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.

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.

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.

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.

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.

Lignan transformation by gut bacteria lowers tumor burden in a gnotobiotic rat model of breast cancer.

High dietary lignan exposure is implicated in a reduced breast cancer risk in women. The bacterial transformation of plant lignans to enterolignans is thought to be essential for this effect. To provide evidence for this assumption, gnotobiotic rats were colonized with the lignan-converting bacteria Clostridium saccharogumia, Eggerthella lenta, Blautia producta and Lactonifactor longoviformis (LCC rats). Germ-free rats were used as the control. All animals were fed a lignan-rich flaxseed diet and breast cancer was induced with 7,12-dimethylbenz(a)anthracene. The lignan secoisolariciresinol diglucoside was converted into the enterolignans enterodiol and enterolactone in the LCC but not in the germ-free rats. This transformation did not influence cancer incidence at the end of the 13 weeks experimental period but significantly decreased tumor numbers per tumor-bearing rat, tumor size, tumor cell proliferation and increased tumor cell apoptosis in LCC rats. No differences between LCC and control rats were observed in the expression of the genes encoding the estrogen receptors (ERs) α, ERß and G-coupled protein 30. The same was true for IGF-1 and EGFR involved in tumor growth. The activity of selected enzymes involved in the degradation of oxidants in plasma and liver was significantly increased in the LCC rats. However, plasma and liver concentrations of reduced glutathione and malondialdehyde, considered as oxidative stress markers, did not differ between the groups. In conclusion, our results show that the bacterial conversion of plant lignans to enterolignans beneficially influences their anticancer effects.

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.

Dextran sodium sulfate-induced inflammation alters the expression of proteins by intestinal Escherichia coli strains in a gnotobiotic mouse model.

To identify Escherichia coli proteins involved in adaptation to intestinal inflammation, mice were monoassociated with the colitogenic E. coli strain UNC or with the probiotic E. coli strain Nissle. Intestinal inflammation was induced by treating the mice with 3.5% dextran sodium sulfate (DSS). Differentially expressed proteins in E. coli strains collected from cecal contents were identified by 2-dimensional difference gel electrophoresis. In both strains, acute inflammation led to the downregulation of pathways involved in carbohydrate breakdown and energy generation. Accordingly, DSS-treated mice had lower concentrations of bacterial fermentation products in their cecal contents than control mice. Differentially expressed proteins also included the Fe-S cluster repair protein NfuA, the tryptophanase TnaA, and the uncharacterized protein YggE. NfuA expression was 3-fold higher in E. coli strains from DSS-treated than from control mice. Reporter experiments confirmed the induction of nfuA in response to iron deprivation, mimicking Fe-S cluster destruction by inflammation. YggE expression, which has been reported to reduce the intracellular level of reactive oxygen species, was 4- to 8-fold higher in E. coli Nissle than in E. coli UNC. This was confirmed by in vitro reporter gene assays indicating that Nissle is better equipped to cope with oxidative stress than UNC. Nissle isolated from DSS-treated and control mice had TnaA levels 4- to 7-fold-higher than those of UNC. Levels of indole resulting from the TnaA reaction were higher in control animals associated with E. coli Nissle. Because of its anti-inflammatory effect, indole is hypothesized to be involved in the extension of the remission phase in ulcerative colitis described for E. coli Nissle.

Daidzein and genistein are converted to equol and 5-hydroxy-equol by human intestinal Slackia isoflavoniconvertens in gnotobiotic rats.

Intestinal conversion of the isoflavone daidzein to the bioactive equol is exclusively catalyzed by gut bacteria, but a direct role in equol formation under in vivo conditions has not yet been demonstrated. Slackia isoflavoniconvertens is one of the few equol-forming gut bacteria isolated from humans and, moreover, it also converts genistein to 5-hydroxy-equol. To demonstrate the isoflavone-converting ability of S. isoflavoniconvertens in vivo, the metabolization of dietary daidzein and genistein was investigated in male and female rats harboring a simplified human microbiota without (control) or with S. isoflavoniconvertens (SIA). Feces, urine, intestinal contents, and plasma of the rats were analyzed for daidzein, genistein, and their metabolites. Equol and 5-hydroxy-equol were found in intestinal contents, feces, and urine of SIA rats but not in the corresponding samples of the control rats. 5-Hydroxy-equol was present at much lower concentrations than equol and the main metabolite produced from genistein was the intermediate dihydrogenistein. The plasma of SIA rats contained equol but no 5-hydroxy-equol. Equol formation had no effect on plasma concentrations of the insulin-like growth factor I. The concentrations of daidzein and genistein were considerably lower in all samples of the SIA rats than in those of the control rats. Male SIA rats had higher intestinal and fecal concentrations of the isoflavones and their metabolites than female SIA rats. The observed activity in the rat model indicates that S. isoflavoniconvertens is capable of contributing in vivo to the bioactivation of daidzein and genistein by formation of equol and 5-hydroxy-equol.

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.

Recent developments and perspectives in the investigation of probiotic effects.

