Ability of Lactobacillus fermentum to overcome host α-galactosidase deficiency, as evidenced by reduction of hydrogen excretion in rats consuming soya α-galacto-oligosaccharides

<p>Abstract</p> <p>Background</p> <p>Soya and its derivatives represent nutritionally high quality food products whose major drawback is their high content of α-galacto-oligosaccharides. These are not digested in the small intestine due to the natural absence of tissular α-galactosidase in mammals. The passage of these carbohydrates to the large intestine makes them available for fermentation by gas-producing bacteria leading to intestinal flatulence. The aim of the work reported here was to assess the ability of α-galactosidase-producing lactobacilli to improve the digestibility of α-galacto-oligosaccharides <it>in situ</it>.</p> <p>Results</p> <p>Gnotobiotic rats were orally fed with soy milk and placed in respiratory chambers designed to monitor fermentative gas excretion. The validity of the animal model was first checked using gnotobiotic rats monoassociated with a <it>Clostridium butyricum </it>hydrogen (H₂)-producing strain. Ingestion of native soy milk by these rats caused significant H₂emission while ingestion of α-galacto-oligosaccharide-free soy milk did not, thus validating the experimental system. When native soy milk was fermented using the α-galactosidase-producing <it>Lactobacillus fermentum </it>CRL722 strain, the resulting product failed to induce H₂emission in rats thus validating the bacterial model. When <it>L. fermentum </it>CRL722 was coadministered with native soy milk, a significant reduction (50 %, <it>P </it>= 0.019) in H₂emission was observed, showing that α-galactosidase from <it>L. fermentum </it>CRL722 remained active <it>in situ</it>, in the gastrointestinal tract of rats monoassociated with <it>C. butyricum</it>. In human-microbiota associated rats, <it>L. fermentum </it>CRL722 also induced a significant reduction of H₂emission (70 %, <it>P </it>= 0.004).</p> <p>Conclusion</p> <p>These results strongly suggest that <it>L. fermentum </it>α-galactosidase is able to partially alleviate α-galactosidase deficiency in rats. This offers interesting perspectives in various applications in which lactic acid bacteria could be used as a vector for delivery of digestive enzymes in man and animals.</p

Butyrate Produced by Commensal Bacteria Down-Regulates Indolamine 2,3-Dioxygenase 1 (IDO-1) Expression via a Dual Mechanism in Human Intestinal Epithelial Cells

Commensal bacteria are crucial for the development and maintenance of a healthy immune system therefore contributing to the global well-being of their host. A wide variety of metabolites produced by commensal bacteria are influencing host health but the characterization of the multiple molecular mechanisms involved in host-microbiota interactions is still only partially unraveled. The intestinal epithelial cells (IECs) take a central part in the host-microbiota dialogue by inducing the first microbial-derived immune signals. Amongst the numerous effector molecules modulating the immune responses produced by IECs, indoleamine 2,3-dioxygenase-1 (IDO-1) is essential for gut homeostasis. IDO-1 expression is dependent on the microbiota and despites its central role, how the commensal bacteria impacts its expression is still unclear. Therefore, we investigated the impact of individual cultivable commensal bacteria on IDO-1 transcriptional expression and found that the short chain fatty acid (SCFA) butyrate was the main metabolite controlling IDO-1 expression in human primary IECs and IEC cell-lines. This butyrate-driven effect was independent of the G-protein coupled receptors GPR41, GPR43, and GPR109a and of the transcription factors SP1, AP1, and PPARγ for which binding sites were reported in the IDO-1 promoter. We demonstrated for the first time that butyrate represses IDO-1 expression by two distinct mechanisms. Firstly, butyrate decreases STAT1 expression leading to the inhibition of the IFNγ-dependent and phosphoSTAT1-driven transcription of IDO-1. In addition, we described a second mechanism by which butyrate impairs IDO-1 transcription in a STAT1-independent manner that could be attributed to its histone deacetylase (HDAC) inhibitor property. In conclusion, our results showed that IDO-1 expression is down-regulated by butyrate via a dual mechanism: the reduction of STAT1 level and the HDAC inhibitor property of SCFAs

A robust and adaptable high throughput screening method to study host-microbiota interactions in the human intestine

The intestinal microbiota has many beneficial roles for its host. However, the precise mechanisms developed by the microbiota to influence the host intestinal cell responses are only partially known. The complexity of the ecosystem and our inability to culture most of these micro-organisms have led to the development of molecular approaches such as functional metagenomics, i.e. the heterologous expression of a metagenome in order to identify functions. This elegant strategy coupled to high throughput screening allowed to identify novel enzymes from different ecosystems where culture methods have not yet been adapted to isolate the candidate microorganisms. We have proposed to use this functional metagenomic approach in order to model the microbiota's interaction with the host by combining this heterologous expression with intestinal reporter cell lines. The addition of the cellular component to this functional metagenomic approach introduced a second important source of variability resulting in a novel challenge for high throughput screening. First attempts of high throughput screening with various reporter cell-lines showed a high distribution of the response and consequent difficulties to reproduce the response, impairing an easy and clear identification of confirmed hits. In this study, we developed a robust and reproducible methodology to combine these two biological systems for high throughput application. We optimized experimental setups and completed them by appropriate statistical analysis tools allowing the use this innovative approach in a high throughput manner and on a broad range of reporter assays. We herewith present a methodology allowing a high throughput screening combining two biological systems. Therefore ideal conditions for homogeneity, sensitivity and reproducibility of both metagenomic clones as well as reporter cell lines have been identified and validated. We believe that this innovative method will allow the identification of new bioactive microbial molecules and, subsequently, will promote understanding of host-microbiota interactions

