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International audience; ContextGut inflammation constitutes a growing health concern in developed countries. It may consist in spontaneous ailments of the gut, involving both the host immune system and microbiota, such as IBD, including ulcerative colitis and Crohn’s disease. It may be caused by a medical treatment, such as mucositis induced by cancer chemotherapy and/or radiotherapy. It coincides with a dysbiosis including a lack of anti-inflammatory bacteria. As an example, propionibacteria are lacking in the microbiota of newborns that develop necrotizing enterocolitis.Methods and resultsWe thus focused on the immunomodulatory properties of GRAS propionibacteria. Selected strains of Propionibacterium freudenreichii induced the regulatory IL-10 cytokine in human immune cells (Foligné et al., 2010, 2013), depending on surface proteins (Le Marechal et al., 2015). Mutation of the slpB gene suppressed this immunomodulatory effect and the resulting slpB mutant induced a rather proinflammatory response (Deutsch et al., 2017). Consumption of wild-type P. freudenreichii protected from colitis induced by both TNBS and by DSS. It alleviated severity of symptoms, modulated local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages (Plé et al., 2015, 2016; Rabah et al., 2020). Accordingly, consumption of Lactococcus lactis NCDO 2118 harboring pXIES-SEC:slpB and expressing the propionibacterial SlpB reduced severity of colitis, lowered weight loss, disease activity index, shortening of the colon length, and histopathological score, compared with mice treated with L. lactis NCDO 2118 wild-type strain.In the context of mucositis induced by the chemotherapy 5-FU, P. freudenreichii prevented weight loss, reduced inflammation and consequently intestinal damages. It regulated key markers, including Claudin-1 and IL-17a genes, as well as IL-12 and IL-1β cytokines levels (Cordeiro et al., 2018). Mutant strain slpB displayed opposite regulatory effect on cld1 expression and on IL-12 levels, and failed to afford protection towards 5-FU-mucositis (do Carmo et al., 2019).ConclusionThis work emphasizes the importance of SlpB in P. freudenreichii ability to reduce both mucositis and colitis inflammation. It opens perspectives for the development of probiotic products aimed at decreasing side effects of chemotherapy and at helping treatment of colitis, thanks to GRAS bacteria.ReferencesCordeiro, B. F., Oliveira, E. R., Silva, D., H, S., Savassi, B. M., Acurcio, L. B., et al. (2018). Whey Protein Isolate-Supplemented Beverage, Fermented by Lactobacillus casei BL23 and Propionibacterium freudenreichii 138, in the Prevention of Mucositis in Mice. Front. Microbiol. 9. doi:10.3389/fmicb.2018.02035.Deutsch, S.-M., Mariadassou, M., Nicolas, P., Parayre, S., Le Guellec, R., Chuat, V., et al. (2017). Identification of proteins involved in the anti-inflammatory properties of Propionibacterium freudenreichii by means of a multi-strain study. Sci Rep 7, 46409. doi:10.1038/srep46409.do Carmo, F. L. R., Rabah, H., Cordeiro, B. F., da Silva, S. H., Pessoa, R. M., Fernandes, S. O. A., et al. (2019). Probiotic Propionibacterium freudenreichii requires SlpB protein to mitigate mucositis induced by chemotherapy. Oncotarget 10, 7198–7219. doi:10.18632/oncotarget.27319.Foligné, B., Breton, J., Mater, D., and Jan, G. (2013). Tracking the microbiome functionality: focus on Propionibacterium species. Gut 62, 1227–1228.Foligné, B., Deutsch, S. M., Breton, J., Cousin, F. J., Dewulf, J., Samson, M., et al. (2010). Promising immunomodulatory effects of selected strains of dairy propionibacteria as evidenced in vitro and in vivo. Appl.Environ.Microbiol. 76, 8259–8264.Le Marechal, C., Peton, V., Ple, C., Vroland, C., Jardin, J., Briard-Bion, V., et al. (2015). Surface proteins of Propionibacterium freudenreichii are involved in its anti-inflammatory properties. J.Proteomics. 113C, 447–461.Plé, C., Breton, J., Richoux, R., Nurdin, M., Deutsch, S.-M., Falentin, H., et al. (2016). Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains: Reverse engineering development of an anti-inflammatory cheese. Mol Nutr Food Res 60, 935–948. doi:10.1002/mnfr.201500580.Plé, C., Richoux, R., Jardin, J., Nurdin, M., Briard-Bion, V., Parayre, S., et al. (2015). Single-strain starter experimental cheese reveals anti-inflammatory effect of Propionibacterium freudenreichii CIRM BIA 129 in TNBS-colitis model. Journal of Functional Foods 18, 575–585. doi:10.1016/j.jff.2015.08.015.Rabah, H., do Carmo, F. L. R., Carvalho, R. D. de O., Cordeiro, B. F., da Silva, S. H., Oliveira, E. R., et al. (2020). Beneficial Propionibacteria within a Probiotic Emmental Cheese: Impact on Dextran Sodium Sulphate-Induced Colitis in Mice. Microorganisms 8, 380. doi:10.3390/microorganisms8030380.- PF induces IL-10 in human PBMCs- Surface layer extraction reduces IL-10 induction- Extracted surface layer induces IL-10- Mutation of the SlpB gene reduces IL-10 induction- PF prevents colitis- PF prevents mucositis- PF slpB mutant does not prevent mucositis- Lactococcus lactis expressing SlpB prevents colitis

