Insights into glycan import by a prominent gut symbiont

Abstract

24In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large 25 nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal 26 bin" transport mechanism. However, many features of SusCD function in glycan uptake remain 27 unclear, including ligand binding, the role of the SusD lid and the size limit for substrate 28 transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from 29Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal 30 structures reveal residues involved in glycan recognition and suggest that the large binding 31 cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding 32 supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies 33 in vivo provide functional insights into the key structural features of the SusCD apparatus and 34 cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, 35directly visualising the dynamics of the pedal bin transport mechanism. 36 37while SusD sits on top of the barrel, opening and closing like a lid to facilitate substrate binding. 59Previous structures of loaded SusCD complexes revealed a bound ligand which was 60 completely encapsulated by the closed pedal bin, indicating that SusCD transporters may 61 have a size limit for transport 15,17 . An investigation on the prototypical SusCD system revealed 62 that a mutant strain lacking the surface endo-amylase preferentially utilised malto-63 oligosaccharides with a degree of polymerisation (DP) of ~5-16 18 , suggesting this is the 64 preferred size range imported by the Bt3701-02 SusCD transporter. Direct evidence for this 65 notion is lacking, however, and this issue has not been explored for any other Sus systems 12 . 66Furthermore, many other key features of SusCD function remain unclear, including the role of 67 the SusD lid and other conserved structural elements, as well as the identity of the glycan 68 recognition elements of both SusC and SusD. 69 70One example of a typical PUL is the B. theta levan utilisation locus, spanning Bt1754-Bt1765 19 . 71Levan is a fructan polysaccharide comprised of β2,6-linked fructose units, with occasional 72 β2,1 fructose decorations and is produced mainly by bacteria as an exopolysaccharide, but 73 also by some cereal plants such as wheat 20,21 . For the levan utilisation system, the cell-surface 74 components are Bt1760 (a GH32 endo-levanase), Bt1761 (a surface glycan binding protein;; 75 SGBP), Bt1762 (SusD) and Bt1763 (SusC) 15,19,22 (Fig. 1a). The cell surface levanase Bt1760 76 and glycan binding proteins (Bt1761 and Bt1762) have been shown to be specific for levan, 77 with no activity against, or binding to, β2,1-linked inulin-type fructans that are common to many 78 plants 19,23 . 79 80In this study we characterise binding and uptake of β2,6 fructo-oligosaccharides (FOS) by the 81 Bt1762-63 SusCD transporter. Co-crystal structures of the cl...

TonB‐dependent transporters in the Bacteroidetes: Unique domain structures and potential functions

“…Indeed, the TBDTs identified by our search demonstrate that most B. theta TBDTs that are devoted to glycan uptake are 200–300 amino acids longer than the well‐studied TBDTs involved in iron uptake in Gram‐negative pathogens. The recent crystal structures of three TBDTs from Bacteroidetes demonstrate that some of this additional sequence is devoted toward elongated loops at the extracellular face of the barrel (Figure 1c) (Glenwright et al., 2017; Gray et al., 2021; Madej et al., 2020). These loops in part engage with the ligand‐binding SusD protein that caps the barrel, as described later.…”

“…Additionally, surveys of TBDTs across bacteria have often noted the key differences between characterized TBDT largely from Enterobacteria and predicted TBDTs in the Bacteroidetes (Blanvillain et al., 2007; Koebnik, 2005). Today, biochemical studies of Bacteroides PUL and the associated TBDTs have begun to give us a better picture of how the TBDTs encoded by the Bacteroidetes share similar structure and function to the classical TBDT while also allowing for novel structures and functions (Glenwright et al., 2017; Gray et al., 2021; Madej et al., 2020). Here, we use Pfam domain analysis of the 121 predicted TBDTs in Bacteroides thetaiotaomicron to review what is currently known and what work remains to be done to better understand sugar transport and the broader role of TBDTs in the Bacteroidetes.…”

“…A surprising feature of the BT2264 and BT1763 structures and subsequent characterization is that these TBDTs are dimers, both in the crystal structures and in size‐exclusion experiments (Glenwright et al., 2017; Gray et al., 2021). The structure of the TBDT/SusD complex RagAB from the oral Bacteroidetes Porphyromonas gingivalis also revealed this complex to be a dimer, which suggests that we may continue to see this trend among the TBDTs of the Bacteroidetes.…”

“…Despite the prevalence of the N‐terminal extension domain, the function of this domain is unknown. Recent characterization of the BT1763 transporter showed that this domain is essential for proper function of the transporter as B. theta is not able to grow on the cognate substrate levan when this domain is removed from BT1763 (Gray et al., 2021). Structural characterization of this domain revealed a small, well‐structured Ig‐like fold (Gray et al., 2021).…”

Communal living: glycan utilization by the human gut microbiota

“…Landmark structural studies revealed that the TBDT–SGBP complexes encoded by pairs of susC / susD gene homologues (viz., SusC H and SusD H proteins) enable polysaccharide breakdown products produced at the cell surface to transit the outer membrane. This protein pair operates through a ‘pedal‐bin’ mechanism, involving the coordinated opening and closing of the lid formed by the SusD H SGBP with movement of the plug domain of the SusC H TBDT (Gray et al ., 2020). Proper assembly of this complex is thus essential to glycan utilization, such that deletion of the SusD H SGBP from a gnPUL abolishes growth on the cognate glycan (Anderson and Salyers, 1989a; 1989b; Tancula et al ., 1992; Reeves et al ., 1996; 1997; Cho and Salyers, 2001; Koropatkin et al ., 2008; 2009; Cameron et al ., 2012; Karunatilaka et al ., 2014; Tauzin et al ., 2016; Tamura et al ., 2019).…”