The quest for a better understanding of how probiotics work has boosted an enormous interest in the molecular processes underlying host-microbe interactions. This review covers recent developments and perspectives in the study of probiotic mechanisms.

Requirement of purine and pyrimidine synthesis for colonization of the mouse intestine by Escherichia coli.

To study the adaptation of an intestinal bacterium to its natural environment, germfree mice were associated with commensal Escherichia coli MG1655. Two-dimensional gel electrophoresis was used to identify proteins differentially expressed in E. coli MG1655 collected from either cecal contents or anaerobic in vitro cultures. Fourteen differentially expressed proteins (>3-fold; P < 0.05) were identified, nine of which were upregulated in cecal versus in vitro-grown E. coli. Four of these proteins were investigated further for their role in gut colonization. After deletion of the corresponding genes, the resulting E. coli mutants were tested for their ability to colonize the intestines of gnotobiotic mice in competition with the wild-type strain. A mutant devoid of ydjG, which encodes a putative NADH-dependent methylglyoxal reductase, reached a 1.2-log-lower cecal concentration than the wild type. Deletion of the nanA gene encoding N-acetylneuraminate lyase affected the colonization and persistence of E. coli in the intestines of the gnotobiotic mice only slightly. A mutant devoid of 5'-phosphoribosyl 4-(N-succinocarboxamide)-5-aminoimidazole synthase, a key enzyme of purine synthesis, displayed intestinal cell counts >4 logs lower than those of the wild type. Deletion of the gene encoding aspartate carbamoyltransferase, a key enzyme of pyrimidine synthesis, even resulted in the washout of the corresponding mutant from the mouse intestinal tract. These findings indicate that E. coli needs to synthesize purines and pyrimidines to successfully colonize the mouse intestine.

Posttranslational inhibition of proinflammatory chemokine secretion in intestinal epithelial cells: implications for specific IBD indications.

BACKGROUND AND AIM: Inflammatory bowel diseases (IBD) are immune-mediated chronic diseases that are characterized by an overreaction of the intestinal immune system to the intestinal microbiota. VSL#3, a mixture of 8 different lactic acid bacteria, is a clinically relevant probiotic compound in the context of IBD, but the bacterial structures and molecular mechanisms underlying the observed protective effects are largely unknown. The intestinal epithelium plays a very important role in the maintenance of the intestinal homeostasis, as the intestinal epithelial cells (IEC) are capable of sensing, processing, and reacting upon signals from the luminal microbiota and the intestinal immune system. This immune regulatory function of the IEC is lost in IBD owing to dysregulated activation of the IEC. Thus, the aim of this study was to reveal protective mechanisms of VSL#3 on IEC function. RESULTS: In vitro, VSL#3 was found to selectively inhibit activation-induced secretion of the T-cell chemokine interferon-inducible protein (IP)-10 in IEC. Cell wall-associated proteins of VSL#3-derived Lactobacillus casei (L. casei) were identified to be the active anti-inflammatory component of VSL#3. Mechanistically, L. casei did not impair initial IP-10 protein production, but induced posttranslational degradation of IP-10 in IEC. Feeding studies in tumor necrosis factor (TNF)Delta ARE/+ mice, a mouse model for experimental ileitis, revealed that neither VSL#3 nor L. casei is capable of reducing ileal inflammation. Even preweaning feeding of VSL#3 did not prevent the development of severe ileitis in TNF Delta ARE/+ mice. In contrast, VSL#3 feeding studies in IL-10-/- mice, a model for experimental colitis, revealed that VSL#3 has local, intestinal compartment-specific protective effects on the development of inflammation. Reduced histopathologic inflammation in the cecum of IL-10-/- mice after VSL#3 treatment was found to correlate with reduced levels of IP-10 protein in primary cecal epithelial cells. CONCLUSION AND OUTLOOK: These results suggest that the inhibitory effect of VSL#3-derived L. casei on IP-10 secretion in IEC is an important probiotic mechanism that contributes to the anti-inflammatory effects of VSL#3 in specific subsets of patients with IBD. An important future aim is the identification of the active probiotic protein, which could serve as a basis for the development of new efficient therapies in the context of IBD.

Absence of intestinal microbiota does not protect mice from diet-induced obesity.

The gut microbiota has been implicated in host nutrient absorption and energy homeostasis. We studied the influence of different diets on body composition in germ-free (GF) and conventional (CV) mice. GF and CV male adult C3H mice were fed ad libitum a semi-synthetic low-fat diet (LFD; carbohydrate-protein-fat ratio: 41:42:17; 19.8 kJ/g), a high-fat diet (HFD; 41:16:43; 21.4 kJ/g) or a commercial Western diet (WD; 41:19:41; 21.5 kJ/g). There was no difference in body weight gain between GF and CV mice on the LFD. On the HFD, GF mice gained more body weight and body fat than CV mice, and had lower energy expenditure. GF mice on the WD gained significantly less body fat than GF mice on the HFD. GF mice on both HFD and WD showed increased intestinal mRNA expression of fasting-induced adipose factor/angiopoietin-like protein 4 (Fiaf/Angptl4), but they showed no major changes in circulating Fiaf/Angptl4 compared with CV mice. The faecal microbiota composition of the CV mice differed between diets: the proportion of Firmicutes increased on both HFD and WD at the expense of the Bacteroidetes. This increase in the Firmicutes was mainly due to the proliferation of one family within this phylum: the Erysipelotrichaceae. We conclude that the absence of gut microbiota does not provide a general protection from diet-induced obesity, that intestinal production of Fiaf/Angptl4 does not play a causal role in gut microbiota-mediated effects on fat storage and that diet composition affects gut microbial composition to larger extent than previously thought.