International audience; IntroductionGut inflammation constitutes a growing health concern in developed countries. It may consist in spontaneous ailments of the gut, involving both the host immune system and microbiota, such as IBD, including ulcerative colitis and Crohn’s disease. It may be caused by a medical treatment, such as mucositis induced by cancer chemotherapy and/or radiotherapy. It coincides with a dysbiosis including a lack of anti-inflammatory bacteria. As an example, propionibacteria are lacking in the microbiota of newborns that develop necrotizing enterocolitis.Methods Dairy propionibacteria strains were tested in vitro on human PBMCs with respect to their ability to induce immunomodulatory cytokines. Selected strains were then tested in vitro with respect to their ability to protect mice from TNBS-induced colitis, from DSS-induced colitis, and from 5FU-induced mucositis.ResultsWe thus focused on the immunomodulatory properties of GRAS propionibacteria. Selected strains of Propionibacterium freudenreichii induced the regulatory IL-10 cytokine in human immune cells (Foligné et al., 2010, 2013), depending on surface proteins (Le Marechal et al., 2015). Mutation of the slpB gene suppressed this immunomodulatory effect and the resulting slpB mutant induced a rather proinflammatory response (Deutsch et al., 2017). Consumption of wild-type P. freudenreichii protected from colitis induced by both TNBS and by DSS. It alleviated severity of symptoms, modulated local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages (Plé et al., 2015, 2016; Rabah et al., 2020). Accordingly, consumption of Lactococcus lactis NCDO 2118 harboring pXIES-SEC:slpB and expressing the propionibacterial SlpB reduced severity of colitis, lowered weight loss, disease activity index, shortening of the colon length, and histopathological score, compared with mice treated with L. lactis NCDO 2118 wild-type strain (Belo et al., 2021).In the context of mucositis induced by the chemotherapy 5-FU, P. freudenreichii prevented weight loss, reduced inflammation and consequently intestinal damages. It regulated key markers, including Claudin-1 and IL-17a genes, as well as IL-12 and IL-1β cytokines levels (Cordeiro et al., 2018). Mutant strain slpB displayed opposite regulatory effect on cld1 expression and on IL-12 levels, and failed to afford protection towards 5-FU-mucositis (do Carmo et al., 2019).P. freudenreichii was further shown to produce extracellular vesicles (EVs), which mimic the immunomodulatory features of propionibacteria in vitro by modulating NF-κB transcription factor activity and IL-8 release in cultured human intestinal epithelial cells (Rodovalho et al., 2020). Proteomic analysis revealed presence of SlpB in these EVs.DiscussionThis work emphasizes the importance of SlpB in P. freudenreichii ability to reduce both mucositis and colitis inflammation. It opens perspectives for the development of probiotic products aimed at decreasing side effects of chemotherapy and at helping treatment of colitis, thanks to GRAS bacteria.

Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines, mitigates the inflammatory response in vitro and in vivo These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e. modulating NF-κB transcription factor activity and IL-8 release) which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and hence the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical and functional properties of EVs produced from cells cultivated on laboratory Yeast Extract Lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant, smaller in diameter and displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacteria interactions.Importance Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in host-microbe interactions. Recently we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems, and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

The role of Propionibacterium freudenreichii in mitigating inflammation has been a subject of study for many years. Lately, it has been shown that immunomodulation properties are strain- specific and that the major responsible for inflammatory modulation ofimmunomodulation by strain CIRM-BIA129 is the presence of the surface layer protein B (SlpB), which has presented immunomodulation even when expressed in other bacteria. Extracellular vesicles (EVs) are nanosized spherical structures, produced by organisms of all kingdoms, including bacteria. They have been associated with inter-organism communication, pathogenesis, competition, and immunomodulation. Recent studies were aiming to address the role of EVs, in the probiotic effects of bacteria. The properties of P. freudenreichii CIRM-BIA129- derived EVs have been investigated. EVs produced by CIRM-BIA129 cultured in milk ultrafiltrate medium (UF) have been characterized by regarding size and morphology. UF-derived EVs displayed a monodisperse pattern with a modal size of 84.80 ± 2.34 nm. They are composed of a wide variety of proteins, mainly involved in metabolic processes, cellular processes and signaling, and storage and processing of information. A, among these proteins, SlpB was found in high abundance. P. freudenreichii EVs were able to inhibit, in a dose dependent manner, the increase of IL-8 production in HT-29 cells induced with LPS, due to NF-KB pathway inhibition, without causing cell cytotoxicity. EVs produced by a CIRM-BIA129 mutant strain with a knockout for SlpB showed a less efficient reduction in IL-8 production. Results have shown that the environmental conditions are able to modify EVs content and, consequently, their immunomodulatory effects. A change in the growth medium, from UF to YEL (yeast extract-lactate) showed a lower production of EVs, with a slightly larger size. EVs produced in YEL did not perform as well in the inhibition of the NF-KB pathway and had no effect on IL-8 production. Recent results have shown that P. freudenreichii EVs were able to protect Caco-2 cells from inflammation-induced excessive increasing permeability. These Altogether, these results show that Evs produced by beneficial propionibacteria are able to generate immunomodulationtrigger immunomodulation, similar to the parental strain. As for the SlpB role in EVs immunomodulation

