Probiotic Bacillus Strains Enhance T Cell Responses in Chicken.

Banning antibiotic growth promotors and other antimicrobials in poultry production due to the increasing antimicrobial resistance leads to increased feeding of potential alternatives such as probiotics. However, the modes of action of those feed additives are not entirely understood. They could act even with a direct effect on the immune system. A previously established animal-related in vitro system using primary cultured peripheral blood mononuclear cells (PBMCs) was applied to investigate the effects of immune-modulating feed additives. Here, the immunomodulation of different preparations of two probiotic Bacillus strains, B. subtilis DSM 32315 (BS), and B. amyloliquefaciens CECT 5940 (BA) was evaluated. The count of T-helper cells and activated T-helper cells increased after treatment in a ratio of 1:3 (PBMCs: Bacillus) with vital BS (CD4+: p < 0.05; CD4+CD25+: p < 0.01). Furthermore, vital BS enhanced the proliferation and activation of cytotoxic T cells (CD8+: p < 0.05; CD8+CD25+: p < 0.05). Cell-free probiotic culture supernatants of BS increased the count of activated T-helper cells (CD4+CD25+: p < 0.1). UV-inactivated BS increased the proportion of cytotoxic T cells significantly (CD8+: p < 0.01). Our results point towards a possible involvement of secreted factors of BS in T-helper cell activation and proliferation, whereas it stimulates cytotoxic T cells presumably through surface contact. We could not observe any effect on B cells after treatment with different preparations of BS. After treatment with vital BA in a ratio of 1:3 (PBMCs:Bacillus), the count of T-helper cells and activated T-helper cells increased (CD4+: p < 0.01; CD4+CD25+: p < 0.05). Cell-free probiotic culture supernatants of BA as well as UV-inactivated BA had no effect on T cell proliferation and activation. Furthermore, we found no effect of BA preparations on B cells. Overall, we demonstrate that the two different Bacillus strains enhanced T cell activation and proliferation, which points towards an immune-modulating effect of both strains on chicken immune cells in vitro. Therefore, we suggest that administering these probiotics can improve the cellular adaptive immune defense in chickens, thereby enabling the prevention and reduction of antimicrobials in chicken farming.

A nutritional supplement based on a synbiotic combination of Bacillus subtilis DSM 32315 and L-alanyl-L-glutamine improves glucose metabolism in healthy prediabetic subjects - A real-life post-marketing study.

Introduction: Impaired glucose homeostasis is a significant risk factor for cardiometabolic diseases, whereas the efficacy of available standard therapies is limited, mainly because of poor adherence. This post-marketing study assessed the glucose-lowering potential of a synbiotic-based formulation. Methods: One hundred ninety-two participants were enrolled in a digital nutrition program with continuous glucose monitoring (CGM) and received a study product comprising Bacillus subtilis DSM 32315 and L-alanyl-L-glutamine. Participants underwent a first sensor phase without supplementation, followed by a 14-day supplementation phase without sensor, and completed by a second sensor phase while continuing supplementation. Fasting glucose levels were determined before and after supplementation by CGM. In addition, the postprandial glycemic response to an oral glucose challenge, body weight, HbA1c concentrations, and BMI was analyzed. Subgroup analyses of subjects with elevated glucose and HbA1c levels vs. normoglycemic subjects were performed. Results: Supplementation with the study product resulted in significant improvements in glucose parameters (delta values: fasting glucose -2,13% ± 8.86; iAUC0-120 -4.91% ± 78.87; HbA1c: -1.20% ± 4.72) accompanied by a significant weight reduction (-1.07 kg ± 2.30) in the study population. Subgroup analyses revealed that the improvements were mainly attributed to a prediabetic subgroup with elevated fasting glucose and HbA1c values before supplementation (delta values: fasting glucose -6.10% 4± 7.89; iAUC0-120 -6.28% ± 115.85; HbA1c -3.31% ± 4.36; weight: -1.47 kg ± 2.82). Conclusion: This study indicates that the synbiotic composition is an effective and convenient approach to counteract hyperglycemia. Further placebo-controlled studies are warranted to test its efficacy in the treatment of cardiometabolic diseases.

Evaluation of commercially available DNA extraction kits for the analysis of the broiler chicken cecal microbiota.

