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The human intestinal mucus layer protects against pathogenic microorganisms and harmful substances, whereas it also provides an important colonization niche for mutualistic microbes. The main functional components of mucus are heavily glycosylated proteins, called mucins. Mucins can be cleaved and utilized by intestinal microbes. The mechanisms between intestinal microbes and the regulation of mucin glycosylation are still poorly understood. In this study, in vitro mucus was produced by HT29-MTX-E12 cells under Semi-Wet interface with Mechanical Stimulation. Cells were exposed to pasteurized nonpathogenic bacteria Akkermansia muciniphila , Ruminococcus gnavus , and Bacteroides fragilis to evaluate influence on glycosylation patterns. Following an optimized protocol, O- and N-glycans were efficiently and reproducibly released, identified, and semiquantified using MALDI-TOF-MS and PGC-LC-MS/MS. Exposure of cells to bacteria demonstrated increased diversity of sialylated O-glycans and increased abundance of high mannose N-glycans in in vitro produced mucus. Furthermore, changes in glycan ratios were observed. It is speculated that bacterial components interact with the enzymatic processes in glycan production and that pasteurized bacteria influence glycosyltransferases or genes involved. These results highlight the influence of pasteurized bacteria on glycosylation patterns, stress the intrinsic relationship between glycosylation and microbiota, and show the potential of using in vitro produced mucus to study glycosylation behavior.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are located in the outer membrane of Gram-negative bacteria and are comprised of three distinctive parts: lipid A, core oligosaccharide (OS), and O-antigen. The structure of each region influences bacterial stability, toxicity, and pathogenesis. Here, we highlight the use of targeted activated-electron photodetachment (a-EPD) tandem mass spectrometry to characterize LPS and LOS from two crucial players in the human gut microbiota, Escherichia coli Nissle and Bacteroides fragilis . a-EPD is a hybrid activation method that uses ultraviolet photoirradiation to generate charge-reduced radical ions followed by collisional activation to produce informative fragmentation patterns. We benchmark the a-EPD method for top-down characterization of triacyl LOS from E. coli R2, then focus on characterization of LPS from E. coli Nissle and B. fragilis . Notably, a-EPD affords extensive fragmentation throughout the backbone of the core OS and O-antigen regions of LPS from E. coli Nissle. This hybrid approach facilitated the elucidation of structural details for LPS from B. fragilis , revealing a putative hexuronic acid (HexA) conjugated to lipid A.

Tumor-resident microbiota in breast cancer promotes cancer initiation and malignant progression. However, targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here, we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF, albeit at low biomass, secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH+ breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation, thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increase the chemosensitivity of breast cancer by impairing BCSCs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Higher Education Press.)