Rôles des sortases A et C de Lactococcus lactis dans l’oligomérisation de pilines

Lactococcus lactis est une bactérie largement utilisée en agroalimentaire. Depuis les années 1990,son étude en a fait un vecteur permettant de délivrer des molécules d’intérêt thérapeutique oubiotechnologique. C’est dans ce cadre de recherche et d’innovation, que nous avons choisi d’étudierla potentialité de L. lactis d’ancrer des protéines à sa surface.L’ancrage covalent à la paroi chez les bactéries à Gram positif peut être réalisé par les sortases. Cestranspeptidases ont été décrites essentiellement chez des bactéries pathogènes. Il a été montré queles sortases de classe A (i) reconnaissent des protéines présentant un domaine d’ancrage, constituéd’un motif LPXTG, (ii) catalysent une réaction de transpeptidation entre ce motif et le pontinterpeptidique de la paroi naissante. Il existe également des sortases de classe C qui reconnaissentdes protéines pilines possédant un motif LPXTG ainsi qu’un motif piline, VYPK. Ces sortases sontcapables d’oligomériser ces pilines en pili, principalement décrits dans la littérature comme facteursde virulence.L. lactis, bactérie non pathogène, possède dans son génome deux gènes codant des sortasespotentiellement responsables de l’ancrage covalent de protéines à la paroi : une première sortase declasse A, ylcC, s’est révélée correspondre à une sortase de ménage (Dieye et al. 2010), et uneseconde sortase, yhhA, possède les propriétés structurales des sortases de classe C.Notre étude a débuté, par l’examen du locus sortase C de L. lactis IL1403. Son analyse in silicomontre qu’il est constitué d’un gène codant une sortase C putative et de trois gènes, yhgD, yhgE etyhhB, codant des pilines putatives. Des anticorps spécifiquement dirigés contre la piline majoritaireputative, YhgE, ont été produits. Ils ont été utilisés pour analyser par Western Blot la présenced’oligomères dans des extraits pariétaux de différentes souches. Aucun oligomère n’a pu êtredétecté à la surface de L. lactis IL1403 cultivé en milieu riche. Chez la même souche surexprimantl’opéron sortase C sous le contrôle d’un promoteur fort constitutif dans un vecteur haut nombre decopies, la formation d’oligomères d’YhgE en surface a été observée. Cela indique la fonctionnalitéde la sortase C et de la piline majoritaire putative YhgE pour former des oligomères à la surface deL. lactis.Dans une seconde étape, nous avons analysé le rôle de la sortase A dans la biogenèse desoligomères de surface. Les résultats montrent que la sortase A n’a pas de rôle dans l’oligomérisationmais qu’elle est responsable de l’ancrage des oligomères à la surface de L. lactis.Les travaux actuels visent à mettre en évidence par microscopie (AFM et MET) la présence de pili àla surface de L. lactis. Nous recherchons aussi le rôle biologique de ces oligomères chez L. lactis etavons donc entrepris d’étudier leur impact sur la formation de biofilm

Butyrate produced by gut commensal bacteria activates TGF-beta1 expression through the transcription factor SP1 in human intestinal epithelial cells

The intestinal microbiota contributes to the global wellbeing of their host by their fundamental role in the induction and maintenance of a healthy immune system. Commensal bacteria shape the mucosal immune system by influencing the proportion and the activation state of anti-inflammatory regulatory T cells (Treg) by metabolites that are still only partially unravelled. Microbiota members such as Clostridiales provide a transforming growth factor beta (TGF beta)-rich environment that promotes the accumulation of Treg cells in the gut. The intestinal epithelial cells (IECs) take a central part in this process, as they are a major source of TGF beta 1 upon bacterial colonisation. In this study, we investigated which gut commensal bacteria were able to regulate the TGFB1 human promoter in IECs using supernatants from cultured bacteria. We reported that Firmicutes and Fusobacteria supernatants were the most potent TGFB1 modulators in HT-29 cells. Furthermore, we demonstrated that butyrate was the main metabolite in bacterial supernatants accounting for TGF beta 1 increase. This butyratedriven effect was independent of the G-protein coupled receptors GPR41, GPR43 and GPR109a, the transporter MCT1 as well as the transcription factors NF-kappa B and AP-1 present on TGFB1 promoter. Interestingly, HDAC inhibitors were inducing a similar TGFB1 increase suggesting that butyrate acted through its HDAC inhibitor properties. Finally, our results showed that SP1 was the main transcription factor mediating the HDAC inhibitor effect of butyrate on TGFB1 expression. This is, to our knowledge, the first characterisation of the mechanisms underlying TGFB1 regulation in IEC by commensal bacteria derived butyrate

Functionality of Sortase A in Lactococcus lactis▿

Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates that carry the canonical LPXTG signature of such substrates. We report here on the functionality of SrtA to anchor five LPXTG substrates to the cell wall, thus suggesting that SrtA is the housekeeping sortase in L. lactis IL1403

La production de pili à la surface de Lactococcus lactis : mise en évidence et impact sur la structure des biofilms: Pili et biofilms chez Lactococcus lactis

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Pilus Biogenesis in Lactococcus lactis: molecular characterization and role in aggregation and biofilm formation

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used

Cellular growth edge effect reduction.

<p>Cell-lines were processes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-g002" target="_blank">Figure 2</a> using crystal violet. Mean p-values +/− SEM comparing borders versus core for different parental cell-lines at 0 or 1 h room temperature incubation are reported.</p

Cellular growth-dependent edge effect reduction applied to three different reporter cell-lines.

<p>Cell-lines were processes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-g003" target="_blank">Figure 3</a>. The table represents the mean of three p-values +/− SEM for comparing borders versus core for the different tested reporter cell-lines.</p