Reduced microbial diversity and high numbers of one single Escherichia coli strain in the intestine of colitic mice.

Commensal bacteria play a role in the aetiology of inflammatory bowel diseases (IBD). High intestinal numbers of Escherichia coli in IBD patients suggest a role of this organism in the initiation or progression of chronic gut inflammation. In addition, some E. coli genotypes are more frequently detected in IBD patients than others. We aimed to find out whether gut inflammation in an IBD mouse model is associated with a particular E. coli strain. Intestinal contents and tissue material were taken from 1-, 8-, 16- and 24-week-old interleukin 10-deficient (IL-10(-/-)) mice and the respective wild-type animals. Caecal and colonic inflammation was observed in IL-10(-/-) animals from the 8 weeks of life on accompanied by a lower intestinal microbial diversity than in the respective wild-type animals. Culture- based and molecular approaches revealed that animals with gut inflammation harboured significantly higher numbers of E. coli than healthy controls. Phylogenetic grouping according to the E. coli Reference Collection (ECOR) system and strain typing by random-amplified polymorphic DNA and pulsed-field gel electrophoresis revealed that all mice were colonized by one single E. coli strain. The strain was shown to have the O7:H7:K1 serotype and to belong to the virulence-associated phylogenetic group B2. In a co-association experiment with gnotobiotic mice, the strain outnumbered E. coli ECOR strains belonging to the phylogenetic group A and B2 respectively. A high number of virulence- and fitness-associated genes were detected in the strain's genome possibly involved in the bacterial adaptation to the murine intestine.

Adaptation of protein expression by Escherichia coli in the gastrointestinal tract of gnotobiotic mice.

The gastrointestinal tract of mammals is inhabited by several hundred bacterial species. While the effects of the gut microbiota upon the host have been widely studied, the microbial response to host factors has only recently attracted attention. In order to investigate the influence of the host on the physiology of gastrointestinal bacteria, a simplified model of host-bacteria interaction was created by associating germfree mice with commensal Escherichia coli. Here we demonstrate the feasibility of analysing the bacterial response to the conditions in the digestive system by a proteomics-based approach. Two-dimensional gel electrophoresis (2D-GE) followed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was used to identify bacterial proteins from caecal and faecal samples. In a set of 60 arbitrarily chosen spots of stably and differentially expressed proteins, 50 different bacterial proteins were identified. Their ascribed functions suggest that the host-associated bacteria adapt their metabolism to the conditions in the intestine by utilizing arginine, asparagine and aspartate as well as glucose/galactose, ribose, maltose, glucuronate, galacturonate and gluconate as substrates. Thirteen proteins not previously detected on 2D-gels and 10 proteins with unknown or poorly characterized physiological function were identified, while the existence of three proteins had so far only been inferred from predictions or by homology.

The bioavailability of apigenin-7-glucoside is influenced by human intestinal microbiota in rats.

We investigated the impact of human intestinal microbiota on bioavailability of the flavone apigenin-7-glucoside (A7G) by comparing germ-free and human microbiota-associated (HMA) rats. First, the ability of the human intestinal microbiota to convert A7G was proven in vitro by incubating A7G with fecal suspensions. Apigenin, naringenin, and 3-(4-hydroxyphenyl)propionic acid were formed as main metabolites. After application of A7G to germ-free rats, apigenin, luteolin, and their conjugates were detected in urine and feces. In HMA rats, naringenin, eriodictyol, phloretin, 3-(3,4-dihydroxyphenyl)propionic acid, 3-(4-hydroxyphenyl)propionic acid, 3-(3-hydroxyphenyl)propionic acid, and 4-hydroxycinnamic acid in their free and conjugated forms were additionally formed. In whole-blood samples from germ-free and HMA rats, only apigenin conjugates and phloretin, respectively, were detected. The total excretion of A7G and its metabolites within 48 h was similarly low in both germ-free and HMA rats, with 11 and 13% of the A7G dose, respectively. In germ-free rats, A7G metabolites dominated by apigenin and its conjugates were mainly excreted with feces. In contrast, the compounds in HMA rats were predominantly recovered from urine, 3-(4-hydroxyphenyl)propionic acid being the main metabolite. The ability of selected gut bacteria and the host intestinal mucosa to deglycosylate A7G was tested using cell extracts. Apigenin was formed by cytosolic extracts of Eubacterium ramulus and Bacteroides distasonis and by the microsomal fraction of the small intestinal mucosa of rats. Overall, human intestinal microbiota largely contributed to A7G metabolism, indicating its influence on the bioactivity of flavones.

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.