16S rRNA gene amplicon sequencing is a state of the art technology to analyze bacterial communities via microbiome profiling. Choosing an appropriate DNA extraction protocol is crucial for characterizing the microbial community and can be challenging, especially when preliminary knowledge about the sample matrix is scarce. The aim of the present study was to evaluate seven commercial DNA extraction kits suitable for 16S rRNA gene amplicon sequencing of the bacterial community of the chicken cecum, taking into account different criteria such as high technical reproducibility, high bacterial diversity and easy handling. The DNA extraction kits differed strongly with respect to extractable DNA quantity, DNA quality, technical reproducibility and bacterial diversity determined after 16S rRNA gene amplicon sequencing and subsequent bioinformatic and biostatistical data processing. While some of the DNA extraction protocols under-represented specific bacterial community members, the removal of PCR inhibitors supported technical reproducibility and subsequently enhanced the recovered bacterial diversity from the chicken cecum community. In conclusion, the removal of PCR inhibitors from the sample matrix seemed to be one of the main drivers for a consistent representation of the bacterial community even of low abundant taxa in chicken cecum samples.

Specific Wheat Fractions Influence Hepatic Fat Metabolism in Diet-Induced Obese Mice.

Low whole grain consumption is a risk factor for the development of non-communicable diseases such as type 2 diabetes. Dietary fiber and phytochemicals are bioactive grain compounds, which could be involved in mediating these beneficial effects. These compounds are not equally distributed in the wheat grain, but are enriched in the bran and aleurone fractions. As little is known on physiological effects of different wheat fractions, the aim of this study was to investigate this aspect in an obesity model. For twelve weeks, C57BL/6J mice were fed high-fat diets (HFD), supplemented with one of four wheat fractions: whole grain flour, refined white flour, bran, or aleurone. The different diets did not affect body weight, however bran and aleurone decreased liver triglyceride content, and increased hepatic n-3 polyunsaturated fatty acid (PUFA) concentrations. Furthermore, lipidomics analysis revealed increased PUFA concentration in the lipid classes of phosphatidylcholine (PC), PC-ether, and phosphatidylinositol in the plasma of mice fed whole grain, bran, and aleurone supplemented diets, compared to refined white flour. Furthermore, bran, aleurone, and whole grain supplemented diets increased microbial α-diversity, but only bran and aleurone increased the cecal concentrations of short-chain fatty acids. The effects on hepatic lipid metabolism might thus at least partially be mediated by microbiota-dependent mechanisms.

Microbial Colonization in Adulthood Shapes the Intestinal Macrophage Compartment.

BACKGROUND AND AIMS: Contact with distinct microbiota early in life has been shown to educate the mucosal immune system, hence providing protection against immune-mediated diseases. However, the impact of early versus late colonization with regard to the development of the intestinal macrophage compartment has not been studied so far. METHODS: Germ-free mice were colonized with specific-pathogen-free [SPF] microbiota at the age of 5 weeks. The ileal and colonic macrophage compartment were analysed by immunohistochemistry, flow cytometry, and RNA sequencing 1 and 5 weeks after colonization and in age-matched SPF mice, which had had contact with microbiota since birth. To evaluate the functional differences, dextran sulfate sodium [DSS]-induced colitis was induced, and barrier function analyses were undertaken. RESULTS: Germ-free mice were characterized by an atrophied intestinal wall and a profoundly reduced number of ileal macrophages. Strikingly, morphological restoration of the intestine occurred within the first week after colonization. In contrast, ileal macrophages required 5 weeks for complete restoration, whereas colonic macrophages were numerically unaffected. However, following DSS exposure, the presence of microbiota was a prerequisite for colonic macrophage infiltration. One week after colonization, mild colonic inflammation was observed, paralleled by a reduced inflammatory response after DSS treatment, in comparison with SPF mice. This attenuated inflammation was paralleled by a lack of TNFα production of LPS-stimulated colonic macrophages from SPF and colonized mice, suggesting desensitization of colonized mice by the colonization itself. CONCLUSIONS: This study provides the first data indicating that after colonization of adult mice, the numeric, phenotypic, and functional restoration of the macrophage compartment requires the presence of intestinal microbiota and is time dependent.

LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells.

B lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.