In a general context of food transition, plant-based fermented products experience a considerable development. This results from evolution of consumers’ habits, demanding healthy products with a low environmental impact. This requires research and development, and thus higher scientific knowledge. Fermented soy milks constitute a favorable alternative source of live probiotics. Lactobacillus delbrueckii subsp. bulgaricus and Propionibacterium freudenreichii are extensively used in probiotic dairy products. Their adaptation to the dairy environment has been extensively studied and documented. However, little is known about their adaptation to the soy environment. We investigated suitability of soymilk as a substratefor growth and acidification by Lactobacillus delbrueckii CIRM-BIA1592 and Propionibacterium freudenreichii CIRM-BIA129. A specific focus was made on changes in term of proteome composition, cellular morphology in comparison with the cultivation in cow milk. In order to understand such adaptation, we explored their growth and survival in soy milk ultrafiltrate and compared it to cow’s milk ultrafiltrate. The two species grew in cow milk,yet not in soy milk. P. freudenreichii did grow in soy milk when co-cultured with the lactic acid bacterium L. plantarum while L. delbrueckii did not, even in co-culture with S. thermophilus. Our results revealed major differences in bacterial morphology and proteome composition. Lactobacilli upregulated stress proteins, and downregulated cell cycle and division machinery. Regarding propionibacteria, differential expression of proteins involved in amino acid metabolism, in carbohydrate metabolism, and in stress remediation was observed. These results suggest that changing the fermented substrate may significantly affect both technological and probiotic properties of conventional dairy starters. This should be considered for the development of new fermented functional foods. Screening of a large collection of lactic and propionic acid bacteria is in progress, based on their technological and probiotic properties. Bacterial stains and consortia thereof will be selected for the production of new plant-based fermented functional foods

International audience; L’intégrité de la barrière épithéliale intestinale (BEI) joue un rôle central dans la pathogenèse des maladies inflammatoires chroniques de l’intestin (MICI). Un intérêt grandissant est porté aux composés alimentaires capables de renforcer ou de restaurer la BEI. C’est le cas de souches de Propionibacterium freudenreichii, une bactérie propionique laitière, capable de restaurer la BEI fragilisée dans un contexte inflammatoire. Egalement, il existe une protection locale de la BEI exercée par les produits du métabolisme des acides gras polyinsaturés oméga-6 (AGPI n-6) par les cellules intestinales. Les omégas 6 sont apportés par l’alimentation, dont le lait. Les enzymes de leur métabolisme sont sous-exprimés chez les patients atteints de MICI, empêchant l’action bénéfique d’une supplémentation en AGPI n-6 chez ces patients. L’augmentation de la biodisponibilité des dérivés AGPI n-6 reste une piste intéressante. Par ailleurs, certaines souches de P. freudenreichii, sont capables de convertir l’acide linoléique, un AGPI n-6, en acide linoléique conjugué (ALC), capable de prévenir des colites induites chez des souris. Ces souches pourraient donc moduler la biodisponibilité et le métabolisme des AGPI n-6. Notre objectif vise à rechercher des effets additifs voire synergiques entre l’effet de 1) P. freudenreichii et 2) des AGPI n-6 sur la BEI. Un criblage de trente-trois souches de P. freudenreichii a été réalisé basé sur les critères suivants : 1) caractérisation de la capacité à induire la production de cytokines anti-inflammatoire (IL-10) par stimulation de PBMCs humaines 2) identification de protéines de surface (S-layer protein) responsable des propriétés anti-inflammatoire par une technique de rasage enzymatique suivis d’une identification par MS/MS 3) mesure de l’activité lipolytique dans la matière grasse laitière et de la conversion de l’acide linoléique en ALC avec une identification des lipides par GC-MS. Toutes les souches sont capables d’induire la production d’IL-10. Seulement, la puissance d’induction d’IL-10 est souche dépendante. Sur les trente-trois souches, dix présentent un profil S-layer anti-inflammatoire. Toutes les souches sont capables de lipolyser la matière grasse laitière, certaines produisent de l’acide linoléique. Toutes les souches exceptées une, sont capable de convertir l’acide linoléique en ALC. De façon similaire aux profils des cytokines, la production d’ALC est souche dépendante. Ces résultats apportent de nouvelles connaissances sur P. freudenreichii, et vont permettre sélectionner des souches qui, ayant un potentiel anti-inflammatoire ainsi que pro-réparateur, seront testées in vivo pour réduire la colite, avec ou sans supplémentation en AGPI n-6.

