additionné d'IPG Buffer (pH 4-7, 2%). Les échantillons sont déposés à l'extrémité acide des bandelettes à l'aide de cupules. La migration s'est effectuée en 24 h selon un programme permettant une augmentation progressive de la tension et une accumulation de 60, Immobiline DryStrip Gels de 13 cm, avec un gradient de pH allant de 4 à 7 (GE Healthcare) sur un système Multiphore II (GE Healthcare

. Kvh, À la fin de la focalisation, les bandelettes sont incubées sous agitation successivement dans les

, Le schéma de fragmentation du HCD est caractérisé par une énergie d'activation plus élevée et un temps d'activation plus court, par comparaison à une fragmentation CID. Les Oglycanes sont habituellement complétement éliminés via un réarrangement en phase gazeuse

, La fragmentation par ETD (Electron-transfer dissociation) permettrait théoriquement d'identifier les modifications post-traductionnelles labiles au CID. Idéalement, elle peut fournir à la fois des informations sur la séquence et sur la localisation des sites de modification

, La fragmentation par ECD (Electron-capture dissociation) est plus préférable lorsqu'il s'agit de peptides ayant des phosphorylations multiples, car elle peut provoquer une fragmentation efficace du squelette peptidique tout en laissant la modification covalente intacte

. Ainsi and . Moradian, identification précise du type de modification et du site de modification peut être très difficile en envisageant un seul type de fragmentation des peptides modifiés, 2014.

, L'analyse de spectrométrie de masse à l'institut Jacques Monod a consisté à combiner les 4 types de fragmentation. Des informations différentes et complémentaires peuvent être ainsi obtenues sur la structure primaire ou les modifications chimiques présentes sur les peptides analysés

, Résultats & Discussion

, Un jeu de sept lectines différentes a été testé, seulement trois d'entre elles ont réagi positivement à la protéine SlpB : la lectin Dolichos biflorus agglutinin (DBA) qui reconnait le motif ?GalNAc, la lectine Ulex europaeus agglutinin 1(UEA 1) qui reconnait le motif ?-fucose, ainsi que la lectine Arachis hypogaea (peanut) agglutinin (PNA) qui reconnait le motif Gal?3GalNAc (Figure 3), Ce volet de travail a consisté à caractériser les modifications covalentes de la protéine SlpB par des outils de protéomique

. Cependant, En effet, la digestion non spécifique par la protéinase K a conduit à la génération de peptides trop courts, ce qui a compliqué l'alignement des séquences peptidiques sur la séquence protéique

. Grange, Il s'agit d'O-glycosylations sur la sérine 283, la sérine 429, la thréonine 415 ; ainsi qu'une N-glycosylation sur l'asparagine 462 (Figure 5). Les types de glycanes mis en évidence sont en cohérence avec les observations faites sur les lectinblot. Cependant, la structure exacte des glycanes reste inconnue, Néanmoins, un niveau d'expression plus bas du gène gtf a été mis en évidence chez les souches présentant le phénotype agglutination (-), ce qui suggère que ces enzymes pourraient être indispensables pour d'autres fonctions bactériennes, 2008.

, En effet, les glycosyltransférases des actinomycètes sont décrites comme étant versatiles, en ce qui concerne le donneur de sucre, mais également dans une moindre mesure, en ce qui concerne l'accepteur des sucres, 2007.

, La coloration au Pro-Q® Diamond des extraits Slps avant ou après le traitement à la phosphatase alkaline montre bien la présence de phosphorylation seulement sur la protéine SlpB

, En ce qui concerne la phosphorylation, seulement des Serine kinases ont été déjà décrites chez les espèces des propionibactéries laitières (Cagen and Friedmann, 1972), mais il n'existe pas d'étude du phosphoprotéome chez P. freudenreichii. Le rôle de la présence d'une telle modification covalente sur la protéine SlpB est inconnu. Néanmoins, l'outil InterPro a prédis la présence d'un domaine dit « Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid » sur la protéine SlpB, L'analyse des données de spectrométrie de masse a également confirmé la présence de phosphorylation sur la thréonine, vol.174

. Parandhaman, En effet, la phosphorylation est décrite comme un mécanisme impliqué dans la dormance, la division cellulaire, l'adaptation aux stress environnementaux, mais également impliquée dans l'interaction avec les cellules hôtes (virulence), chez de nombreuses espèces d'actinobactéries (Mijakovic and Macek, Les auteurs attribuent à cette modification un rôle structural probable ou de transduction de signaux environnementaux, 2012.

, Pour la protéine SlpB, il est probable que la phosphorylation a un rôle dans l'activation ou la désactivation de son activité enzymatique prédite

, Les méthodes d'enrichissement des glycopeptides par chromatographie d'interaction hydrophobe (HILIC) ou des phosphopeptides par chromatographie d'affinité (IMAC) permettra d'augmenter le rapport signal sur bruit, Néanmoins, quand bien même l'ensemble des résultats obtenus semblent cohérents; il faut prendre en compte ces résultats avec quelques précautions, 2001.

