The microbial community that inhabits the human distal gut increases our ability to digest complex carbohydrates (glycans). Bacteria in this community have evolved several strategies to metabolize the many diet- and host-derived glycans that inundate their habitat. Members of the Bacteroidetes, one of two numerically dominant phyla of gut bacteria, possess a series of homologous outer membrane protein systems (Sus-like systems) that bind and enzymatically degrade glycans. These species ubiquitously produce multiple capsular polysaccharides (CPS) on their cell surfaces. The role of these capsules remains undefined, although some studies point to evasion or manipulation of host immunity. We have shown that CPS expression in the abundant human gut symbiont Bacteroides thetaiotaomicron (Bt) is coordinated with expression of some Sus- like systems involved in degrading host-derived mucus glycans. Moreover, Bt populations that are forced to rely exclusively on host glycans in the intestines of gnotobiotic mice express different CPS relative to Bt living in mice fed a diet rich in plant glycans. These observations lead to our central hypothesis that Bt coordinates expression of its surface CPS structures with the particular glycan that it is catabolizing because the biochemical properties of each individual capsule are compatible with a specific subset of glycan nutrients. Several properties could contribute to this phenomenon, including increased miscibility of exogenous glycans with some CPS structures or recycling of sugars derived from degraded glycans into new capsules. The Bt type strain (VPI-5482) encodes eight different gene clusters for producing CPS. Our preliminary data suggest that alterations in CPS gene expression by this strain decrease its growth rate on some substrates such as mucus O-glycans, while rendering it identical to or faster than wild-type on others. To extend these findings, we have constructed a series of eight mutants that are each deficient in all but one CPS locus. Each strain produces only a single surface capsule, allowing us to isolate its contribution to growth on different glycans in vitro and in vivo. We wil use these eight strains, in conjunction with a custom growth array containing 47 different carbohydrates, to measure the effect of individual capsules on Bt glycan metabolism in vitro. In addition, these strains provide a unique opportunity to isolate each CPS polymer and explore its glycochemical structure, which we will perform in collaboration Dr. Bradley Reuhs from Purdue University. Finally, we will introduce nucleotide signature-tagged variants of these eight strains into germfree mice to measure their ability to compete against each other in vivo. We will manipulate the type and abundance of dietary glycans fed to mice, and examine colonization of the mucus layer by each strain, as two variables that we hypothesize will influence the fitness of individual CPS-expressing strains. Together, the data gathered in the proposed experiments will allow us to integrate the role of variable CPS expression into a growing understanding of how bacteria assemble into a complex and physiologically active community in the human intestinal tract.
The hundreds of bacterial species that colonize the human intestinal tract are essential for the digestion of dietary carbohydrates and also create numerous capsular polysaccharides on their cell surface that protect them from host immune responses. We will measure the interactions between bacterial cell surface capsules and the carbohydrates that intestinal bacterial help us to digest. The findings will shed light on how the many related bacteria that normally live in our intestine influence human health by assisting in carbohydrate digestion or, in some cases, degrading glycans contained in the protective mucus barrier.