International audience; Introduction and Aims. Inflammatory Bowel Diseases (IBD), including Ulcerative Colitis (UC), coincide with alterations in the gut microbiota. Consumption of immunomodulatory strains of probiotic bacteria may induce or prolong remission in UC patients. Fermented foods, including cheeses, constitute major vectors for bacteria consumption. New evidences revealed anti-inflammatory effects of selected strains within the species P.freudenreichii, S.thermophilus and L.delbrueckii. We thus hypothesized that consumption of a functional cheese, fermented by selected strains of these three species, may exert a positive effect on IBD.Methods. Strains of each bacterial species were screened based on the ability to induce regulatory IL-10 in human immune PBMC cells, or to downregulate nuclear factor kappa B (NF-κB) activation in a cultured HT-29 human intestinal epithelial cell line. We then investigated the effect of monostrain experimental cheese. We further investigated the impact two-strains experimental cheese. Finally, we produced in industrial conditions an Emmental cheese using one selected strain of each of the three species, i.e. P. freudenreichii CIRM-BIA 129 in combination with Lactobacillus delbrueckii CNRZ327 and Streptococcus thermophilus LMD-9. Consumption of all cheeses was investigated with respect to prevention of chemically induced colitis in mice.Results. Consumption of the experimental cheeses, or of the industrial Emmental, reduced the severity of subsequent chemically induced colitis, weight loss, disease activity index and histological score, in mice. Furtherrmore, consumption of the Emmental cheese, in a preventive way, reduced small bowel Immunoglobulin A (IgA) secretion, restored occludin gene expression and prevented induction of Tumor Necrosis Factor α (TNFα), Interferon γ (IFNγ) and Interleukin-17 (IL-17).Conclusion. Assembling immunomodulatory strains of both lactic acid and propionic acid starter bacteria leads to an anti-inflammatory Emmental cheese, as revealed in vivo. This opens new perspectives for the development of functional fermented food products for personalised nutrition in the context of IBD.

International audience; Introduction Propionibacterium freudenreichii, is a probiotic bacterium, with an established immunomodulatory role. Selected strains indeed induce the release of regulatory IL-10. Extractable surface proteins of the S-layer type (Slp) are involved in such modulation, but the exact mode of interaction with the host remains unknown. Mucositis is a common side effect of cytotoxic chemotherapy characterized by mucosal injury, inflammation, diarrhea, and weight loss. We hypothesized that immunomodulatory P. freudenreichii may mitigate inflammation and that SlpB may be required for such an effect.Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. We hypothesized that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs and thus export immunomodulatory proteins to interact with the host.MethodsP. freudenreichii CIRM-BIA 129, previously selected for its ability to induce IL-10 release by PBMCs, was administered orally to mice, prior to colitis induction using TNBS.The slpB gene was inactivated by insertional inactivation. Both parental wild type and mutant strains were administered orally to mice, prior to induction of mucositis using 5-FU. The SlpB protein was purified and its effect on cultured human intestinal epithelial cells investigated with respect to cytokines expression.EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, prior to physicochemical and functional characterization.ResultsConsumption of P. freudenreichii CIM-BIA129 strain protected mice against TNBS-induced colitis in mice, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages.This strain prevented 5-FU-induced mucositis in mice, regulated key markers, including the expression of Claudin-1 (Cld1) and IL-17a (Il17a), as well as the levels of IL-12 and IL-1β cytokines.The slpB gene was mutated by insertional inactivation and the absence of SlpB protein in the resulting mutant was verified. The mutant strain displayed opposite immunomodulatory effect and failed to mitigate induced mucositis.In HT-29 human intestinal epithelial cells P. freudenreichii reduced expression of IL-8 and TNF-α cytokines in LPS-stimulated cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. In the same cells, purified SlpB was shown to induce expression of IL-10 in a dose-dependent manner.Extracellular vesicles (EVs), they were purified from cell-free culture supernatants of P. freudenreichii. They showed typical shapes and sizes of EVs and contained a broad range of proteins, including SlpB. EVs modulated inflammatory responses, IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells, as well as the NF-κB pathway.DiscussionThis work emphasizes the importance of SlpB in immunomodulatory P. freudenreichii. It constitutes the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. It opens perspectives for the development of functional foods and other bioactive products in order to help preventing and/or treating inflammatory diseases, and to decrease side effects of chemotherapy.