Analysis of factors contributing to variation in the C57BL/6J fecal microbiota across German animal facilities.

The intestinal microbiota is involved in many physiological processes and it is increasingly recognized that differences in community composition can influence the outcome of a variety of murine models used in biomedical research. In an effort to describe and account for the variation in intestinal microbiota composition across the animal facilities of participating members of the DFG Priority Program 1656 "Intestinal Microbiota", we performed a survey of C57BL/6J mice from 21 different mouse rooms/facilities located at 13 different institutions across Germany. Fresh feces was sampled from five mice per room/facility using standardized procedures, followed by extraction and 16S rRNA gene profiling (V1-V2 region, Illumina MiSeq) at both the DNA and RNA (reverse transcribed to cDNA) level. In order to determine the variables contributing to bacterial community differences, we collected detailed questionnaires of animal husbandry practices and incorporated this information into our analyses. We identified considerable variation in a number of descriptive aspects including the proportions of major phyla, alpha- and beta diversity, all of which displayed significant associations to specific aspects of husbandry. Salient findings include a reduction in alpha diversity with the use of irradiated chow, an increase in inter-individual variability (beta diversity) with respect to barrier access and open cages and an increase in bacterial community divergence with time since importing from a vendor. We further observe a high degree of facility-level individuality, which is likely due to each facility harboring its own unique combination of multiple varying attributes of animal husbandry. While it is important to account and control for such differences between facilities, the documentation of such diversity may also serve as a valuable future resource for investigating the origins of microbial-driven host phenotypes.

Alleviation of high fat diet-induced obesity by oligofructose in gnotobiotic mice is independent of presence of Bifidobacterium longum.

SCOPE: Diet-induced obesity is associated with changes in the gut microbiota and low-grade inflammation. Oligofructose was reported to ameliorate high fat diet-induced metabolic disorders in mice by restoring the number of intestinal bifidobacteria. However, this has not been experimentally demonstrated. METHODS AND RESULTS: We fed conventional mice, germfree mice, mice associated with a simplified human gut microbiota composed of eight bacterial species including Bifidobacterium longum (SIHUMI), and mice associated with SIHUMI without B. longum a low fat diet (LFD), a high fat diet (HFD), or a HFD containing 10% oligofructose (HFD + OFS) for five weeks. We assessed body composition, bacterial cell numbers and metabolites, markers of inflammation, and gut permeability. Conventional mice fed HFD or HFD + OFS did not differ in body weight gain and glucose tolerance. The gnotobiotic mouse groups fed LFD or HFD + OFS gained less body weight and body fat, and displayed an improved glucose tolerance compared with mice fed HFD. These differences were not affected by the presence of B. longum. Mice fed HFD showed no signs of inflammation or increased intestinal permeability. CONCLUSION: The ability of oligofructose to reduce obesity and to improve glucose tolerance in gnotobiotic mice fed HFD was independent of the presence of B. longum.

Effects of dietary inulin on bacterial growth, short-chain fatty acid production and hepatic lipid metabolism in gnotobiotic mice.

In literature, contradictory effects of dietary fibers and their fermentation products, short-chain fatty acids (SCFA), are described: On one hand, they increase satiety, but on the other hand, they provide additional energy and promote obesity development. We aimed to answer this paradox by investigating the effects of fermentable and non-fermentable fibers on obesity induced by high-fat diet in gnotobiotic C3H/HeOuJ mice colonized with a simplified human microbiota. Mice were fed a high-fat diet supplemented either with 10% cellulose (non-fermentable) or inulin (fermentable) for 6 weeks. Feeding the inulin diet resulted in an increased diet digestibility and reduced feces energy, compared to the cellulose diet with no differences in food intake, suggesting an increased intestinal energy extraction from inulin. However, we observed no increase in body fat/weight. The additional energy provided by the inulin diet led to an increased bacterial proliferation in this group. Supplementation of inulin resulted further in significantly elevated concentrations of total SCFA in cecum and portal vein plasma, with a reduced cecal acetate:propionate ratio. Hepatic expression of genes involved in lipogenesis (Fasn, Gpam) and fatty acid elongation/desaturation (Scd1, Elovl3, Elovl6, Elovl5, Fads1 and Fads2) were decreased in inulin-fed animals. Accordingly, plasma and liver phospholipid composition were changed between the different feeding groups. Concentrations of omega-3 and odd-chain fatty acids were increased in inulin-fed mice, whereas omega-6 fatty acids were reduced. Taken together, these data indicate that, during this short-term feeding, inulin has mainly positive effects on the lipid metabolism, which could cause beneficial effects during obesity development in long-term studies.

