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The impact of Functional foods on the gut microbiota is a fundamental question since shifts in bacterial composition are associated with inflammation progression. Therefore, this study investigated the effects of two types of cheeses fermented by probiotic bacteria in C57BL6 mice either in healthy conditions or in dextran sulfate sodium (DSS)-induced colitis. One cheese was fermented by Propionibacterium freudenreichiiCIRM-BIA129. A second kind of cheese was made in industrial conditions to reproduce Emmental but using the above-mentioned strain in combination with Lactobacillus delbrueckiiCNRZ327 and Streptococcus thermophilusLMD-9 as starters. The gut microbiota was investigated by shotgun metagenomic sequencing and the taxonomic and functional profiles were assessed through sequence alignment to NCBI taxonomy and KEGG pathways databases. Our results suggest that both treatments did not disturb the microbial community ecology. Emmental intake increased symbionts as Romboutsia. and Akkermansia muciniphila. Furthermore, metabolic pathway analysis suggests that A. muciniphilamay produce bioactive metabolites as acetate and cooperate with other commensal species to produce indole and gamma-aminobutyric acid. Mice with colitis have restored some of the impaired microbiome metabolic functions when treated with cheese fermented only by Propionibacterium, while the Emmental cheese promoted the increase of Ligilactobacillus murinus. This taxon presented several genes with immunomodulatory activity potential. This study provides insights for engineering functional cheeses to increase beneficial bacteria in the gut. However, further studies are required to investigate their role in regulating the gut-brain axis. Moreover, possible anti-inflammatory mechanisms based on the promotion of Lg. murinusinteraction with the host was suggested.

ABSTRACTThe role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psmmutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureuswild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis.

The presence of Staphylococcus intermediusin food remains unclear because routine laboratory analysis does not discriminate between S. intermediusand Staphylococcus aureus, a major cause of food poisoning. Both species share many phenotypic characteristics, including coagulase and thermonuclease production. In both species, some strains can produce enterotoxin and therefore can be the cause of food poisoning outbreaks. Although the ID32 Staph System (bioMérieux, SA, Marcy l'Etoile, France), based on a miniaturized phenotypic characterization, gives satisfactory results for discriminating between these two species, some rapid molecular PCR-based methods have been developed to identify S. aureusspecifically, but they do not identify S. intermedius. Here, we developed a rapid, accurate, and discriminative multiplex PCR method that targets species-specific sequences in the nucgene, which encodes thermonuclease in the two species. The test includes an internal positive control that targets a highly conserved region of 16S ribosomal RNA gene (rDNA). A total of 116 strains were used to validate our test. The test gave no signal on the following Staphylococcusspecies: S. epidermidis, S. chromogenes, S. hyicus, S. warneri, S. xylosus, S. lentus, and S. sciuri. It allowed a 100% successful discrimination between S. aureus(44 strains tested) and S. intermedius(57 strains) isolated from different origins.

Intranasal immunization in mice with recombinant lactococci expressing the interleukin-12 and the HPV-16 E7 antigen. Interleukin-12 (IL-12), a heterodimeric cytokine, plays an important role in cellular immunity against several bacterial, viral and parasitic infections, and has adjuvant activity when co-delivered with DNA vaccines. IL-12 has also been used with success in cancer immunotherapy treatments. However, systemic IL-12 therapy has been limited by high-level toxicity. Here, we describe the inducible expression and secretion of IL-12 performed in the food-grade model lactic acid bacterium (LAB), Lactococcus lactis. IL-12 was expressed as 2 separate polypeptides (p35-p40), or as a single recombinant polypeptide (scIL-12). Local administration of IL-12 producing strains at the intranasal mucosal surface resulted in an antigen-specific cellular response (i.e., secretion of Th1 cytokines, IL-2 and IFN-?) elicited by a recombinant L. lactis strain displaying a cell wall-anchored HPV-16 E7 antigen was dramatically increased by co-administration with an L. lactis strain secreting IL-12 protein. This work shows that IL-12 is produced and secreted in active form by L. lactis and that this strategy can be used successfully to enhance an antigen-specific immune response and to stimulate local mucosal immunity. L'interleukine-12 (IL-12) est une cytokine h?t?rodim?rique qui joue un r?le important dans la r?ponse immunitaire cellulaire lors d'infections d'origine virale, bact?rienne ou parasitaire. Son adjuvanticit? a ?t? d?montr?e lors de co-administration avec des vaccins ? ADN et son utilisation est envisag?e pour le d?veloppement de vaccins contre diverses maladies infectieuses. Elle a ?galement ?t? utilis?e avec succ?s dans le traitement de cancers par immunoth?rapie. Cependant, son utilisation en traitement syst?mique reste d?licat du fait d'effets secondaires li?s ? une haute toxicit?. Le travail pr?sent? ici d?crit la construction de souches de Lactococcus lactis permettant (i) l'expression monocistronique d'une prot?ine de fusion regroupant les deux sous-unit?s de l'IL-12 et (ii) l'expression bicistronique des deux sous-unit?s, p35 et p40, donnant la forme h?t?rodim?rique native de l'IL-12. Nos r?sultats montrent que L. lactis est capable de s?cr?ter les deux formes d'IL-12. L'analyse des surnageants de culture par ?lectrophor?se en conditions non-d?naturantes et par test ELISA montre que les sous-unit?s sont correctement assembl?es pour former une IL-12 native. Ceci d?montre la capacit? de L. lactis ? former des ponts disulfures. Par ailleurs, nous d?crivons ici la construction d'une souche de L. lactis produisant une forme ancr?e de la prot?ine E7 du papillomavirus de type 16. Des essais de co-administration de souches de L. lactis produisant l'IL-12 et de souches ancrant l'E7 d?montrent l'effet adjuvant de l'IL-12 lors d'immunisation intranasale chez la souris. Ces souches recombinantes de L. lactis pourraient ?tre utilis?es pour la production industrielle de cytokine ou pour l'?laboration de cocktail de souches dans le d?veloppement de vaccins vivants.