, Annexe 3 : publications scientifiques ? Publications publiées dans des journaux à comité de lecture : -Rabah H, Do Carmo FLR, Jan G. Dairy Propionibacteria: Versatile Probiotics. Microorganisms, vol.5, p.24, 2017.

H. -rabah, O. Ménard, F. Gaucher, F. Do-carmo, D. Dupont et al., Cheese matrix protects the immunomodulatory surface protein SlpB of Propionibacterium freudenreichii during in vitro digestion, Food Res Int, vol.106, pp.712-733, 2018.

-. Rabah, H. , D. Oliveira-carvalho, R. D. Gaucher, F. Cordeiro et al., Extractable Bacterial Surface Proteins in Probiotic-Host Interaction. Front Microbiol, vol.9, p.645, 2018.
URL : https://hal.archives-ouvertes.fr/hal-01763214

H. --rabah, S. Ferret-bernard, S. Huang, L. Normand, L. Cousin et al., The Cheese Matrix Modulates the Immunomodulatory Properties of Propionibacterium freudenreichii CIRM-BIA 129 in Healthy Piglets, Front. Microbiol, vol.9, p.2584, 2018.

S. Huang, H. Rabah, S. Ferret-bernard, L. Normand, L. Gaucher et al., Propionic fermentation by the probiotic Propionibacterium freudenreichii to functionalize whey, Journal of Functional Foods
URL : https://hal.archives-ouvertes.fr/hal-02043531

?. Publications-en-préparation-:--rabah, H. Jardin, J. Briard-bion, V. Maillard, M. B. Gaucher et al., The growth medium influence on in vitro Propionibacterium freudenreichii adaptation ability to caecal environment, Target Journal: Applied Microbiology and Biotechnology

H. Rabah, D. Carmo, and F. , Effect of probiotic Emmental cheese on DSS-induced colitis on mice, Target Journal: Applied Microbiology and Biotechnology

H. Rabah, O. Ménard, F. Gaucher, F. Do-carmo, D. Dupont et al., Cheese matrix protects the immunomodulatory surface protein SlpB of Propionibacterium freudenreichii during in vitro digestion, 12th International Scientific Conference on Probiotics, Prebiotics, Gut Microbiota and Health, 2018.
URL : https://hal.archives-ouvertes.fr/hal-01707548

L. , young scientist award" à IPC2018 pour la présentation orale, ex aequo avec Stefano Colombo (Italie)

, ? Communications par poster

H. Rabah, F. Do-carmo, and G. Jan, Is cheese a good delivery vehicle for Propionibacterium freudenreichii anti-inflammatory property?, Symposium on Propionibacteria and Bifidobacteria, 2016.

H. Rabah, O. Ménard, F. Gaucher, F. Do-carmo, D. Dupont et al., Cheese matrix as a delivery vehicle for immunomodulatory proteins of Propionibacterium freudenreichii, International Probiotics and Antimicrobial Proteins Conference, 2017.

H. Rabah, S. Ferret-bernard, S. Huang, L. Le-normand, F. J. Cousin et al., The delivery vehicle modulates the immunomodulatory effect of Propionibacterium freudenreichii in healthy piglets. The 10th cheese symposium, 2018.
URL : https://hal.archives-ouvertes.fr/hal-01781893

H. Rabah, F. Gaucher, F. Do-carmo, M. B. Maillard, and G. Jan, In vitro assessment of Propionibacterium freudenreichii adaptation to caecal environment. Congrès national de la société française de microbiologie, 2018.

H. Rabah, S. Ferret-bernard, S. Huang, L. Le-normand, F. J. Cousin et al., The cheese matrix modulates the immunomodulatory effect of Propionibacterium freudenreichii in healthy piglets. les 4èmes rencontres du GIS Nutrition Alimentation Métabolisme et Santé, 2018.

, Annexe 5 : publications et communications scientifiques en collaboration ? Articles scientifiques publiées dans des revues à comité de lecture

D. Carmo, F. Rabah, H. Huang, S. Gaucher, F. Deplanche et al.,

V. and J. G. , Propionibacterium freudenreichii Surface Protein SlpB is involved in adhesion to Intestinal HT-29 Cells, Front Microbiol, vol.8, 2017.

S. Huang, H. Rabah, J. Jardin, V. Briard-bion, S. Parayre et al., Hyperconcentrated Sweet Whey, a New Culture Medium That Enhances Propionibacterium freudenreichii stress tolerance, Appl Environ Microbiol, vol.82, issue.15, pp.4641-51, 2016.
URL : https://hal.archives-ouvertes.fr/hal-01454632

S. Huang, S. Méjean, H. Rabah, A. Dolivet, L. Loir et al., , 2016.

, Double use of concentrated sweet whey for growth and spray drying of probiotics: Towards maximal viability in pilot scale spray dryer, Journal of Food Engineering, vol.196, pp.11-17, 2017.

. ?-chapitre-de-livre,

D. Carmo, F. Rabah, H. , F. Cordeiro, B. et al.,

, Applications of Probiotic Bacteria and Dairy Foods in Health. Current Research in Microbiology, Open Access eBooks 919 North Market Street Suite 425 Wilmington, 2017.