ABSTRACT: Raw LCMS spectra data showing the activity of genetically engineered E. coli expressing bile acid metabolic enzymes when fed with specific bile acid substrates.
Project description:Transcriptomic studies of high-cell density fermentations of E. coli producing chemicals of relevance for biotechnology are limited. The aim of this study was to perform fermentations of an E. coli strain that had been engineered to produce styrene and examine the transcriptomic response. This was compared to an identical strain of E. coli that had the styrene pathway inactivated by the introduction of amino acid mutations in PAL2 and fdc1, the two enzymes required for styrene production. A further comparison was done by exposing E. coli cells to styrene added externally to the fermentation broth.
Project description:Bile acid diarrhoea is a chronic condition caused by increased delivery of bile acids to the colon. The underlying mechanisms remain to be elucidated. To investigate genes involved in bile acid diarrhoea, systems-level analyses were employed on a rat bile acid diarrhoea model. Twelve male Wistar Munich rats, housed in metabolic cages, were fed either control or bile acid-mixed (1% w/w) diets for ten days. Food intake, water intake, urine volume, bodyweight and faecal output were monitored daily. After euthanasia, colonic epithelial cells were isolated using calcium-chelation and processed for systems-level analyses, i.e. RNA-sequencing transcriptomics and mass spectrometry proteomics. Bile acid-fed rats suffered diarrhoea, indicated by increased drinking, faeces weight and faecal water content compared with control rats. Urine output was unchanged. With bile acid-feeding, RNA-sequencing revealed 204 increased and 401 decreased mRNAs; mass spectrometry 183 increased and 111 decreased proteins. Among the altered genes were genes associated with electrolyte and water transport (including Slc12a7, Clca4 and Aqp3) and genes associated with bile acid transport (Slc2b1, Abcg2, Slc51a, Slc51b and Fabps). Correlation analysis showed a significant positive correlation (Pearson’s r=0.28) between changes in mRNA-expression and changes in protein-expression. However, caution must be exercised in making a direct correlation between experimentally determined transcriptomes and proteomes. Genes associated with bile acid transport responded to bile acid-feeding, suggesting that colonic bile acid transport occur by regulated protein facilitated mechanisms rather than passive diffusion. In addition, the study provides annotated rat colonic epithelial cell transcriptome and proteome with response to bile acid-feeding.
Project description:Abstract: Interruption of bile acid recirculation through inhibition of the apical sodium-dependent bile acid transporter (ASBT) is a promising strategy to alleviate hepatic cholesterol accumulation in non-alcoholic steatohepatitis, and improve the metabolic aspects of the disease. Putative disease-attenuating effects of the ASBT inhibitor volixibat (5, 15, and 30 mg/kg) were investigated in high-fat diet (HFD)-fed Ldlr-/-.Leiden mice over 24 weeks. Plasma and fecal bile acid levels, plasma insulin, lipids, and liver enzymes were monitored. Final analyses included liver histology, intrahepatic lipids, mesenteric white adipose tissue mass, and liver gene profiling. Consistent with its mechanism of action, volixibat significantly increased total bile acid excretion. At the highest dose, volixibat significantly attenuated the HFD-induced increase in hepatocyte hypertrophy, hepatic triglyceride and cholesteryl ester levels, and mesenteric white adipose tissue deposition, while total plasma bile acid levels remained constant. Non-alcoholic fatty liver disease activity score was significantly lower in volixibat-treated mice than in the HFD controls. Gene profiling showed that volixibat reversed the inhibitory effect of the HFD on metabolic master regulators, including peroxisome proliferator-activated receptor-γ coactivator-1β, insulin receptor, and sterol regulatory element-binding transcription factor 2. Volixibat may have beneficial effects on physiological and metabolic aspects of non-alcoholic steatohepatitis pathophysiology.
