Synthetic Mucins as Glycan-Defined Prebiotics

Abstract The human microbiome contains at least as many bacterial cells as human cells. The mucosal layer that lines all epithelial cells organizes, cultivates, and regulates these bacterial inhabitants. Some commensal bacteria offer benefits, like improving gut barrier function, suppressing pathobiont growth, and modulating host immunity. These health benefits have fueled the popularity of probiotics, but their retention is often hindered by their low colonization efficiency and mucosal adhesion. Mucins, the primary structural components of the mucosal layer, are essential for the organization and regulation of microbial populations, promoting growth and offering sites for adhesion through their multivalent presentation of O-glycosylation. The molecular mechanisms of mucin– probiotic interactions remain understudied due, in part, to the inability to incisively manipulate native mucin sequences or the glycans they bear. In this investigation, we developed synthetic mucins with defined glycan presentations to interrogate glycan-dependent interactions between mucus and probiotic Lactobacillus species. Though synthetic mucins can dampen the effects of pathogens, toxins, and viruses, their impact on probiotic bacteria as prebiotics or binding sites is unclear. We synthesized mucin surrogates that bind to three investigated Lactobacillus species. The nutrient conditions under which bacteria were cultured influenced glycan binding preferences, suggesting mucin–probiotic interactions change with nutrient availability. The addition of synthetic mucins to native mucin increased Lactobacillus fermentum adherence. Additionally, an increase in Lactobacillus glycosidase activity indicated that native and synthetic mucins both function as prebiotics, as probiotic bacteria can cleave the displayed O-glycans. Thus, synthetic mucins can cultivate target probiotic bacteria and increase adhesion as binding sites, highlighting their value as tools for elucidating native mucin functions and as promising agents for promoting human health. Competing Interest Statement The authors have declared no competing interest. Abbreviations - 4-MU - 4-methylumberriferyl - AF405 - AlexaFluor 405 - α-ArMan - aryl α-mannosylated - cFDA - carboxyfluorescein diacetate - DMSO - dimethylsulfoxide - α-Fuc - fucosylated - β-Gal - β-galactosylated - β-GalNAc - N-acetyl galactosaminated - β-GlcNAc - N-acetyl glucosaminated - HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer - β-Lac - lactosylated - MRS - Man,Rogosa, and Sharpe broth - OD600 - optical density at 600 nm - PEG3 - triethylene glycol - qPCR - quantitative polymerase chain reaction