The present invention relates to the field of probiotics. More particularly, the present invention relates to a composition comprising at least one bacterial strain pertaining to the genus Propionibacterium for use in the treatment or prevention of mucositis in a subject, preferably for the treatment or prevention of the alteration of tight junctions of epithelial tissue associated with mucositis.; La présente invention concerne le domaine des probiotiques. Plus particulièrement, la présente invention concerne une composition comprenant au moins une souche bactérienne appartenant au genre Propionibacterium pour une utilisation dans le traitement ou la prévention de la mucite chez un sujet, de préférence pour le traitement ou la prévention de l'altération des jonctions serrées du tissu épithélial associé avec mucite.

National audience

Mucositis, a common and often debilitating side effect of cancer chemotherapy, is a clinically important gastrointestinal inflammatory disease. It consists in a painful inflammation and ulceration of the mucosa lining the digestive tract, which may compromise proper nutrition of the patient, as well as termination of the treatment. It thus increases mortality and morbidity and contributes to rising health care costs. Its treatment is mainly supportive and often fails to relieve symptoms. Several studies indicate a role of the microbiota and suggest a healing effect of probiotic ingestion. Probiotic bacteria may maintain homeostasis and reduce side effects of chemotherapy toxicity. A major limit to probiotic bacteria anti-inflammatory effect is their susceptibility to digestive stresses and several studies indicate the key protective role of food matrices in increasing probiotics’ effect via the protection of bacteria against digestive constraints. In this study, we investigated the role of fermented milk, using Lactobacillus casei or Propionibacterium freudenreichii as probiotic starter, and of its fortification via addition of whey proteins.Matériels et Méthodes/Materials and methodsWe evaluated the role of whey protein isolate (WPI), when added to skim milk fermented by L. casei BL23 or by P. freudenreichii 138, as a protective matrix against in vitro stress challenges. In addition, we investigated in vivo the therapeutic effect of these fermented beverages in a murine model of mucositis induced by 5-Fluorouracil (5-FU). The monitored outcomes included weight loss, ileum histopathological score, villus height, crypt depth and number of mucus-producing goblet cells.Résultats principaux/Main ResultsThis study demonstrated that milk supplementation with 30% (w/v) of WPI increases the survival rate of both strains when challenged with acid, bile salts, high temperature and cold storage stresses, compared to fermented skim milk without the addition of WPI. Moreover, treatment with the probiotic beverages prevented weight loss and intestinal damages in mice receiving 5-FU. All symptoms of mucositis were drastically reduced by the consumption of developed probiotic fermented milks.ConclusionThis study evidenced the protective effect of selected strains of both lactic and propionic acid bacteria, in the context of induced mucositis. It confirmed that selected strains might be used bot as starter and probiotic. It opens new avenues for the development of fermented functional foods for target populations.

Introduction Propionibacterium freudenreichii (PF) is a Swiss-type cheeses starter and has versatile probiotic properties due to various beneficial metabolites. PF recently emerged as a suitable candidate to develop fermented functional foods. Delivery of viable bacteria to the colon is crucial for in situ beneficial metabolites production. The delivery vehicle was shown to influence P. freudenreichii tolerance towards digestive stresses. It is also suggested to influence PF adaptation to the colon environment. In addition, during digestion, the delivery vehicle may prevent or allow proteolysis of surface proteins. These surface proteins are generally thought to contribute to bacteria tolerance towards environmental stresses. Aim This study aimed at evaluating PF (strain CIRM-BIA 129) adaptation to the caecal environment, according to the delivery vehicle. Besides, we evaluated PF survival in the caecal environment after removal of bacterial surface proteins by enzymatic proteolysis. We address the plausible role of S-layer protein SlpB in PF adaptation and survival. Methods Caecal medium was prepared from caecal contents recovered from piglets. PF pellets were harvested from cultures in Yeast Extract-Lactate (YEL), in Milk ultrafiltrate, or from cheese. Wild-type and SlpB KO mutant PF were further compared with respect to survival in caecal medium. Bacterial pellets were also submitted to surface proteins proteolysis to mimic digestion. Cultures were incubated 50 h at 37°C in anaerobiosis. Survival was monitored by plate counting, optical density and by live/dead fluorescence labeling. Results PF survival decreased in the caecal medium with a similar rate, whatever the growth medium tested. However, optical density was stable and the epifluorecence images did not show dead bacteria. Reduced survival rate was not due to significant cell death.PF seemed rather to enter a viable but nonculturable state in the caecal medium. Surprisingly, proteolytic removal of surface layer proteins enhanced PF culturable state in caecal medium. In addition, the mutation of SlpB did not change PF culturable state but surface proteins proteolysis enhanced also PF KO SlpB s culturable state. Conclusion These results indicate that surface proteins proteolysis during digestion may enhance PF survival and metabolic activity within the caecal medium. A comparative proteomic analysis will address the molecular mechanisms involved in enhanced survival.

