We report that commensal bacteria can influence goblet cells and mucin composition in the gut, providing new information about the relation among mucus, bacteria and intestinal homeostasis. B. thetaiotaomicron enhances goblet cell differentiation leading to an increase of goblet cell number and mucin gene expression in the colon of gnotobiotic rats. The presence of B. thetaiotaomicron also affects the composition of mucin O-glycans, with relative decreases in sulfated and neutral oligosaccharides in favor of sialylated oligosaccharides. B. thetaiotaomicron, therefore, appears to provoke modifications in the secretory lineage compared to GF rats, favoring mucus production and we could hypothesize that this is possibly for its own benefit. When B. thetaiotaomicron is associated with F. prausnitzii, which is an acetate consumer and a butyrate producer, the effects on both goblet cells and mucin O-glycosylation are attenuated. Using a novel gnotobiotic model, which is the first described involving F. prausnitzii, we showed that two major species of commensal bacteria can modulate the effects of the bacteria on goblet cells and thus on mucus production and mucin glycosylation.
The GIT is an environment with harsh constraints (pH, oxygen, access to nutrients) which shape the microbial composition. The genome of Bacteroides species possesses gene clusters for capsular polysaccharide synthesis [17, 18] and these polysaccharides are important for bacterial colonization of the GIT: they improve survival in the GIT, and also stimulate the immune system [35–37]. Scanning electron microscopy revealed differences in the B. thetaiotaomicron cell wall in Bt-2d and Bt-30d rats. Possibly, during the first days of adaptation to the GIT, B. thetaiotaomicron produce capsular polysaccharides which collapse through a dehydration process, as previously described [38, 39]. The transcriptomic profile of B. thetaiotaomicron before and after its passage through GIT shows that B. thetaiotaomicron adapts its metabolism to survive in the GIT . When B. thetaiotaomicron is associated with Eubacterium rectale, a representative of the C. coccoides group, both bacteria adapt their own metabolism to the presence of the other . Thus, bacteria in the GIT presumably adapt to the host, and also to other bacteria . In this study, we observed that in mono-associated rats, B. thetaiotaomicron induces goblet cell differentiation leading to an increase in mucin gene expression and goblet cell number, and a parallel decrease in chromogranine produced by enteroendocrine cell. These observations are in accordance with previous work showing that the colonic epithelium also adapts to the presence of bacteria [41–43]; these adaptations are parts of the mutual interactions essential for human health.
We show that, following colonization of the GIT by B. thetaiotaomicron, the pattern of mucin glycosylation changes, with increased expression of glycans carrying sialic acid residues (either NeuAc or NeuGc), correlated with decreased expression of sulfated and neutral oligosaccharides. A previous immunohistochemical study described similar alterations in the glycosylation pattern in the mouse intestine, induced by B. thetaiotaomicron implantation or by soluble factors produced by this microorganism . B. thetaiotaomicron also induces fucosylation of intestinal epithelial cells, which is linked to its ability to use fucose, a component of glycosylated mucin . Thus, glycan production or distribution is modulated by B. thetaiotaomicron, and this may involve processes important for signaling and mediating the host mucosal response. According to the enhancement of the expression of st3gal4 in the presence of B. thetaiotaomicron, we can assume that there is a mechanism by which this bacterium modulates the composition of mucus chain O-glycans by impacting directly enzyme expression of the host. B. thetaiotaomicron may require high levels of expression of sialylated mucins for attachment and colonization of the GIT. Indeed, B. thetaiotaomicron may express adhesins specifically recognizing and binding to sialic acid residues and may also use sialic acid residues as host substrates. B. thetaiotaomicron possesses 28 predicted sulfatases and only 1 single predicted anaerobic sulfatase-maturating enzyme which allow bacteria to adapt to and forage on host sulfated glycans as nutrients . Our observations of a substantial decrease in sulfated oligosaccharides in the rats colon two days after B. thetaiotaomicron colonization are consistent with this enzymatic potential. Presumably, B. thetaiotaomicron uses these sulfate residues rapidly not only as nutrient sources but also to gain access to other monosaccharides, such as N-acetylglucosamine (GlcNAc) and galactose (Gal), the two monosaccharides substituted by sulfate residues on mucin O-glycans. In a favorable ecosystem (that is, in the absence of other microorganisms), there is probably no need for the bacteria or the host to maintain a high level of expression of sulfated glycans. However, when B. thetaiotaomicron is associated with F. prausnitzii, the proportion of sulfated glycans is greater than that observed in the presence of B. thetaiotaomicron alone. This suggests that, in a more complex environment involving a competitive ecosystem, sulfated glycans may confer an ecological advantage on B. thetaiotaomicron.