Project description:the commensal gut microbiota modulates multiple sclerosis with unknown mechanisms. Commensal bacteria producing bile acid-deconjugating enzymes are fundamental to generate secondary bile acid metabolites (BAM) with immunoregulatory function. We show that immune regulatory BAM prevent autoimmunity in the central nervous system by inducing immune tolerance at the intestinal level and peripherally limiting effector function and aggressiveness of myelin-reactive T cells. We validated the key role of microbiota-induced BAM in humans by showing that relapsing-remitting multiple sclerosis patients have significantly reduced level of an important immune regulatory BAM (deoxycholic acid) and lower abundance of BAM-producing bacteria that correlate with increased percentages of peripheral effector Th17 cells. Our data indicate that the immune regulatory biliary network is crucial for the prevention of central nervous system autoimmunity.
Project description:Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis, and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG(+/-) mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR.
Project description:Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis, and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG(+/-) mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR
Project description:Intestinal lipid absorption, the entry-point for fats into the body, requires the coordinated actions of bile acids and lipases. Here, we uncover distinct yet cooperative roles of bile acids in driving the differential uptake of dietary fatty acids. We first decreased bile acid pool size by disrupting the rate-limiting enzyme in bile acid synthesis, Cyp7a1, using liver-directed gene editing in mice. Compared to lipase inhibition, reduced bile acids prevented diet-induced obesity, increased anorectic hormones, suppressed excessive eating, and improved systemic lipid metabolism. Remarkably, decreasing bile acids selectively decreased absorption of saturated fatty acids, but preserved polyunsaturated fatty acids. By targeting additional bile acid enzymes, we identified specific functions of individual bile acid species. Mechanistically, we show that cholic acid preferentially solubilizes polyunsaturated fatty acids into mixed micelles for intestinal uptake. Our studies demonstrate that bile acids can selectively control fatty acid uptake, revealing insights for future interventions in metabolic disease.
Project description:Commensal microbes are exposed to enterohepatically circulated steroids such as bile acids and hormones in the gastrointestinal, vaginal, and urinary tracts. Since commensal microbes are exposed to these molecules exclusively in association with their host, we hypothesized that they may serve as effectors to identify and characterize genetic pathways involved in the commensal-host relationship. Host specific and some ingested steroids (phytoestrogens) are enterohepatically circulated through the lumen of the small intestine. Bile acid steroids are present at high concentrations, approximating 4 to 20 mM in the duodenum, and are released in bile from the common bile duct. Steroid hormones are secreted in bile at levels approximating 6 to 13 mg per day once conjugated to glucuronide or sulfate by the liver. Re-absorption of steroids by the terminal ileum is an incomplete process: for example, 200 to 600 mg of bile acid steroids per day in humans escape to the colon where complex microbial populations exist. We have shown that steroid hormones estradiol, progesterone, and hydrocortisone serve as strong substrates for the major RND- and MFS-type (except hydrocortisone) tripartite multidrug efflux systems, AcrAB-TolC and EmrAB-TolC respectively, even though such molecules fail to demonstrate antimicrobial properties in this bacterial background (Elkins and Mullis [2006] J. Bacteriol. 188:1191-1195). Although bile acids are subject to efflux, they are also known to directly interact with intracellular global regulators such as MarR (Prouty et al. [2004] Microbiol. 150:775-783) and Rob (Rosenberg et al., [2003] Mol. Microbiol. 48:1609-1619) consequently altering antimicrobial and bile resistance profiles. In our present study, whole-genome DNA microarrays of E. coli were used to determine what general effect steroids, both bile acid and hormones, may have on the transcriptome in relation to the human commensal environment. Keywords: Comparative Chemical Class Treatment
Project description:Bile acid diarrhoea is a chronic condition with increased delivery of bile acids to the colon causing diarrhoea. In this study we used a rodent model of bile acid malabsorption to investigate the general impact on increased levels of bile acids on mRNA- and protein-expression in colon epithelial cells. Twelve male Wistar Munich rats were housed in metabolic cages and fed control or bile acid-mixed (1% w/w) diets for ten days. After euthanasia, colonic epithelial cells were isolated using Ca2+ chelation and processed for mass spectrometry-based proteomics to quantify changes in protein expressions.