Thèse en Cotutelle : Université Fédérale du Minas Gerais; Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese ripening starter, with the GRAS status (Generally Recognized As Safe). P. freudenreichii has revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. The anti- inflammatory effect is however highly strain-dependent and due, at least in part, to key surface compounds favouring probiotic effects. Gram-positive bacteria, including P. freudenreichii, can be covered with an external proteinaceous layer called a surface-layer paracrystalin layer and formed by the self-assembly of surface-layer-proteins (Slps). Slps were shown, in different bacteria, to be involved in several probiotics traits, such as adhesion to host cells and mucus, persistence within the gut, or immunomodulation. The aim of this study is to investigate, in a P. freudenreichii probiotic strain, the surface protein that plays the main role in the probiotic interaction with the host. The P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties and possesses several Slps, including SlpB. In the presented work, inactivation of the corresponding gene, CB129ΔslpB a mutant strain, caused a drastic decrease in adhesion to intestinal epithelial HT-29 cells, further evidencing the key role of Slps in cell adhesion. we investigated immune response of HT-29 cells towards P. freudenreichii CIRM-BIA 129 and CB129ΔslpB. The wild type strain mainly induced expression of the immunomodulatory IL-10 by the cells. Interestingly, the mutant strain induced decreased expression of IL-10, yet increased expression of IL-8. To finally evaluate the role of the key surface protein SlpB in the ant-inflammatory property of P. freudenreichii, we examined both strains in a murine mucositis model induced by 5-Fluorouracil (5-FU). P. freudenreichii wild type strain was able to prevent weight loss, to preserve villous architecture and to preserve secretory granules density inside Paneth cells in mucositis–affected mice. By contrast, the mutant CB129ΔslpB strain failed to exert such protective effect. This study evidenced the involvement of P. freudenreichii SlpB protein in its anti-inflammatory properties and confirmed the versatile role fulfilled by Slps in probiotic host interactions.; Propionibacterium freudenreichii est une bactérie Gram-positive bénéfique, traditionnellement utilisée comme levain d’affinage fromager, qui bénéficie du statut GRAS (Generally Recognized As Safe). P. freudenreichii a révélé un effet immunomodulateur qui a été confirmé in vivo par la capacité à protéger des souris d’une colite aigüe induite. L’effet anti-inflammatoire est cependant hautement souche-dépendant. Il est dû, au moins en partie, à des composés de surface clés qui favorisent ces effets probiotiques. Les bactéries Gram- positives, y compris P. freudenreichii, peuvent être recouvertes d’une couche extérieure protéique, appelée « surface-layer », paracristalline, et formée par l’autoassemblage de protéines dites de S-layer (Slps). Les Slps, dans différentes bactéries, sont impliquées dans plusieurs caractéristiques probiotiques, telles que l’adhésion aux cellules de l’hôte et au mucus, la persistance dans l’intestin, ou encore l’immunomodulation. Le but de cette étude est d’étudier, chez une souche probiotique de P. freudenreichii, la protéine de surface qui joue le principal rôle dans les interactions probiotiques avec l’hôte. La souche P. freudenreichii CIRM-BIA 129, récemment reconnue comme immunomodulatrice prometteuse, possède plusieurs protéines de surface Slps, y compris SlpB. Dans la présente étude, l’inactivation du gène correspondant, dans la souche mutante CB129ΔslpB, a provoqué une baisse drastique de l’adhésion aux cellules intestinales épithéliales HT-29, confirmant le rôle clé des Slps dans l’adhésion aux cellules. P. freudenreichii CIRM-BIA 129. Nous avons étudié la réponse immune de cellules HT-29 stimulées par le sauvage ou par le mutant. Le sauvage induisait principalement l’expression de la cytokine immunomodulatrice IL-10. Le mutant induisait, de façon intéressante, moins d’IL-10, mais plus d’IL-8. Afin d’évaluer the rôle de cette protéines clé SlpB dans les effets antiinflammatoires de P. freudenreichii, nous avons testé ces deux souches dans un modèle murin de mucosite induite par le 5-Fluorouracile. La consommation de la souche sauvage prévenait la perte de poids, préservait l’architecture des villosités, ainsi que la densité des granules de sécrétion au sein des cellules de Paneth, chez les souris atteintes de mucosite. Cependant, le mutant n’avait pas cet effet protecteur. Cette étude a mis en évidence l’implication de la protéine SlpB de P. freudenreichii dans les propriétés anti-inflammatoires de cette bactérie et confirme le rôle versatile joué par les Slps dans les interactions probiotique/hôte.

Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese ripening starter, with the GRAS status (Generally Recognized As Safe). P. freudenreichii has revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. The anti-inflammatory effect is however highly strain-dependent and due, at least in part, to key surface compounds favouring probiotic effects. Gram-positive bacteria, including P. freudenreichii, can be covered with an external proteinaceous layer called a surface-layer paracrystalin layer and formed by the self-assembly of surface-layer-proteins (Slps). Slps were shown, in different bacteria, to be involved in several probiotics traits, such as adhesion to host cells and mucus, persistence within the gut, or immunomodulation. The aim of this study is to investigate, in a P. freudenreichii probiotic strain, the surface protein that plays the main role in the probioticinteraction with the host. The P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties and possesses several Slps, including SlpB. In the presented work, inactivation of the corresponding gene, CB129¿slpBa mutant strain, caused a drastic decrease in adhesion to intestinal epithelial HT-29 cells, further evidencing the key role of Slps in cell adhesion. we investigated immune response of HT-29 cells towards P. freudenreichii CIRM-BIA 129 and CB129¿slpB. The wild type strain mainly induced expression of the immunomodulatory IL-10 by the cells. Interestingly, th; Propionibacterium freudenreichii est une bactérie Gram-positive bénéfique, traditionnellement utilisée comme levain d’affinage fromager, qui bénéficie du statut GRAS (Generally Recognized As Safe). P. freudenreichii a révélé un effet immunomodulateur qui a été confirmé in vivo par la capacité à protéger des souris d’une colite aigüe induite. L’effet anti-inflammatoire est cependant hautement souche-dépendant. Il est dû, au moins en partie, à des composés de surface clés qui favorisent ces effets probiotiques. Les bactéries Gram-positives, y compris P. freudenreichii, peuvent être recouvertes d’une couche extérieure protéique, appelée « surface-layer », paracristalline, et formée par l’autoassemblage de protéines dites de S-layer (Slps). Les Slps, dans différentes bactéries, sont impliquées dans plusieurs caractéristiques probiotiques, telles que l’adhésion aux cellules de l’hôte et au mucus, la persistance dans l’intestin, ou encore l’immunomodulation. Le but de cette étude est d’étudil’immunomodulation. Le but de cette étude est d’étudier, chez une souche probiotique de P. freudenreichii, la protéine de surface qui joue le principal rôle dans les interactions probiotiques avec l’hôte. La souche P. freudenreichii CIRM-BIA 129, récemment reconnue comme immunomodulatrice prometteuse, possède plusieurs protéines de surface Slps, y compris SlpB. Dans la présente étude, l’inactivation du gène correspondant, dans la souche mutante CB129¿slpB, a provoqué une baisse drastique de l’adhésion aux cellules intestinales épithéliales HT-29, confirmant le rôle clé des Slps dans l’adhési

INTRODUCTION Propionibacterium freudenreichii is an emergent probiotic bacterium, belonging to dairy propionibacteria, within the group of Actinomycetales, and possessing the GRAS (Generally Recognized As Safe, USA, FDA) and QPS (Qualified Presumption of Safety, EFSA, European Union) status. It is a traditional Swiss-type cheeses starter and an emergent probiotic, exerting several beneficial effects, including anti-inflammatory modulation of gut inflammation. This feature relies on several metabolites and on surface proteins including in particular the surface protein SlpB. METHOD In the present study, we assessed first the impact of proteolytic degradation of SlpB on its immunomodulatory effect on colon epithelial HT-29 cells. Intact P. freudenreichii cells, purified surface layer proteins (SLPs), as well as trypsin-proteolyzed SLPs, were used to stimulate HT-29 cells, in the presence or absence of pro-inflamatory LPS. Cytokines were then quantified to evaluate the inflammatory response. We then investigated the potential of cheese matrix as a delivery vehicle of anti-inflammatory P. freudenreichii MAMP to the colon, by comparing dairy liquids matrices to a solid Swiss-type cheese matrix, using two in vitro digestion models. Integrity of SlpB was monitored using western blotting. RESULTS In this study, we firstly investigated the relevance to avoid SlpB digestive proteolysis, by comparing the effect of i) P. freudenreichii CIRM-BIA 129, ii) its native SLPs, or iii) peptides resulting from SLPs stimulations, with respect to modulation of HT-29 cells response to lipopolysaccharide (LPS) challenge. The anti-inflammatory effect exerted by P. freudenreichii CIRM-BIA 129 and by native surface proteins (SLPs) on HT-29 cells was abolished by digestive proteolysis. This result confirmed the importance to protect immunomodulatory surface proteins from digestive proteolysis in order to allow gut immune system modulation. Thus, we examined the effect of dairy matrices on P. freudenreichii viability and on SlpB integrity during digestion. In comparison with liquid matrices, the cheese matrix provided an enhanced tolerance to digestive stresses and protection of SlpB towards proteolysis, during two in vitro digestion models: static and dynamic. CONCLUSION These in vitro results provide new insights into the matrix effect on P. freudenreichii probiotic functionalities. The cheese matrix offers significant protection against the digestion stresses by enhancing survival, and by protecting surface layer proteins from proteolysis. However, in vivo investigations are necessary to study the matrix effect during digestion, to follow SLPs expression and to evaluate the matrix effect on immunomodulation by P. freudenreichii within the gut. Such data accordingly will allow the development of new functional foods for the delivery of P. freudenreichii to the gastrointestinal tract of humans in the aim to help prevention or treatment of life-style related diseases with an inflammatory component.