We were unable to obtain, on a large scale and reproducibly, F. prausnitzii mono-associated rats; the implantation of F. prausnitzii required the prior presence of B. thetaiotaomicron. Furthermore, F. prausnitzii was unable to colonize the GIT early after the introduction of B. thetaiotaomicron. This may have been due to the physicochemical environment. F. prausnitzii became established in the gut only after a decrease in the oxidoreduction potential, caused by the presence of B. thetaiotaomicron, suggesting that B. thetaiotaomicron “prepares” the GIT to accommodate sustainably more oxygen-sensitive bacteria. F. prausnitzii and, to a larger extent, the C. leptum group, arrive in the GIT late after birth, presumably because they are sensitive to oxygen. Moreover, after intestinal resection, disrupting the gut structure and exposing the colon to oxygen, the C. leptum group disappears from the microbiota . In the case of strict anaerobes like F. prausnitzii, their ability to colonize is governed by the physicochemical constraints of gut rather than by any lack of metabolic substrates. This contrasts with Lactobacillus bulgaricus, which is an aerobic-tolerant lactic acid bacterium, and which does not colonize the gut of GF rats in the absence of lactose . That colonization by B. thetaiotaomicron precedes the implantation of F. prausnitzii gives clues about mechanisms governing microbial ecology and processes of colonization.
The two bacteria were metabolically complementary both in vitro and in vivo. F. prausnitzii consumes acetate produced by B. thetaiotaomicron and in turn produces butyrate. Short-chain fatty acids (SCFA) trigger pleiotropic signals in the host, including signals regulating mucin synthesis and secretion [33, 49–51]. SCFA, in particular butyrate and to a lesser extent acetate, are reported to have inducer effects on mucin synthesis and production in vitro[33, 49, 51]. Butyrate also increases the expression of the transcription factor klf4 in HT29 cells . On the contrary, another experiment reported an inhibitor effect of butyrate on mucin synthesis in vitro. All these data show the potential role of SCFA in modulating the mucus pathway and it may be a mechanism by which they affect the goblet cell differentiation pathway in our gnotobiotic models. Using HT29-MTX, a mucus producing cell line, we showed that acetate increases KLF4 abundance. Understanding the effects of the microbiota on mucus production and goblet cell differentiation is of major importance as mucus acts as a protective barrier and disruption of this mucus layer leads to inflammation ; abnormalities of the mucus layer, and modulations of goblet cells and mucin secretion have been described in inflammatory bowel diseases . In a murine model of DSS-induced colitis, the mucus layer is altered before the induction of any inflammation evidencing the importance of mucus layer integrity to prevent inflammation . The O-glycosylation pattern of mucins has also a determinant role in health, with sulfated colonic mucins playing a protective role . Indeed, mucins are poorly sulfated in patients with ulcerative colitis . Butyrate induces the expression of sulfotransferases, the enzymes catalyzing sulfatation of mucin in the mouse colon  and also up-regulates expression of galactose-3-O-sulfotransferase 4 in human intestinal epithelial goblet cells . The increase in sulfated mucins observed in our B. thetaiotaomicron and F. prausnitzii di-associated rats is consistent with these previous studies. We, therefore, suggest that metabolites, mainly acetate and butyrate, produced by B. thetaiotaomicron and F. prausnitzii, respectively, may be responsible, at least in part, for the modifications that we observed in goblet cells and mucin O-glycosylation.