Structural and functional investigations of the dextran utilisome of Bacteroides thetaiotaomicron

Abstract

The Gram-negative symbiont Bacteroides thetaiotaomicron (Bt) is an abundant member of the human gut microbiome (HGM), and one of the most studied bacteria within the HGM. Bt has long been known as a specialist in glycan degradation, with at least 86 polysaccharide utilisation loci (PULs), co-regulated operons that minimally encode a SusCD gene pair. PUL proteins are involved in the degradation and import of glycans that derive from host proteins, host diet, or other microbiota members. The polysaccharide dextran, a glycan secreted by lactic acid bacteria, is used as a food industry additive and also found in natural foods such as wine and honey. Previous work established that the outer membrane proteins of the levan (lev) and dextran (dex) PULs form heteromeric complexes (“utilisomes”) that cooperate at the cell surface to degrade and import their target glycan utilising TonB-dependent transport. Both utilisomes include a TonB-dependent transporter (SusCdex/lev), and three surface lipoproteins (SLPs); a closely associated “lid” protein (SusDdex/lev), a glycoside hydrolase (GHdex/lev) and a surface glycan binding protein (SGBPdex/lev). Here, to better understand how Bt utilises dextran, we investigated the dextran utilisome in more detail through structural and functional characterisation. X-ray crystallography and cryo-EM were used to solve the structures of the lipoprotein components and the whole liganded utilisome, respectively. This revealed that the SusCDdex core was similar to the levan utilisome in arrangement. SusCdex, like SusClev, contained “aromatic lock” residues which likely produce an allosteric cascade after substrate binding, enabling the release of the N- terminus of SusCdex and allowing TonB to bind with the TonB box of SusC. Additionally, a possible “ionic lock” within SusCdex was identified that may be involved in this process. SGBPdex and GHdex were divergent in fold and arrangement between the two utilisomes. Three occupied binding sites were observed for dextran oligosaccharides on GHdex, SusDdex and the SusCdex plug domain, which helped illustrate how dextran is transferred between utilisome components. Unlike previously characterised SusCD complexes, the dextran utilisome remained dynamic in the presence of its cognate substrate. SusDdex was also observed in both “open” and “closed” lidded states, with dextran bound in both. Putative ionic interactions between SusCdex and SusDdex were identified that may stabilise the closed state when substrate was bound. A movie demonstrated the transition between the two states and showed that SGBPdex may supply glycans to GHdex during the SusDdex-open state. Growth curves showed that GHdex activity is essential for utilisation of dextrans ≥10 kDa, while the presence of SGBPdex provided Bt a minor growth benefit, particularly with larger dextrans. Dextran binding affinities of the SLPs were in the micromolar range, and GHdex had the highest affinities, while SGBPdex had a notable preference for larger dextrans. Point mutations in all three SLPs identified key dextran binding residues. Using Bt and E. coli TonBs, the SusCdex TonB box:TonB interaction was found to have affinity in the low micromolar range, consistent with the literature on the interaction in E. coli. Kinetics of dissociation were particularly fast, perhaps denoting a feature that allows quick recycling for TonB-dependent transport. Overall, these data illustrate how dextran is acquired in Bt, building on our understanding of the utilisome model of glycan processing and transport in human gut Bacteroides.