Some Gram-positive bacteria, including probiotic ones, are covered with an external proteinaceous layer called a surface-layer. Described as a paracrystalline layer and formed by the self-assembly of a surface-layer-protein (Slp), this optional structure is peculiar. The surface layer per se is conserved and encountered in many prokaryotes. However, the sequence of the corresponding Slp protein is highly variable among bacterial species, or even among strains of the same species. Other proteins, including surface layer associated proteins (SLAPs), and other non-covalently surface-bound proteins may also be extracted with this surface structure. They can be involved a various functions. In probiotic Gram-positives, they were shown by different authors and experimental approaches to play a role in key interactions with the host. Depending on the species, and sometime on the strain, they can be involved in stress tolerance, in survival within the host digestive tract, in adhesion to host cells or mucus, or in the modulation of intestinal inflammation. Future trends include the valorization of their properties in the formation of nanoparticles, coating and encapsulation, and in the development of new vaccines.

Propionibacterium freudenreichii is an emergent probiotic, presenting several beneficial effects including anti-inflammatory properties, mediated by surface layer proteins (SLPs) belonged to the S-layer lattice, in particularly the protein SlpB. We hypothesize cheese matrix may be the best delivery vehicle for P. freudenreichii’s anti-inflammatory potential, by enhancing propionibacteria survival to digestives stresses, and by allowing undamaged SLPs to reach the digestive tract. Firstly, we compared the immunomodulatory effects of P. freudenreichii and intact SLPs, to SLPs digested by trypsin, i.e SLPs-peptides on HT29-cells. In contrast to P. freudenreichii and SLPs, SLPs peptides don’t reduce pro-inflammatory cytokines expression during cells co-stimulation with lipopolysaccharide. This result confirmed the importance to protect SLPs from proteolysis to permit them to trigger gut immune system. Secondly, we evaluated propionibacteria resistance and slpB proteolysis during in vitro static digestion, in different delivery vehicles increasingly concentrated in dairy proteins: milk ultrafiltrate, milk and cheese. The experiment clearly showed the protective effect of the Slp proteins by the cheese matrix and better bacterial viability. Then, we carried out in vitro a dynamic digestion using Didgi® system. We selected two matrices only: the milk ultrafiltrate and the cheese. We decided to apply fixed digestion parameters for both matrices: the digestive parameters of cheese. The results show a protective effect of the cheese matrix on the viability of the propionic bacteria and on SLPs against digestive proteolysis. Taken together, those results show that cheese is an adequate delivery vehicle for P. freudenreichii immunomodulatory proteins.

The intestinal microbiota composition has a great impact on physiology and health, since commensal bacteria are crucial to maintain homeostasis and immune regulation of the gut. Consequently, disturbances of this microbiota, a process known as dysbiosis, have severe implications for the host health such as the rise of many gastrointestinal (GI) problems; including inflammatory disorders like the Inflammatory Bowel Diseases (IBD), mucositis, as well as colorectal cancer (CRC). The consumption of probiotics with beneficial effects is a promising tool to help treating such disorders. Indeed, they modulate diverse biological mechanisms involved in GI homeostasis and have been commonly used to reduce such disorders. In this chapter, we present the molecular mechanisms triggered by probiotic bacteria to modulate the gut physiology during gastrointestinal disorder and the importance of the gastrointestinal stresses tolerance as a limiting factors for probiotic application. Moreover, we focus on the emergence of functional probiotic foods, which can act as excellent vehicles, by enhancing stress tolerance and providing a protective matrix towards digestive stresses.

Propionibacterium freudenreichii is a beneficial Actinobacterium, (GRAS & QPS status). It is exploited in the food industry for vitamins production and in Swiss type cheeses manufacturing, such as Emmental (1). Dairy propionibacteria have several probiotic features, including strain-dependent anti-inflammatory properties (Interleukine 10 induction by immune cells) (2). Previous studies showed surface protein (S- layer proteins) are involved in these properties (2), particularly the protein SlpB (Data no published).A monoxenic cheese, fermented by P.freudenreichii only, was developed. Consumption of this cheese reduced severity of chemically induced colitis in mice model (3). This shows the probiotic potential of P.freudenreichii for helping of Inflammatory Bowel Diseases (IBD) treatment. We assume cheese may be the best delivery vehicle of anti-inflammatory potential of P.freudenreichii to digestive tract. Indeed, we presume cheese enhances propionibacteria resistance to stress conditions during digestion. The presence of caseins matrix may exert a protective effect, allowing undamaged bacteria and surface proteins to reach the digestive tract.The experimental strategy chosen was to evaluate propionibacteria resistance and slpB protein proteolysis during in vitro digestion, in different delivery vehicles increasingly concentrated in dairy proteins: culture on milk ultrafiltrate (UF), culture on milk and a monoxenic cheese. P.freudenreichii viability and S-layer proteins susceptibility to proteolysis was followed in the different matrix during in vitro digestion. Two anti-inflammatory strains were used for this experiment: CIRM-BIA 129 and CIRM-BIA 118 (4).This will be correlated with in vivo experiments in the perspective to promote dairy products fermented by propionibacteria as probiotics for target populations.