Understanding nutrient transport across the outer membrane by members of the human gut microbiota

Abstract

The human gut contains a dense group of microbes termed the microbiota, which has been shown to play a major role in health and disease. Despite significant diversity at species level, the microbiota is dominated by only two phyla, the Gram-negative Bacteroidetes and Gram-positive Firmicutes. The Bacteroidetes are able to use a wide range of different complex glycans from both dietary and host sources. Bacteroidetes express groups of co-regulated, cell envelope associated proteins termed polysaccharide utilisation loci (PULs). Each PUL is specific for a different glycan with some species of Bacteroides, one of the major genera of the gut, encoding >100 predicted PULs. PULs encodes enzymes, binding proteins, a regulator and a transporter which are localised to the outer-membrane or periplasm for the complete degradation and transport of the target glycan. Following initial, partial degradation of the target polysaccharide at the cell surface, the resulting oligosaccharides are transported into the periplasm. This process involves an outer membrane complex consisting of a substrate binding lipoprotein (SusD-like) and a β-barrel TonB dependent transporter (SusC-like). This SusCD transporter complex is vital to utilisation of glycans by Bacteroidetes as oligosaccharide breakdown to monosaccharides occurs in the periplasm. Despite the importance of this process to microbiota function, the mechanism of SusCD function is unclear. How extracellular substrate binding by the SusD-like protein is coupled to import by SusC is unknown as other classes of TonB dependent transporters do not have a partner lipoprotein. A SusCD complex with two auxiliary lipoproteins, BT2261-4, was expressed natively and purified directly from Bacteroides thetaiotaomicron. The X-ray crystal structure shows the SusC transporters form a homodimer with a SusD-like binding protein capping each barrel like a lid. The structure also shows a linear peptide bound at the interface of the SusC and SusD proteins via interactions with the peptide backbone. Expression levels of the BT2261-4 complex indicated that the proteins were required for growth under nutrient stress conditions which suggests a possible role in peptide scavenging. A classical glycan targeting SusCD complex, BT1762-3 from the Bacteroides thetaiotaomicron levan PUL, was targeted by adding a His-tag to the genomic copy of the SusD-like binding protein BT1762. The His-tag allowed purification of the BT1762-3 complex which led to two further SusCD structures; apo and with a levan oligosaccharide bound. BT1762-3 has the same overall conformation as the peptide importing SusCD suggesting the dimeric SusC transporter and SusD protein ‘cap’ general structure is conserved across SusCD complexes. MD simulations and electrophysiology experiments allowed us to propose a model for SusCD function where the SusD-like binding protein sits on top of the SusC-like transporter like a lid and is able to open like a pedal bin to allow oligosaccharide binding and uptake.