264 Bacteroides Thetaiotaomicron and Faecalibacterium prausnitzii Shape the Mucus Production and Mucin O-Glycosylation in Colon Epithelium

Abstract

The intestinal mucus layer plays a key role in themaintenance of host-microbiota homeostasis. The production of goblet cells, which secrete mucus, is modulated by microbiota, but neither the species nor the mechanisms involved in this process are still unknown. We studied how two prominent commensal bacteria may influence the mucus production by goblet cells and the profile of mucin glycosylation in gnotobiotic rats. We have chosen Bacteroides thetaiotaomicron, which is characterized by its high mucus-polysaccharides degrading potential, and Faecalibacterium prausnitzii, which is a sensor of intestinal health. Germ free rats (GF) were orally inoculated with B. thetaiotaomicron either alone or with a mix of B. thetaiotaomicron and F. prausnitzii leading respectively to mono-associated (Bt-rats) and di-associated rats (Bt+Fp-rats). A panel of goblet cells markers was analyzed by histological staining, immunohistochemistry, quantitative PCR and Western blot in colon epithelium. The mucin O-glycosylation was determined by MALDI TOF mass spectrometry. In Bt-rats, the goblet cells number and the expression of mucus-related genes (muc2, muc4, klf4, c1galt1 and b4galt4 mRNAs) were increased compared to GF ones. KLF4 protein, a transcription factor involved in goblet cell terminal differentiation, was also increased in Bt-rats, whereas a decrease in Chromogranin A protein, a marker of enteroendocrine cells was observed. We propose that B. thetaiotaomicron provokes an imbalance inside the secretory lineage by favoring mucus production at the expense of enteroendocrine cells. When B. thetaiotaomicron was associated to F. prausnitzii, the effects on goblet cells were reduced/ decreased/diminished. Indeed, the number of goblet cells per crypt and the amount of KLF4 protein were lower in Bt+Fp-rats than in Bt-rats. We then analyzed the mucus quality by studying the profile of mucin O-glycosylation. In Bt-rats, a decrease in the production of sulfated (4.5% of total oligosaccharides instead of 12.9%) and neutral (40.1% instead of 52.8%) oligosaccharides was observed and was correlated to an increased proportion of sialylated O-glycans carrying NeuAc (24.2% instead of 18.9%) or NeuGc (31.2% instead of 15.4%) residues compared to GF rats. Thus, B. thetaiotaomicron impacts the composition of mucin O-glycans, with a decrease in sulfated and neutral oligosaccharides in favor of sialylated ones. Furthermore, glycosylation of mucins from Bt+Fp-rats resembled to those of GF rats. As previously observed for goblet cells, F. prausnitzii seemed to decrease the effect of B. thetaiotaomicron on mucus. Using a novel gnotobiotic model, which is the first described with F. prausnitzii, we showed how the balance between B. thetaiotaomicron and F. prausnitzii plays a key role in protecting epithelium via their respective effects on mucus.