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

Abstract

Glycosaminoglycans (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 Δ^4,5UA-GlcNAc, Δ^4,5UA-GlcNAc-GlcA-GlcNac, Δ^4,5UA-[GlcNAc-GlcA]₂-GlcNac, and Δ^4,5UA-[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.

Keywords: CAZymes; PL29; bacterial metabolism; carbohydrate metabolism; cell surface enzyme; chondroitin sulfate; glycosaminoglycan degradation; human gut microbiota; hyaluronan; hyaluronate lyase; polysaccharide.

Figure 1.

Modular architecture of native BtCDH and phylogenetic analyses. A, BtCDH (GenBank^TM accession number AAO78434) contains an N-terminal lipoprotein signal peptide sequence (SP) and a large central region sandwiched by two domains of unknown function (DUF4988 and DUF4955). B, phylogenetic tree showing the distribution of BtCDH homologs in selected species from various environments.

Figure 2.

Analyses of chondroitin 4-sulfate (CSA) degradation by BtCDH lyase. A, chemical structure of intact CSA. B, TLC of CSA before and after digestion by BtCDH lyase. For the reaction, 13.3 mg/ml of CSA from bovine trachea was treated with 0.3 μm of BtCDH lyase overnight. The reaction was stopped by boiling and 53.4 μg of digested CSA analyzed by TLC. Lane 1, CSA; lane 2, CSA treated with BtCDH lyase. C, HPAEC chromatogram of CSA digestion. Half the amount (26.7 μg) of sample in A was analyzed by HPAEC. D, ¹H NMR results of CSA digestion by BtCDH lyase. Digested CSA products were purified by SEC and later analyzed by NMR. Panel 1, NMR spectrum of undigested CSA. Panel 2, spectra of species from all SEC fractions combined (fractions 1–43 in Fig. S1). Panel 3, spectra of species from selected homogenous fractions (pooled fractions 37–39 in in Fig. S1). Both spectra show resonance peaks for H1 and H4 (black and red) at 5.1 and 5.9 ppm, respectively, consistent with the formation of terminal unsaturated CSA oligosaccharides. Panel 4 (inset), a typical chemical rearrangement of components of the nonreducing end sugar in CSA after lyase digestion. H4 and H1 are marked with red and black circles, respectively.

Figure 3.

Evidence of BtCDH endo-lyase activity. A, TLC showing progressive degradation of hyaluronanic acid (HA_L, 10–20 kDa) by BtCDH lyase over time. For the reaction, 32 mg/ml of HA_L was digested with 2 μm of enzyme. The reaction was stopped at the various time points indicated, and 64 μg of each sample was analyzed by TLC. B, mass spectrometry data of purified HA_L oligosaccharide species. Panel 1, spectrum for Δ^4,5UA-disaccharide (Δ^4,5UA-Di or band A). Panel 2, spectrum for Δ^4,5UA-octasaccharide (Δ^4,5UA-Octa or band B). Panel 3, spectrum for Δ^4,5UA-tetrasaccharide (Δ^4,5UA-Tetra). Panel 4, spectrum for Δ^4,5UA-hexasaccharide (Δ^4,5UA-Hexa). C, structural representation of various species detected in B.

Figure 4.

Activity of BtCDH against various chondroitin sulfate oligosaccharides. Substrates (1 mg/ml each) were treated with 0.5 μm of BtCDH (shown as DPx +, with x being the number of constituent monosaccharides) overnight or diluted in equivalent volume of buffer as control (DPx). HPAEC chromatograms show major shifts in the signals of sulfated DP10 and DP12 oligosaccharides following treatment, accompanied by the appearance of a strong signals for the Δ^4,5UA-GalNAc disaccharide in each case.

Figure 5.

Effect of temperature and pH and metals on BtCDH lyase activity. A, effect of temperature. Enzymatic activity was measured using 40 μm of CSA as substrate in 20 mm NaH₂PO₄ at pH 7.0 over different temperatures. B, effect of pH. BtCDH activity was measured at different pH levels using 20 mm of various buffers at 37 °C. C, effect of various metals. The reactions were carried out in 20 mm NaH₂PO₄ buffer containing 5 mm EDTA, Mg²⁺, Ca²⁺, Mn²⁺, and Co²⁺ and 1 mm Zn²⁺ and Ni²⁺ in separate experiments.

Figure 6.

Localization of BtCDH and evidence of extracellular chondroitin lyase activity. A, Western blots showing the detection of full and truncated versions of BtCDH (BtCDH_H and BtCDH_L, respectively) in B. thetaiotaomicron WT, ΔBtCDH, and BtCDH FLAG cell extracts using polyclonal rabbit anti-BtCDH (top panel) and anti-FLAG tag antibodies (bottom panel). B, Western blotting analyses of proteinase K–treated and control cells using anti-BtCDH antibodies. The top results show loss of BtCDH_H and BtCDH_L after the treatment of whole cells with proteinase K. The bottom results show detection of an intact periplasmic control protein, the BT4657 heparinase of B. thetaiotaomicron (16). C, immunofluorescence detection of BtCDH in B. thetaiotaomicron WT and ΔBtCDH cells. The top panel shows green fluorescence signals of BtCDH. The middle panel shows blue signals from stained nuclei. The bottom panel is a merger of both signals. D, evidence of extracellular CS degradation in B. thetaiotaomicron by TLC. Top gel, TLC analyses of CS degradation when whole cells of B. thetaiotaomicron WT, ΔBtCDH, and BtCDH FLAG are incubated with CSA. Bottom gel, TLC analyses of CS degradation when whole cells of B. thetaiotaomicron WT, ΔBtCDH, and BtCDH FLAG are incubated with CSC.

Figure 7.

Proposed model of CS metabolism showing the context of BtCDH activity. BtCDH orchestrates extracellular degradation of sulfated CS (DP ≥ 10) to generate oligosaccharide products that are imported through an outer membrane protein complex (encoded by BT_3331 and BT_3332 genes) into the periplasmic space. They are further degraded by periplasmic sulfatases, lyases, and unsaturated glycoside hydrolase enzymes yielding end products 5-keto, 4-deoxyuronate, and GalNAc, which can be metabolized to provide energy for cell growth. Disaccharide intermediates generated during the process serve as signaling molecules activating expression of locus genes through binding to a hybrid two-component sensor protein (encoded by BT_3334) when CS is present.