The bacteria that inhabit the human intestinal tract are essential for immune and gastrointestinal development, pathogen protection, and complex carbohydrate digestion. Their ability to thrive in this niche is dependent upon their ability to extract carbohydrate nutrition from this highly competitive ecosystem. Bacteroidetes are numerically dominant Gram-negative members of the human gut microbiota that all rely upon similarly patterned outermembrane protein systems termed starch utilization (Sus)-like systems to capture carbohydrate nutrition. Every Sus-like system targets a unique glycan, and some species devote nearly 20% of their genomes towards encoding these proteins. Sus-like systems are only found in the Bacteroidetes, making these proteins attractive targets for manipulating the metabolism of these organisms to support human health. Our long-term goal is to understand the molecular events that support glycan utilization via Sus-like systems. In this proposal we will focus on the molecular interactions among the outermembrane proteins SusCDEFG in the prototypical starch utilization system (Sus) of Bacteroides thetaiotaomicron (Bt). We have determined the molecular structures of the starch-binding lipoproteins SusDEFG, but how these proteins interact with the TonB-dependent transporter SusC to facilitate glycan import is unknown. All Sus-like systems have homologs of SusC, and of SusD, and so understanding how SusCD interact with each other and with SusEFG will inform a general model of how Sus-like systems facilitate glycan uptake.
In Specific Aim 1 we will determine the nature and stoichiometry of the SusCD interaction, as well as how SusEFG affect this assembly. Our working hypothesis is that SusD facilitates interactions between SusC and the SusEFG proteins. We will identify interacting Sus proteins via co-immunoprecipitation and proteomics. In parallel, we will examine the co- localization and stoichiometry of the Sus proteins in live cells y single molecule imaging.
In Specific Aim 2 we will create a functional map of the SusC structure. Our working hypothesis is that the extracellular loops of SusC bind SusD. Although recombinant SusD has weak affinity for starch, the SusCD interaction may create a higher affinity site for maltooligosaccharides that enhances import. We will test targeted mutants of susC and susD in Bt and E. coli for their ability to interact with each other and starch. We will incorporate unnatual amino acids into SusC to introduce fluorescent tags and photo-crosslinkable residues that can map the interaction between SusC and SusD. Finally, we will determine the x-ray crystal structure of SusC with or without SusD to understand its topology and function. Together, these data will reveal the molecular details of the Sus complex assembly, and allow us to generate a working model of a conserved glycan acquisition paradigm that is unique to the Bacteroidetes. With these details we can design new and selective strategies to manipulate microbial metabolism in the human gut.
Bacteroidetes are dominant bacterial members of the human gut microbiota and essential for maintaining health. Here we will determine the assembly of a model carbohydrate uptake system, the starch utilization system (Sus), found in gut Bacteroides species. This work will reveal the molecular details of a conserved carbohydrate uptake pathway unique to the Bacteroidetes, and provide new strategies for manipulating microbial metabolism in the human gut.
| Tuson, Hannah H; Foley, Matthew H; Koropatkin, Nicole M et al. (2018) The Starch Utilization System Assembles around Stationary Starch-Binding Proteins. Biophys J 115:242-250 |
| Foley, Matthew H; Martens, Eric C; Koropatkin, Nicole M (2018) SusE facilitates starch uptake independent of starch binding in B. thetaiotaomicron. Mol Microbiol 108:551-566 |
| Arnal, Gregory; Cockburn, Darrell W; Brumer, Harry et al. (2018) Structural basis for the flexible recognition of ?-glucan substrates by Bacteroides thetaiotaomicron SusG. Protein Sci 27:1093-1101 |
| Cockburn, Darrell W; Koropatkin, Nicole M (2016) Polysaccharide Degradation by the Intestinal Microbiota and Its Influence on Human Health and Disease. J Mol Biol 428:3230-3252 |
| Foley, Matthew H; Cockburn, Darrell W; Koropatkin, Nicole M (2016) The Sus operon: a model system for starch uptake by the human gut Bacteroidetes. Cell Mol Life Sci 73:2603-17 |