Interleukin-12 (IL-12), a heterodimeric cytokine, plays an important role in cellular immunity to several bacterial, viral, and parasitic infections and has adjuvant activity when it is codelivered with DNA vaccines. IL-12 has also been used with success in cancer immunotherapy treatments. However, systemic IL-12 therapy has been limited by high levels of toxicity. We describe here inducible expression and secretion of IL-12 in the food-grade lactic acid bacterium Lactococcus lactis. IL-12 was expressed as two separate polypeptides (p35-p40) or as a single recombinant polypeptide (scIL-12). The biological activity of IL-12 produced by the recombinant L. lactis strain was confirmed in vitro by its ability to induce gamma interferon (IFN-gamma) production by mouse splenocytes. Local administration of IL-12-producing strains at the intranasal mucosal surface resulted in IFN-gamma production in mice. The activity was greater with the single polypeptide scIL-12. An antigen-specific cellular response (i.e., secretion of Th1 cytokines, IL-2, and IFN-gamma) elicited by a recombinant L. lactis strain displaying a cell wall-anchored human papillomavirus type 16 E7 antigen was dramatically increased by coadministration with an L. lactis strain secreting IL-12 protein. Our data show that IL-12 is produced and secreted in an active form by L. lactis and that the strategy which we describe can be used to enhance an antigen-specific immune response and to stimulate local mucosal immunity.

ABSTRACTInterleukin-12 (IL-12), a heterodimeric cytokine, plays an important role in cellular immunity to several bacterial, viral, and parasitic infections and has adjuvant activity when it is codelivered with DNA vaccines. IL-12 has also been used with success in cancer immunotherapy treatments. However, systemic IL-12 therapy has been limited by high levels of toxicity. We describe here inducible expression and secretion of IL-12 in the food-grade lactic acid bacterium Lactococcus lactis.IL-12 was expressed as two separate polypeptides (p35-p40) or as a single recombinant polypeptide (scIL-12). The biological activity of IL-12 produced by the recombinant L. lactisstrain was confirmed in vitro by its ability to induce gamma interferon (IFN-γ) production by mouse splenocytes. Local administration of IL-12-producing strains at the intranasal mucosal surface resulted in IFN-γ production in mice. The activity was greater with the single polypeptide scIL-12. An antigen-specific cellular response (i.e., secretion of Th1 cytokines, IL-2, and IFN-γ) elicited by a recombinant L. lactisstrain displaying a cell wall-anchored human papillomavirus type 16 E7 antigen was dramatically increased by coadministration with an L. lactisstrain secreting IL-12 protein. Our data show that IL-12 is produced and secreted in an active form by L. lactisand that the strategy which we describe can be used to enhance an antigen-specific immune response and to stimulate local mucosal immunity.

