Vitamin B12 Deficiency Alters the Gut Microbiota in a Murine Model of Colitis

Purpose: Inflammatory bowel disease (IBD) refers to a spectrum of autoimmune diseases, which result in chronic intestinal inflammation. Previous findings suggest a role for diet, nutrition and dysbiosis of the gut microbiota in both the development and progression of the condition. Vitamin B12 is a key cofactor of methionine synthase and is produced solely by microbes. Previous work links increased levels of homocysteine, a substrate of methionine synthase, MetH, to IBD indicating a potential role for vitamin B12 deficiency in intestinal injury and inflammation. This study assessed the role of vitamin B12 in shaping the gut microbiota and determining responses to intestinal injury using a reproducible murine model of colitis. Methods: The effects of vitamin B12 supplementation and deficiency were assessed in vivo; 3-week-old post-weanling C57Bl/6 mice were divided into three dietary treatment groups: (1) sufficient vitamin B12 (50 mg/Kg), (2) deficient vitamin B12 (0 mg/Kg) and (3) supplemented vitamin B12 (200 mg/Kg) for a period of 4 weeks. Intestinal injury was induced with 2% dextran sodium sulphate (DSS) via drinking water for 5 days. The impact of varying levels of dietary vitamin B12 on gut microbiota composition was assessed using 16S rRNA gene sequencing from fecal samples collected at day 0 and day 28 of the dietary intervention, and 7 days following induction of colitis on day 38, when blood and colonic tissues were also collected. Results: No significant alterations were found in the gut microbiota composition of disease-free animals in response to dietary interventions. By contrast, after DSS-induced colitis, >30 genera were significantly altered in vitamin B12 deficient mice. Altered B12 levels produced no significant effect on composite disease-activity scores; however, administration of a B12 deficient diet resulted in reduced DSS-induced epithelial tissue damage. Conclusions: Vitamin B12 supplementation does not alter the gut microbiota composition under healthy conditions, but does contribute to differential microbial responses and intestinal dysbiosis following the induction of experimental colitis

From mouth to macrophage: mechanisms of innate immune subversion by Mycobacterium avium subsp. paratuberculosis

International audienceJohne’s disease (JD) is a chronic enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP). The high economic cost and potential zoonotic threat of JD have driven efforts to develop tools and approaches to effectively manage this disease within livestock herds. Efforts to control JD through traditional animal management practices are complicated by MAP’s ability to cause long-term environmental contamination as well as difficulties associated with diagnosis of JD in the pre-clinical stages. As such, there is particular emphasis on the development of an effective vaccine. This is a daunting challenge, in large part due to MAP’s ability to subvert protective host immune responses. Accordingly, there is a priority to understand MAP’s interaction with the bovine host: this may inform rational targets and approaches for therapeutic intervention. Here we review the early host defenses encountered by MAP and the strategies employed by the pathogen to avert or subvert these responses, during the critical period between ingestion and the establishment of persistent infection in macrophages

Crystal structure of the bb ' domains of the protein disulfide isomerase ERp57

The synthesis of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a family of ER proteins which includes general enzymes such as PDI that recognize unfolded proteins and others that are selective for specific proteins or classes. Using small-angle X-ray scattering and X-ray crystallography, we report the structure of a selective isomerase, ERp57, and its interactions with the lectin chaperone calnexin. Using isothermal titration calorimetry and NMR spectroscopy, we show that the b' domain of ERp57 binds calnexin with micromolar affinity through a conserved patch of basic residues. Disruption of this binding site by mutagenesis abrogates folding of RNase B in an in vitro assay. The relative positions of the ERp57 catalytic sites and calnexin binding site suggest that activation by calnexin is due to substrate recruitment rather than a direct stimulation of ERp57 oxidoreductase activity200639NRC publication: Ye

Non-digestible oligosaccharides directly regulate host kinome to modulate host inflammatory responses without alterations in the gut microbiota

<p>Data repository for:</p><p> </p><p>Wu RY, Määttänen P, Napper S, Scruten E, Li B, Koike Y, Johnson-Henry KC, Pierro A, Rossi L, Botts SR, Surette MG, Sherman PM. Non-digestible oligosaccharides directly regulate host kinome to modulate host inflammatory responses without alterations in the gut microbiota. Microbiome. 2017;5:135.</p><p> </p><p>Available at: <a href="https://doi.org/10.1186/s40168-017-0357-4">https://doi.org/10.1186/s40168-017-0357-4</a></p><p>Repository information:</p><p> </p><p>PSWY FASTQ sequence files - 16S rRNA gene sequence files for P10 mice exposed to lipopolysaccharide (LPS) and non-digestible oligosaccharides (inulin and scFOS)</p><p> </p><p>16S_metadata.txt - describes 16S rRNA gene sequence files</p><p> </p><p>16S_preprocessed_sequences.fna - preprocessed sequence file for 16S rRNA gene data (quality filtering, chimera checking, etc.)</p><p> </p><p>16S_otu_table_host_singleton_filtered_unrarified.biom - OTU table for 16S rRNA gene sequence analysis before rarefaction</p><p> </p><p>16S_otu_table_host_singleton_filtered_sd9194.biom - OTU table for 16S rRNA gene sequence analysis after rarefaction to 9,194 OTU counts/sample</p><p> </p><p>16S_PICRUSt_analysis_code.txt - commands for PICRUSt functional metagenome prediction</p><p> </p><p>kinome_array_data.txt - raw kinome array data for Caco-2Bbe1 cells exposed to non-digestible oligosaccharides (inulin and scFOS) and challenged with enterohemorrhagic <i>Escherichia coli</i> (EHEC)</p><p> </p><p>kinome_array_metadata.txt - describes raw kinome array data</p><p> </p><p> </p><p>kinome_array_PIIKA_2_parameters.txt - PIIKA 2 parameters for kinome array analysis</p

ERp57 does not require interactions with calnexin and calreticulin to promote assembly of class I histocompatibility molecules and it enhances peptide loading independently of its redox activity

ERp57 is a thiol oxidoreductase that catalyzes disulfide formation in heavy chains of class I histocompatibility molecules. It also forms a mixed disulfide with tapasin within the class I peptide loading complex, stabilizing the complex and promoting efficient binding of peptides to class I molecules. Since ERp57 associates with the lectin chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, we examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells. Moreover, ERp57 formed a mixed disulfide with tapasin and promoted efficient peptide loading in the absence of interactions with calnexin and calreticulin. These findings suggest that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin chaperones. We also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide binding site in substrate recognition. Finally, by inactivating the redox-active sites of ERp57, we demonstrate that its enzymatic activity is dispensable in stabilizing the peptide loading complex and in supporting efficient peptide loading. Thus, ERp57 appears to play a structural rather than catalytic role within the peptide loading complex