Clostridium ramosum promotes high-fat diet-induced obesity in gnotobiotic mouse models.

UNLABELLED: The intestines of obese humans and mice are enriched with Erysipelotrichi, a class within the Firmicutes. Clostridium ramosum, a member of the Erysipelotrichi, is associated with symptoms of the metabolic syndrome in humans. To clarify the possible obesogenic potential of this bacterial species and to unravel the underlying mechanism, we investigated the role of C. ramosum in obesity development in gnotobiotic mice. Mice were associated with a simplified human intestinal (SIHUMI) microbiota of eight bacterial species, including C. ramosum, with the SIHUMI microbiota except C. ramosum (SIHUMIw/oCra), or with C. ramosum only (Cra) and fed a high-fat diet (HFD) or a low-fat diet (LFD). Parameters related to the development of obesity and metabolic diseases were compared. After 4 weeks of HFD feeding, the mouse groups did not differ in energy intake, diet digestibility, gut permeability, and parameters of low-grade inflammation. However, SIHUMI and Cra mice fed the HFD gained significantly more body weight and body fat and displayed higher food efficiency than SIHUMIw/oCra mice fed the HFD. Gene expression of glucose transporter 2 (Glut2) in jejunal mucosa and of fatty acid translocase (CD36) in ileal mucosa was significantly increased in the obese SIHUMI and Cra mice compared with the less obese SIHUMIw/oCra mice. The data demonstrate that the presence of C. ramosum in SIHUMI and Cra mice enhanced diet-induced obesity. Upregulation of small intestinal glucose and fat transporters in these animals may contribute to their increased body fat deposition. IMPORTANCE: Obesity is a growing health problem worldwide. Changes in the proportions of Bacteroidetes and Firmicutes, the two dominant phyla in the human and the murine intestinal tract, link the intestinal microbiota to obesity. Erysipelotrichi, a class within the Firmicutes, increase in response to high-fat feeding in mice. Clostridium ramosum, a member of the Erysipelotrichi, has been linked to symptoms of the metabolic syndrome. We hypothesized that C. ramosum promotes obesity development and related pathologies. Our experiments in gnotobiotic mice show that C. ramosum promoted diet-induced obesity, probably by enhancing nutrient absorption. Identification of obesogenic bacteria and understanding their mode of action enable the development of novel strategies for the treatment of this epidemic disease. Pharmaceuticals that target obesogenic bacteria or their metabolism could help to prevent and treat obesity and related disorders in the future.

Association of germ-free mice with a simplified human intestinal microbiota results in a shortened intestine.

Genetic, nutritional, and gut microbiota-derived factors have been proposed to play a role in the development of the whole intestine that is around 40% longer in PRM/Alf mice compared with other mouse strains. The PRM/Alf genotype explains 60% of this length difference. The remaining 40% are due to a maternal effect that could depend on the gut microbiota transmitted by the mother to their pups. Germ-free PRM/Alf mice and C3H/He mice were associated with a simplified human microbiota (SIHUMI) to study its impact on gut length. The small intestines of the SIHUMI-associated mice were 16.4% (PRM/Alf) and 9.7% (C3H/He) shorter than those of the corresponding germ-free counterparts. Temporal temperature gradient gel electrophoresis and quantitative real-time PCR revealed differences in microbiota composition between both SIHUMI-associated mouse strains. Anaerostipes caccae was one log lower in PRM/Alf mice than in C3H/He mice. Since polyamines and short-chain fatty acids (SCFAs) are important intestinal growth factors, their concentrations were explored. Cecal concentrations of putrescine, spermine, spermidine, and N-acetylspermine were 1.5-fold, 3.7-fold, 2.2-fold, and 1.4-fold higher, respectively, in the SIHUMI-C3H/He mice compared with the SIHUMI-PRM/Alf mice. In addition, cecal acetate, propionate, and butyrate concentrations in SIHUMI-C3H/He mice were 1.4-fold, 1.1-fold, and 2.1-fold higher, respectively, than in SIHUMI-PRM/Alf mice. These results indicate that polyamines and SCFAs did not promote gut lengthening in any of the two mouse strains. This suggests that as yet unknown factors provided by the SIHUMI prevented gut lengthening in the SIHUMI-associated mice compared with the germfree mice.

