Structural and functional characterisation of the dextran utilisome from <i>Bacteroides thetaiotaomicron</i>.
This study offers detailed structural insights into the dextran utilisome of Bacteroides thetaiotaomicron, advancing our understanding of bacterial glycan utilization mechanisms.
Where it sits
this study against the rest of the ss-31 corpusSummary and findings
The study characterizes the dextran utilisome of Bacteroides thetaiotaomicron using structural biology techniques. High-resolution structures of SBGPdex, SusDdex, and GHdex bound to dextran oligosaccharides were solved. Isothermal titration calorimetry quantified ligand binding, and cryo-EM visualized open and closed states of the complex.
Abstract
<i>Bacteroides thetaiotaomicron</i> (<i>B. theta</i>) is a model Bacteroidota of the healthy human gut microbiota and a specialist in glycan utilisation. Like other <i>Bacteroides</i>, <i>B. theta</i> has many highly regulated polysaccharide utilisation loci (PUL) that encode outer membrane (OM) TonB-dependent transporters (SusC), closely associated "lid" lipoproteins (SusD), and additional surface-exposed lipoproteins (SLPs) that bind and partially degrade specific glycans derived from host cells, diet, or other microbiota members. The canonical starch PUL products are thought to form a dynamic complex in the presence of starch. However, other PULs form stable complexes in the absence of substrate (recently named "utilisomes"), with additional surface lipoproteins tightly associated with the core SusCD complex. In this study, we characterised the <i>B. theta</i> dextran utilisome, with a SusCD<sup>dex</sup> core and an associated glycoside hydrolase (GH<sup>dex</sup>) and surface glycan binding protein (SBGP<sup>dex</sup>). Via X-ray crystallography we solved high-resolution structures of SBGP<sup>dex</sup> in isolation and SusD<sup>dex</sup> and GH<sup>dex</sup> bound to dextran oligosaccharides. We used isothermal titration calorimetry (ITC) to quantify ligand binding of wild type and mutant SLPs. We further used single particle cryo-EM of the catalytically inactive dextran utilisome to visualise open and closed states of the complex. Three occupied dextran binding sites were observed across SusC<sup>dex</sup>, SusD<sup>dex</sup> and GH<sup>dex</sup>, with substrate observed in both open and closed states of SusD<sup>dex</sup>. 3D variability analysis showed a minority of particles in the process of SusD<sup>dex</sup> lid closure. Together our work defines commonalities and differences across utilisomes dedicated to the import of simple glycans.
Background
This study addresses the structural and functional characterization of the dextran utilisome in Bacteroides thetaiotaomicron, a model organism for studying the human gut microbiota. B. theta is known for its ability to utilize glycans, with polysaccharide utilization loci (PUL) encoding transporters and proteins involved in glycan degradation. Understanding the structure and function of these complexes is important for elucidating how gut bacteria process dietary and host-derived glycans.
Methods
The study employed X-ray crystallography to solve high-resolution structures of the surface glycan binding protein (SBGPdex) and components of the dextran utilisome bound to dextran oligosaccharides. Isothermal titration calorimetry was used to quantify ligand binding of wild type and mutant surface lipoproteins. Single particle cryo-EM was utilized to visualize the dextran utilisome in open and closed states, and 3D variability analysis was conducted to observe the dynamics of the complex.
Results
High-resolution structures of SBGPdex, SusDdex, and GHdex bound to dextran oligosaccharides were obtained. Isothermal titration calorimetry provided quantitative data on ligand binding for both wild type and mutant surface lipoproteins. Cryo-EM revealed the open and closed states of the dextran utilisome, with three occupied dextran binding sites observed across SusCdex, SusDdex, and GHdex. 3D variability analysis indicated that a minority of particles were in the process of SusDdex lid closure.
Interpretation
The study provides detailed structural insights into the dextran utilisome of B. theta, highlighting commonalities and differences in glycan import mechanisms. While the findings are significant for understanding bacterial glycan utilization, their direct clinical relevance is limited. The study's reliance on a model organism and structural techniques means that the results may not fully translate to human biology or clinical applications.
Key findings
- High-resolution structures of SBGPdex, SusDdex, and GHdex bound to dextran oligosaccharides were solved.
- Isothermal titration calorimetry quantified ligand binding of wild type and mutant SLPs.
- Single particle cryo-EM visualized open and closed states of the dextran utilisome.
- Three occupied dextran binding sites were observed across SusCdex, SusDdex, and GHdex.
- 3D variability analysis showed a minority of particles in the process of SusDdex lid closure.
Limitations
- Model organism study, not directly translatable to humans.
- Structural focus with no direct clinical implications.
- No therapeutic or clinical outcomes assessed.