The human gut microbe Bacteroides thetaiotaomicron encodes the founding member of a novel glycosaminoglycan-degrading polysaccharide lyase family PL29

Abstract

Edited by Chris WhitfieldGlycosaminoglycans (GAGs) and GAG-degrading enzymes have wide-ranging applications in the medical and biotechnological industries. The former are also an important nutrient source for select species of the human gut microbiota (HGM), a key player in host-microbial interactions. How GAGs are metabolized by the HGM is therefore of interest and has been extensively investigated in the model human gut microbe Bacteroides thetaiotaomicron. The presence of as-yet uncharacterized GAG-inducible genes in its genome and of related species, however, is testament to our incomplete understanding of this process. Nevertheless, it presents a potential opportunity for the discovery of additional GAG-degrading enzymes. Here, we investigated a gene of unknown function (BT_3328) from the chondroitin sulfate (CS) utilization locus of B. thetaiotaomicron. NMR and UV spectroscopic assays revealed that it encodes a novel polysaccharide lyase (PL), hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron (Bt)) and its ability to degrade the GAGs CS, dermatan sulfate (DS), and hyaluronic acid (HA). When incubated with HA, BtCDH generated a series of unsaturated HA sugars, including ⌬ 4,5 UA-GlcNAc, ⌬ 4,5 UA-GlcNAc-GlcA-GlcNac, ⌬ 4,5 UA-[GlcNAc-GlcA] 2 -GlcNac, and ⌬ 4,5 UA-[GlcNAc-GlcA] 3 -GlcNac, as end products and hence was classed as endo-acting. A combination of genetic and biochemical assays revealed that BtCDH localizes to the cell surface of B. thetaiotaomicron where it enables extracellular GAG degradation. BtCDH homologs were also detected in several other HGM species, and we therefore propose that it represents the founding member of a new polysaccharide lyase family (PL29). The current discovery also contributes new insights into CS metabolism by the HGM.

Metabolism of multiple glycosaminoglycans by Bacteroides thetaiotaomicron is orchestrated by a versatile core genetic locus

“…BT3331 susD demonstrated a very strict substrate specificity binding only to CS-A with a measurable affinity, similar to that of BT3329 SGBP (Supplementary Table 1). The surface lyase, BT3328 PL29 , showed its highest enzymatic activity and affinity against CS-A > CS-C > HA and little activity on DS, differing from previous data 18 . End point assays, the total number of glycosidic bonds cleaved, followed the relationship of HA > CS-C > CS-A ( Fig.…”

“…Bacteroides culture and genetic manipulation: Various Bacteroides strains and B. theta VPI-5482 were cultured anaerobically in a Whitley A35 Workstation (Don Whitley) at 37°C in either tryptone yeast extract glucose medium or minimal medium (MM) containing GAGs (described below). B. theta strains containing specific gene deletions or inactivated versions of enzymes were made by counter selectable allelic exchange as described below 18 . All genetic mutants, except Δbt3328, were provided by Dr Guy E. Townsend and Prof. Eduardo Groisman 13 .…”

Health effects of dietary sulfated polysaccharides from seafoods and their interaction with gut microbiota

“…Two types of enzymes that cleave glycosidic bonds in the CAZymes database include GHs and PLs (El Kaoutari et al., 2013), which may be used to cleave the glycosidic bonds of SPs (Cartmell et al., 2017; Cockburn & Koropatkin, 2016). Notably, endogenous host HS and CS have been confirmed to be degraded by PLs belonging to families PL‐8, PL‐12, PL‐13, and PL‐29 (El Kaoutari et al., 2013; Ndeh et al., 2018). With regard to the distribution of CAZymes in the gut microbiota, Bacteroidetes encode 137.1 CAZymes per genome on an average, whereas Firmicutes only encode 39.6 CAZymes per genome (El Kaoutari et al., 2013).…”

Research and Application of Chondroitin Sulfate/Dermatan Sulfate-Degrading Enzymes

“…Sugiura et al identified two highly homologous occlusion-derived variants of virus envelope protein 66s from Autographa californica nucleopolyhedrovirus and Bombyx mori nucleopolyhedrovirus, and both of the variants showed specific lyase activity towards non-sulfated and 6-O-sulfated CS (Sugiura et al, 2011;Sugiura et al, 2013). BtCDH from the human gut microbe Bacteroides thetaiotaomicron belongs to a new PL family (PL29) and shows optimum endolytic activity toward HA and CS, particularly large chains longer than decasaccharide, at a very high temperature of 60 • C (Ndeh et al, 2018). Two novel CS/DS lyases have been found from Acinetobacter sp.…”