High-fat diet alters gut microbiota physiology in mice.

The intestinal microbiota is known to regulate host energy homeostasis and can be influenced by high-calorie diets. However, changes affecting the ecosystem at the functional level are still not well characterized. We measured shifts in cecal bacterial communities in mice fed a carbohydrate or high-fat (HF) diet for 12 weeks at the level of the following: (i) diversity and taxa distribution by high-throughput 16S ribosomal RNA gene sequencing; (ii) bulk and single-cell chemical composition by Fourier-transform infrared- (FT-IR) and Raman micro-spectroscopy and (iii) metaproteome and metabolome via high-resolution mass spectrometry. High-fat diet caused shifts in the diversity of dominant gut bacteria and altered the proportion of Ruminococcaceae (decrease) and Rikenellaceae (increase). FT-IR spectroscopy revealed that the impact of the diet on cecal chemical fingerprints is greater than the impact of microbiota composition. Diet-driven changes in biochemical fingerprints of members of the Bacteroidales and Lachnospiraceae were also observed at the level of single cells, indicating that there were distinct differences in cellular composition of dominant phylotypes under different diets. Metaproteome and metabolome analyses based on the occurrence of 1760 bacterial proteins and 86 annotated metabolites revealed distinct HF diet-specific profiles. Alteration of hormonal and anti-microbial networks, bile acid and bilirubin metabolism and shifts towards amino acid and simple sugars metabolism were observed. We conclude that a HF diet markedly affects the gut bacterial ecosystem at the functional level.

Mild gut inflammation modulates the proteome of intestinal Escherichia coli.

Using interleukin 10-deficient (IL-10(-/-) ) and wild-type mice monoassociated with either the adherent-invasive Escherichia coli UNC or the probiotic E. coli Nissle, the effect of a mild intestinal inflammation on the bacterial proteome was studied. Within 8 weeks, IL-10(-/-) mice monoassociated with E. coli UNC exhibited an increased expression of several proinflammatory markers in caecal mucosa. Escherichia coli Nissle-associated IL-10(-/-) mice did not do so. As observed previously for E. coli from mice with acute colitis, glycolytic enzymes were downregulated in intestinal E. coli UNC from IL-10(-/-) mice. In addition, the inhibitor of vertebrate C-type lysozyme, Ivy, was upregulated on messenger RNA (mRNA) and protein level in E. coli Nissle from IL-10(-/-) mice compared with E. coli UNC from these mice. Higher expression of Ivy in E. coli Nissle correlated with an improved growth of this probiotic strain in the presence of lysozyme-ethylenediaminetetraacetic acid (EDTA). By overexpressing Ivy, we demonstrated that Ivy contributes to a higher lysozyme resistance of E. coli, supporting the role of Ivy as a potential fitness factor. However, deletion of Ivy did not alter the growth phenotype of E. coli Nissle in the presence of lysozyme-EDTA, suggesting the existence of additional lysozyme inhibitors that can take over the function of Ivy.

Commensal Akkermansia muciniphila exacerbates gut inflammation in Salmonella Typhimurium-infected gnotobiotic mice.

