Characterization of Gastrointestinal Mucin-degrading Systems in Gut Bacteria

Abstract

Mucus is a protective barrier secreted in the gastrointestinal tract to promote healthy separation between host tissues and the resident gut microbiota. Mucus forms two distinct layers covering the epithelium and is mainly composed of complex, highly glycosylated mucin monomers. Interestingly, some bacterial species occupy the outer mucus layer and utilize it as a nutrient source. While this is a feature of a healthy microbiota, mucin-degrading bacteria have been implicated in contributing to disease, such as inflammatory bowel disease, when present with other risk factors. Bacterial enzymes that target highly complex mucin glycoproteins have been identified, but a single enzymatic repertoire that enables a species to degrade specific components of mucins has not been identified. Thus, mucin-degrading mechanisms must be empirically characterized in individual species to understand how these bacteria access different components of mucin and how this activity influences the gut bacterial community.

Here, I further characterize the mucin-degrading mechanisms of two gut bacteria: Ruminococcus torques and Bacteroides thetaiotaomicron. I found that R. torques degrades both intact mucin glycoproteins and free mucin glycans, predominantly using constitutively expressed, secreted enzymes. This mechanism allows R. torques to cross-feed degraded mucin products to B. thetaiotaomicron, which can only utilize free mucin glycans, as demonstrated in in vitro co-culture experiments and growth curves on R. torques pre-digested mucin. Thus, I have established that R. torques is a keystone mucin-degrader, which acts as a primary degrader of this complex substrate and releases simpler products that become available to species that cannot access mucin glycoprotein alone.

While examining interspecies interactions is important to understand how mucin is degraded within the gut bacterial community, identifying and characterizing individual mucin-degrading enzymes is important to identify potential therapeutic targets to block bacterial mucin-degradation in vivo. To this end, we identified the activity of 36 putative mucin glycan-degrading glycoside hydrolase enzymes in B. thetaiotaomicron, including the discovery of novel endo-glycanase activity in three GH18 family enzymes. Interestingly, B. thetaiotaomicron encodes a redundant mucin-degrading enzyme repertoire, expressing multiple enzymes from the same glycoside hydrolase family or with the same substrate specificities. I assessed the contributions of a subset of these enzymes to the mucin-degrading ability and fitness of B. thetaiotaomicron by testing gene deletion mutant strains in in vitro growth assays on purified mucin substrates and in vivo competitions against the parent strain. Indeed, enzymatic redundancy did protect against loss of some enzymes, such as fucosidases. However, in other cases, such as with a mutant lacking three mucin-degrading loci, complementation with a single sulfatase enzyme was sufficient to rescue the loss of the other enzymes in these loci. Identification of these key enzymes critical to the ability of B. thetaiotaomicron to degrade mucin substrates will inform future experiments to develop approaches to blocking mucin-degradation.

Together, these results underscore the importance of continued investigation and characterization of mucin-degrading mechanisms in additional species to understand both community interactions and individual enzyme contributions to this phenotype, which will facilitate the development of tools to block mucin-degradation in cases where it contributes to disease.