Secretion of heterologous proteins of therapeutical interest in Lactococcus lactis . The GRAS (for Generally Regarded As Safe) bacterium, Lactococcus lactis, is a good candidate to produce heterologous proteins of vaccinal, medical or technological interest. The secretion of such proteins would allow a direct contact between the protein and its target and therefore would be a better mode of production than intracellular production. To develop heterologous protein export systems in L. lactis, we used the staphylococcal nuclease. We combined these secretion tools to the nisin-inducible controlled expression system. We first developed two new expression vectors allowing the inducible production of Nuc intra- and extracellularly. We then used them to produce in L. lactis two heterologous proteins of therapeutical interest: (i) the ovine omega interferon which possesses antiviral properties and which will be useful to enhance immune response in the case of the use of L. lactis as antigen delivery vehicle and (ii) a fusion protein between the Blg41-60 epitope and the mature part of Nuc. Lactococcus lactis, bact?rie lactique mod?le, est un micro-organisme GRAS (Generally Regarded As Safe). Sa parfaite innocuit? et le d?veloppement des connaissances sur ses capacit?s de production et de s?cr?tion de prot?ines h?t?rologues en font un bon candidat pour la s?cr?tion de prot?ines d'int?r?t th?rapeutique. Notre ?quipe a d?velopp? de nombreux outils d'optimisation de la s?cr?tion de prot?ines h?t?rologues chez L. lactis que nous avons combin?s au syst?me d'expression inductible par la nisine. Les deux nouveaux vecteurs d'expression d?crits permettent, en fonction des signaux choisis, une expression inductible et une localisation intra- ou extracellulaire de notre prot?ine mod?le, la nucl?ase de Staphylococcus aureus (Nuc). Nous pr?sentons ici deux exemples d'application de ces outils de s?cr?tion ? des prot?ines d'int?r?t th?rapeutique. Les souches d?crites permettent : (i) la production d'interf?ron omega ovin, mol?cule stimulant la r?ponse immunitaire, utile pour d?velopper des vaccins vivants et (ii) la production d'une prot?ine de fusion ?pitope 41-60 de la ?-lactoglobuline:Nuc, utile pour ?tudier les ph?nom?nes allergiques li?s ? la ?-lactoglobuline.

Characterization of host factors affecting heterologous protein secretion in Lactococcus lactis. Lactococcus lactis is the model organism for Lactic Acid Bacteria (LAB) and is a GRAS bacterium. It is therefore a good candidate for the secretion of heterologous proteins of therapeutic interest. In order to investigate new potential uses for LAB, we study host factors involved in the production, the stability and/or the secretion of heterologous proteins in L. lactis. Our strategy uses a random insertional mutagenesis performed on a L. lactis MG1363 derivative strain carrying a uspnuc cassette in its chromosome. The uspnuc cassette encodes a hybrid precursor composed of the signal peptide of the Usp45 secreted protein and the staphylococcal nuclease (Nuc) which is used here as a reporter protein for the secretion process. Nuc activity of bacterial colonies is readily detectable in vivo and this provides an efficient screening for the selection of secretion mutants. We thus selected and analyzed six L. lactis mutants affected in Nuc secretion. Five out of the 6 corresponding genes were identified. Three of them seem to be involved in the production and/or secretion of proteins: 2 genes are homologous to unknown peptidases and one is homologous to a gene located downstream of a transcription regulator. Combination of different strategies should provide new insight of the secretion process in L. lactis and could result in the construction of secretion mutants with enhanced capacities. Lactococcus lactis, bact?rie lactique mod?le, pr?sente une parfaite innocuit? hygi?nique. Elle constitue, de ce fait, un bon candidat pour la s?cr?tion de prot?ines d'int?r?t th?rapeutique. Une partie de nos travaux dans ce domaine concerne la caract?risation des facteurs d'h?te impliqu?s dans la production, la stabilit? et/ou la s?cr?tion de prot?ines h?t?rologues chez L. lactis. La strat?gie employ?e consiste en une mutagen?se par transposition al?atoire sur le chromosome. Cette mutagen?se est men?e sur une souche de L. lactis portant une cassette uspnuc int?gr?e sur le chromosome. La cassette uspnuc code pour un pr?curseur hybride compos? du peptide signal d'Usp45, prot?ine s?cr?t?e par L. lactis, et de la partie mature de la nucl?ase de Staphylococcus aureus (Nuc) utilis?e ici comme prot?ine rapporteur de la s?cr?tion. L'activit? de Nuc, facilement d?tectable in vivo sur les colonies bact?riennes, constitue un crible efficace pour la s?lection de mutants affect?s dans la s?cr?tion. Six mutants de L. lactis affect?s dans la s?cr?tion de Nuc ont ainsi ?t? s?lectionn?s et analys?s. Cinq des 6 g?nes correspondants ont ?t? identifi?s. Trois semblent particuli?rement impliqu?s dans la production et la s?cr?tion de prot?ines : 2 sont homologues ? des g?nes de peptidases inconnues et un est homologue ? un g?ne situ? dans une r?gion potentiellement r?gulatrice d'un facteur de transcription. Diff?rentes approches par mutagen?se devraient d?boucher sur une meilleure connaissance de la machinerie de s?cr?tion de L. lactis et la construction de mutants hyperproducteurs ou hypers?cr?teurs.