Glycan Catabolism by Human Gut Symbionts involves Dynamic Protein Interactions

Abstract

The human gut hosts trillions of bacteria that directly influence human health. The majority of gut microbiota play an important role in nutrition by metabolizing host-indigestible complex glycans into short-chain fatty acids. Bacteroides thetaiotaomicron (Bt), a prominent bacterial symbiont in the distal gut, metabolizes over a dozen complex glycans using membrane-associated protein complexes. The Starch Utilization System (Sus), a multi-protein complex in Bt that is essential for growth on starch, uses eight proteins (SusRABCDEFG) to process starch. SusCDEFG localize in the outer membrane and likely form a complex to facilitate starch binding, degradation and import. However, conventional biochemical methods have been unable to completely reveal the assembly and dynamics of these proteins in response to starch. We have applied single-molecule super-resolution imaging to characterize the Sus complex response to different sugars in live Bt under anaerobic conditions. Protein correlation studies performed with HaloTag-labeled SusG and other fluorescent antibody-labeled Sus proteins demonstrated that simple sugars such as glucose or maltose do not induce Sus complex assembly. Conversely, incubation of Bt cells with starch enhanced the co-localization of Sus proteins, suggesting starch-induced assembly of the complex. Furthermore, single-molecule tracking revealed that in the absence of starch, SusG predominately moves in a fast diffusion mode corresponding to freely diffusing SusG. In contrast, two-color single-molecule experiments performed with fluorophore-labeled starch and SusG showed that starch confines the free motion of SusG and induces the assembly of Sus proteins. Overall, our results suggest that starch catabolism involves dynamic interactions of Sus proteins, which assemble as a complex in the presence of starch in live cells.