Bifidobacterium dentium Fortifies the Intestinal Mucus Layer via Autophagy and Calcium Signaling Pathways

Abstract

Much remains unknown about how the intestinal microbiome interfaces with the protective intestinal mucus layer. Bifidobacterium species colonize the intestinal mucus layer and can modulate mucus production by goblet cells. However, select Bifidobacterium strains can also degrade protective glycans on mucin proteins. We hypothesized that the human-derived species Bifidobacterium dentium would increase intestinal mucus synthesis and expulsion, without extensive degradation of mucin glycans. In silico data revealed that B. dentium lacked the enzymes necessary to extensively degrade mucin glycans. This finding was confirmed by demonstrating that B. dentium could not use naive mucin glycans as primary carbon sources in vitro. To examine B. dentium mucus modulation in vivo, Swiss Webster germfree mice were monoassociated with live or heat-killed B. dentium. Live B. dentium-monoassociated mice exhibited increased colonic expression of goblet cell markers Krüppel-like factor 4 (Klf4), Trefoil factor 3 (Tff3), Relm-β, Muc2, and several glycosyltransferases compared to both heat-killed B. dentium and germfree counterparts. Likewise, live B. dentium-monoassociated colon had increased acidic mucin-filled goblet cells, as denoted by Periodic Acid-Schiff-Alcian Blue (PAS-AB) staining and MUC2 immunostaining. In vitro, B. dentium-secreted products, including acetate, were able to increase MUC2 levels in T84 cells. We also identified that B. dentium-secreted products, such as γ-aminobutyric acid (GABA), stimulated autophagy-mediated calcium signaling and MUC2 release. This work illustrates that B. dentium is capable of enhancing the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways, with a net increase in mucin production. IMPORTANCE Microbe-host interactions in the intestine occur along the mucus-covered epithelium. In the gastrointestinal tract, mucus is composed of glycan-covered proteins, or mucins, which are secreted by goblet cells to form a protective gel-like structure above the epithelium. Low levels of mucin or alterations in mucin glycans are associated with inflammation and colitis in mice and humans. Although current literature links microbes to the modulation of goblet cells and mucins, the molecular pathways involved are not yet fully understood. Using a combination of gnotobiotic mice and mucus-secreting cell lines, we have identified a human-derived microbe, Bifidobacterium dentium, which adheres to intestinal mucus and secretes metabolites that upregulate the major mucin MUC2 and modulate goblet cell function. Unlike other Bifidobacterium species, B. dentium does not extensively degrade mucin glycans and cannot grow on mucin alone. This work points to the potential of using B. dentium and similar mucin-friendly microbes as therapeutic agents for intestinal disorders with disruptions in the mucus barrier.

Oxyresveratrol Induces Autophagy via the ER Stress Signaling Pathway, and Oxyresveratrol-Induced Autophagy Stimulates MUC2 Synthesis in Human Goblet Cells

“…Mucin is known as the major component in the intestinal mucus layer, and the intestinal mucosal barrier plays a critical role in maintaining intestinal homeostasis and preventing external damage. Autophagy promotes expression of MUC2 [19,50,51], and OXY induces MUC2 expression in intestinal goblet cells [17]. In this study, we found that OXY activates autophagy and that OXY-induced MUC2 expression is decreased by an ER inhibitor.…”

Reuterin disrupts Clostridioides difficile metabolism and pathogenicity through reactive oxygen species generation

“… 78 Mucin proteins from LS174 T supernatants were collected by ETOH precipitation with protease inhibitors as previously described. 79 MUC2 was purified from crude mucin by guanidinium chloride and cesium chloride gradient ultracentrifugation on a Beckman Coulter Ultra-Centrifuge (30.2 Ti rotor) as previously described. 80 MUC2-containing fractions were identified by slot blot immunostaining with a MUC2 antibody (cat# NBP1-31231, 1:100 dilution, Novus), LI-COR Odyssey Blotting reagents, Odyssey imaging system, and Image Studio software (LI-CORE Biosciences).…”

Role of Protein Glycosylation in Host-Pathogen Interaction

“…This is because the attack of most pathogens is blocked by glycosylated proteins covering the host cell surface as barriers. Such barriers may act in two manners in host defense, (I) physically separate pathogens and host cells [75,76]; (II) chemically inhibit or kill pathogens [16,[77][78][79].…”