The overall goal of this application is to define mechanisms of glycan metabolism by bacteria inhabiting the human gut microbiome so as to establish novel therapeutic pathways to manipulating the composition of the gut microbial community to treat myriad human diseases. The gut microbiota has a profound effect on human health and physiology, providing the host benefits such as modulation of immune development, inhibition of pathogen colonization, digestion of dietary fibers and absorption of nutrients. Abnormalities in microbiota composition, or dysbiosis, however, have been implicated in numerous and diverse disease states. A critical variable that dictates the composition and physiology of the microbiota is the influx of glycans into the intestine, mostly from diet and host mucosal secretions. Given the broad diversity of glycans that enter and exist in the gut, microorganisms must possess varied and efficient strategies for competing for these nutrients, on which they depend as a critical energy source. Despite the prominence of glycans in the human intestine and its documented role in controlling important aspects of health and disease, the molecular mechanisms of glycan breakdown and import employed by gut microbes remain poorly understood. This severely limits our ability to build an intellectual framework for the design of methods and molecules with which to remodel the composition of the gut microbiota in order to improve human health – for instance, to favor commensal over pathogenic bacteria or to limit the growth of inflammatory or antibiotic-resistant bacteria. Here, we propose mechanistic studies using biochemical, biophysical and genetic techniques to define pathways employed by gut microbes for the degradation and import of various glycans, and to determine the structures and functions of key enzymes that release glycans, as well as their interactions and functional cooperation with proteins that capture these released glycans. This work is significant because it addresses molecular mechanisms involved in the major human health burden of dysbiosis. The proposed studies are innovative both technically and conceptually – from the development and employment of novel mass spectrometry-based methods for measuring the specificity and kinetics of protein deglycosylation to the hypotheses that a single bacterium can express multiple enzymes with the same glycan specificity in order to survive in distinct environments and that glycan-hydrolyzing enzymes have evolved to become a new class of cell surface glycan-binding proteins. The research plan will be accomplished through ongoing and new collaborations between the Sundberg, Koval and Mallagaray labs, who bring non-overlapping, complementary and synergistic strengths in structural glycobiology and super-resolution microscopy, resulting in a collaborative team ideally suited to this project.