Project description:Aided by deep sequencing techniques, recent studies suggest the existence of distinct breast microbiota and a shift in microbial community composition in diseased breast compared to normal breast. However, their functional impact and underlying mechanisms are unknown. Present study examines the contribution of pro-carcinogenic bacteria in breast cancer initiation, growth and progression. Extensive data mining and metagenomic analyses of existing datasets revealed the presence of toxin producing Bacteriodes fragilis in malignant breast. B. fragilis is a pro-carcinogenic bacterium known for its potential to initiate and promote colon cancer; its pathogenicity has been attributed to its unique toxin ‘BFT’. About 35% of human population asymptomatically harbor ETBF colonization in the gut. Mice with enteric B fragilis infection exhibited a significant amount of circulating BFT and distinct morphological alterations in mammary gland similar to focal hyperplasia. Histological analysis revealed inflammation, fibrosis, breast duct thickening and hyperproliferation of breast epithelial cells. In vitro, upon treatment with BFT, prominent cytoskeletal reorganization, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells were observed along with molecular markers of epithelial-to-mesenchymal transition. Decreased expression of epithelial marker, E-cadherin along with elevated levels of mesenchymal markers, N-cadherin and vimentin were observed. BFT also increased the expression of EMT-related transcription factors, Snail, Slug and Twist. BFT-treated cells attained stem cell-like phenotype exhibiting an increased ability to form secondary and tertiary mammospheres. Mechanistic studies showed that BFT induced expression and nuclear translocation of NICD and β-catenin resulting in activation of downstream targets. Inhibition of Notch1 and β-catenin using γ-secretase and β-catenin inhibitors successfully inhibited functional effects of BFT. Mammary gland implantation and in vivo limiting dilution assays were utilized to corroborate the in vitro findings. BFT-pretreated MCF7 cells exhibit increased tumor growth and form multifocal tumors in mice. In vivo limiting dilution assay using breast tumors from BFT-pretreated MCF7 cells exhibited a striking increase in tumor-initiating cells. Follow-up analyses of these tumors demonstrated increased migratory, invasive, and -mammospheres-forming behavior confirming that brief BFT exposure elicits long-term molecular changes. Altered expression of stemness markers was also confirmed by RNA sequencing.

Project description:Background & Aims: Alcoholic liver disease (ALD) is a major public health concern. Gut dysbiosis plays a critical role in its pathogenesis, yet the specific microbial and functional shifts driving ALD progression require further elucidation. This study aims to characterize these alterations and their mechanisms.Methods: We recruited 28 male patients with alcoholic fatty liver (AFL), 22 with alcoholic cirrhosis (ALC), and 25 healthy male controls. Fecal samples were analyzed via shotgun metagenomic sequencing and targeted short-chain fatty acid (SCFA) metabolomics. Serum samples underwent untargeted metabolomics. Key findings were validated in mouse models through fecal microbiota transplantation (FMT), bacterial gavage, and integrated transcriptomic and metabolomic analyses.Results: Metagenomic analysis revealed a depletion of SCFA-producing bacteria (e.g., Faecalibacterium prausnitzii, Fusicatenibacter saccharivorans) and an enrichment of enterotoxigenic Bacteroides fragilis (ETBF) in ALD patients. Fecal concentrations of acetate, propionate, and butyrate were significantly decreased and negatively correlated with serum ALT, AST, and GGT levels. Serum metabolomics identified a distinct profile in ALD, featuring elevated levels of metabolites such as 9,10-DHOME and reduced levels of specific lysophosphatidylcholines. Multi-omics integration suggested microbial regulation of ALD via bile acid and SCFA pathways. FMT from ALD patients exacerbated alcohol-induced liver injury in mice. Gavage with ETBF significantly worsened ALD phenotypes in mice, likely mediated by Bacteroides fragilis toxin (BFT)-induced gut barrier damage and inflammation. Mouse multi-omics further demonstrated that ETBF perturbation enhanced the tryptophan-kynurenine metabolic pathway and upregulated hepatic retinol metabolism, contributing to increased liver injury, inflammation, and fibrosis.Conclusion: Gut dysbiosis characterized by the loss of beneficial SCFA producers and the rise of pathogenic ETBF is pivotal in ALD progression. ETBF exacerbates disease via toxin-driven barrier dysfunction and the dysregulation of tryptophan and retinol metabolic pathways, revealing novel mechanistic insights and potential therapeutic targets.

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Effect of co-culture with ETBF in NOZ cell line on gene expreesion difference

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