Excessive mucin degradation by intestinal bacteria may contribute to inflammatory bowel diseases because access of luminal antigens to the intestinal immune system is facilitated. This study investigated how the presence of a mucin degrading commensal bacterium affects the severity of an intestinal Salmonella enterica Typhimurium-induced gut inflammation. Using a gnotobiotic C3H mouse model with a background microbiota of eight bacterial species (SIHUMI) the impact of the mucin-degrading commensal bacterium Akkermansia muciniphila (SIHUMI-A) on inflammatory and infectious symptoms caused by S. Typhimurium was investigated. Presence of A. muciniphila in S. Typhimurium-infected SIHUMI mice caused significantly increased histopathology scores and elevated mRNA levels of IFN-γ, IP-10, TNF-α, IL-12, IL-17 and IL-6 in cecal and colonic tissue. The increase in pro-inflammatory cytokines was accompanied by 10-fold higher S. Typhimurium cell numbers in mesenteric lymph nodes of SIHUMI mice associated with A. muciniphila and S. Typhimurium (SIHUMI-AS) compared to SIHUMI mice with S. Typhimurium only (SIHUMI-S). The number of mucin filled goblet cells was 2- to 3-fold lower in cecal tissue of SIHUMI-AS mice compared to SIHUMI-S, SIHUMI-A or SIHUMI mice. Reduced goblet cell numbers significantly correlated with increased IFN-γ mRNA levels (r(2) = -0.86, ***P<0.001) in all infected mice. In addition, loss of cecal mucin sulphation was observed in SIHUMI mice containing both A. muciniphila and S. Typhimurium compared to other mouse groups. Concomitant presence of A. muciniphila and S. Typhimurium resulted in a drastic change in microbiota composition of SIHUMI mice: the proportion of B. thetaiotaomicron in SIHUMI-AS mice was 0.02% of total bacteria compared to 78%-88% in the other mouse groups and the proportion of S. Typhimurium was 94% in SIHUMI-AS mice but only 2.2% in the SIHUMI-S mice. These results indicate that A. muciniphila exacerbates S. Typhimurium-induced intestinal inflammation by its ability to disturb host mucus homeostasis.

Differences in mucosal gene expression in the colon of two inbred mouse strains after colonization with commensal gut bacteria.

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.

The peptide transporter PEPT1 is expressed in distal colon in rodents and humans and contributes to water absorption.

The peptide transporter PEPT1, expressed in the brush border membrane of enterocytes, mediates the uptake of di- and tripeptides from luminal protein digestion in the small intestine. PEPT1 was proposed not to be expressed in normal colonic mucosa but may become detectable in inflammatory states such as Crohn's disease or ulcerative colitis. We reassessed colonic expression of PEPT1 by performing a systematic analysis of PEPT1 mRNA and protein levels in healthy colonic tissues in mice, rats, and humans. Immunofluorescence analysis of different mouse strains (C57BL/6N, 129/Sv, BALB/c) demonstrated the presence of PEPT1 in the distal part of the colon but not in proximal colon. Rat and human intestines display a similar distribution of PEPT1 as found in mice. However, localization in human sigmoid colon revealed immunoreactivity present at low levels in apical membranes but substantial staining in distinct intracellular compartments. Functional activity of PEPT1 in colonic tissues from mice was assessed in everted sac preparations using [¹4C]Gly-Sar and found to be 5.7-fold higher in distal compared with proximal colon. In intestinal tissues from Pept1-/- mice, no [¹4C]Gly-Sar transport was detectable but feces samples revealed significantly higher water content than in wild-type mice, suggesting that PEPT1 contributes to colonic water absorption. In conclusion, our studies unequivocally demonstrate the presence of PEPT1 protein in healthy distal colonic epithelium in mice, rats, and humans and proved that the protein is functional and contributes to electrolyte and water handling in mice.

Novel insights into E. coli's hexuronate metabolism: KduI facilitates the conversion of galacturonate and glucuronate under osmotic stress conditions.

Using a gnotobiotic mouse model, we previously observed the upregulation of 2-deoxy-D-gluconate 3-dehydrogenase (KduD) in intestinal E. coli of mice fed a lactose-rich diet and the downregulation of this enzyme and of 5-keto 4-deoxyuronate isomerase (KduI) on a casein-rich diet. The present study aimed to define the role of the so far poorly characterized E. coli proteins KduD and KduI in vitro. Galacturonate and glucuronate induced kduD and kduI gene expression 3-fold and 7 to 11-fold, respectively, under aerobic conditions as well as 9 to 20-fold and 19 to 54-fold, respectively, under anaerobic conditions. KduI facilitated the breakdown of these hexuronates. In E. coli, galacturonate and glucuronate are normally degraded by UxaABC and UxuAB. However, osmotic stress represses the expression of the corresponding genes in an OxyR-dependent manner. When grown in the presence of galacturonate or glucuronate, kduID-deficient E. coli had a 30% to 80% lower maximal cell density and 1.5 to 2-fold longer doubling times under osmotic stress conditions than wild type E. coli. Growth on lactose promoted the intracellular formation of hexuronates, which possibly explain the induction of KduD on a lactose-rich diet. These results indicate a novel function of KduI and KduD in E. coli and demonstrate the crucial influence of osmotic stress on the gene expression of hexuronate degrading enzymes.

Contribution of gut bacteria to the metabolism of cyanidin 3-glucoside in human microbiota-associated rats.

Cyanidin 3-glucoside (C3G) is one of the major dietary anthocyanins implicated in the prevention of chronic diseases. To evaluate the impact of human intestinal bacteria on the fate of C3G in the host, we studied the metabolism of C3G in human microbiota-associated (HMA) rats in comparison with germ-free (GF) rats. Urine and faeces of the rats were analysed for C3G and its metabolites within 48 h after the application of 92 µmol C3G/kg body weight. In addition, we tested the microbial C3G conversion in vitro by incubating C3G with human faecal slurries and selected human gut bacteria. The HMA rats excreted with faeces a three times higher percentage of unconjugated C3G products and a two times higher percentage of conjugated C3G products than the GF rats. These differences were mainly due to the increased excretion of 3,4-dihydroxybenzoic acid, 2,4,6-trihydroxybenzaldehyde and 2,4,6-trihydroxybenzoic acid. Only the urine of HMA rats contained peonidin and 3-hydroxycinnamic acid and the percentage of conjugated C3G products in the urine was decreased compared with the GF rats. Overall, the presence of intestinal microbiota resulted in a 3·7% recovery of the C3G dose in HMA rats compared with 1·7% in GF rats. Human intestinal bacteria rapidly degraded C3G in vitro. Most of the C3G products were also found in the absence of bacteria, but at considerably lower levels. The higher concentrations of phenolic acids observed in the presence of intestinal bacteria may contribute to the proposed beneficial health effects of C3G.

Role of commensal gut bacteria in inflammatory bowel diseases.

Aberrant immune responses toward commensal gut bacteria can result in the onset and perpetuation of inflammatory bowel diseases (IBD). Reduced microbiota diversity in conjunction with lower proportion of Gram positive and higher proportion of Gram negative bacteria than in healthy subjects is frequently reported in IBD patients. In a subset of IBD patients, E. coli strains with specific features trigger disease. Important molecular mechanisms underlying this effect have been identified. However, in the majority of patients the exact nature of host-microbe interactions that contribute to IBD development has so far not been defined. The application of metagenomic techniques may help to identify bacterial functions that are involved in the aggravation or alleviation of IBD. Subsequently, the relevance for disease development of bacterial candidate genes may be tested taking advantage of reductionist animal models of chronic gut inflammation. This approach may help to identify bacterial functions that can be targeted in future concepts of IBD therapy.

Impact of nutritional factors on the proteome of intestinal Escherichia coli: induction of OxyR-dependent proteins AhpF and Dps by a lactose-rich diet.

To study the impact of nutritional factors on protein expression of intestinal bacteria, gnotobiotic mice monoassociated with Escherichia coli K-12 were fed three different diets: a diet rich in starch, a diet rich in nondigestible lactose, and a diet rich in casein. Two-dimensional gel electrophoresis and electrospray-tandem mass spectrometry were used to identify differentially expressed proteins of bacteria recovered from small intestine and cecum. Oxidative stress response proteins such as AhpF, Dps, and Fur, all of which belong to the oxyR regulon, were upregulated in E. coli isolates from mice fed the lactose-rich diet. Luciferase reporter gene assays demonstrated that osmotic stress caused by carbohydrates led to the expression of ahpCF and dps, which was not observed in an E. coli ΔoxyR mutant. Growth of ahpCF and oxyR deletion mutants was strongly impaired when nondigestible sucrose was present in the medium. The wild-type phenotype could be restored by complementation of the deletions with plasmids containing the corresponding genes and promoters. The results indicate that some OxyR-dependent proteins play a major role in the adaptation of E. coli to osmotic stress. We conclude that there is an overlap of osmotic and oxidative stress responses. Mice fed the lactose-rich diet possibly had a higher intestinal osmolality, leading to the upregulation of OxyR-dependent proteins, which enable intestinal E. coli to better cope with diet-induced osmotic stress.