Novel glycan-targeted extracellular proteases from divergent mucosal microbes

PhD ThesisTrillions of microorganisms inhabit mucosal surfaces of the human body. Despite increasing evidence of their impact on human health, many of the molecular mechanisms underlying hostmicrobial interactions (HMI) are poorly understood. To contribute to our understanding of HMI at mucosal surfaces, we investigated the novel family of M60-like/PF13402 domain-containing proteins and their putative functional partners. M60-like domains are shared by proteins from several mucosal microbes including two important human mucosal microbes; the bacterial mutualist Bacteroides thetaiotaomicron and the protist pathogen Trichomonas vaginalis, suggesting these proteins are important for interaction with the mucosal layer. We initially tested our hypothesis that these are glycoprotein-targeted metal dependent proteases in both these organisms. The three M60-like domains of B. thetaiotaomicron proteins (BT4244, BT3015 and BT4272) exhibited mucin protease activity. This proteolytic activity was shown to be inhibited in a mutant version of the protein (BT4244-FL-E575D) as well as in the presence of Ethylenediaminetetraacetic acid (EDTA), implying BT4244 and its relatives are metal dependent proteases. All M60-like proteins from B. thetaiotaomicron contained a carbohydrate binding module (CBM) from family 32 and these were shown to be capable of binding galacto-configured sugars that are common to mucin glycans, while in contrast the putative carbohydrate binding PA14 domain of the T. vaginalis TVAG339720 M60-like protein interacted with heparin and its sulphated derivatives. Mucins are glycoproteins and prominent components of the mucus secreted at mucosal surfaces while heparin is a close relative of heparan sulphate which typically exists as part of proteoglycans in the glycocalyx of mucosal epithelia. Although the actual target of the M60-like domain of TVAG339720 and its relatives in T. vaginalis are not currently known, the interaction of the TVAG339720 PA14 domain with heparin suggests that these may be proteases targeting proteoglycans and play a role in adhesion of the pathogen to the epithelial layer, a key initial step in pathogenesis. M60-like domain-containing proteins of B. thetaiotaomicron are also components of Sus-like systems. Sus-like systems are Bacteroidetes specific machinery that comprise a suite of cellenvelope located carbohydrate-active enzymes and sugar binding proteins that target complex glycans, with each Sus-like system tuned to the degradation of a specific glycan. The Sus-like system containing the BT4244 enzyme (BT4240-50), encoded by the polysaccharide locus (PUL) PULBT_4240-50 was characterised in this study. The results demonstrated that BT4244 is a surface protein and that its proteolytic activity is part of a concerted action of BT4240-50 components to utilise complex mucin glycoproteins containing the T (Galβ1-3GalNAc) and F (GalNAcα1-3GalNAc) antigens. Gene deletion studies revealed that PULBT_4240-50 provides a competitive advantage to the organism when grown on mucins, probably through its possession of the N-acetylgalactosamine (GalNAc) kinase BT4240, which was shown to be crucial for GalNAc utilisation. Finally, although variably conserved in closely related Bacteroides, the high frequency of PULBT_4240-50 components in this group of organisms suggests it may be an important evolutionary adaptation for survival at mucosal surfaces. Our findings not only set the stage for future functional studies on the novel M60-like/PF13402 family of proteins and their functional partners, but also further our understanding of host-microbial interactions at mucosal surfaces.Cameroonian government, the UK department for international development (DFID) and Newcastle University through the UK Commonwealth scholarships

A Novel Extracellular Metallopeptidase Domain Shared by Animal Host-Associated Mutualistic and Pathogenic Microbes

The mucosal microbiota is recognised as an important factor for our health, with many disease states linked to imbalances in the normal community structure. Hence, there is considerable interest in identifying the molecular basis of human-microbe interactions. In this work we investigated the capacity of microbes to thrive on mucosal surfaces, either as mutualists, commensals or pathogens, using comparative genomics to identify co-occurring molecular traits. We identified a novel domain we named M60-like/PF13402 (new Pfam entry PF13402), which was detected mainly among proteins from animal host mucosa-associated prokaryotic and eukaryotic microbes ranging from mutualists to pathogens. Lateral gene transfers between distantly related microbes explained their shared M60-like/PF13402 domain. The novel domain is characterised by a zinc-metallopeptidase-like motif and is distantly related to known viral enhancin zinc-metallopeptidases. Signal peptides and/or cell surface anchoring features were detected in most microbial M60-like/PF13402 domain-containing proteins, indicating that these proteins target an extracellular substrate. A significant subset of these putative peptidases was further characterised by the presence of associated domains belonging to carbohydrate-binding module family 5/12, 32 and 51 and other glycan-binding domains, suggesting that these novel proteases are targeted to complex glycoproteins such as mucins. An in vitro mucinase assay demonstrated degradation of mammalian mucins by a recombinant form of an M60-like/PF13402-containing protein from the gut mutualist Bacteroides thetaiotaomicron. This study reveals that M60-like domains are peptidases targeting host glycoproteins. These peptidases likely play an important role in successful colonisation of both vertebrate mucosal surfaces and the invertebrate digestive tract by both mutualistic and pathogenic microbes. Moreover, 141 entries across various peptidase families described in the MEROPS database were also identified with carbohydrate-binding modules defining a new functional context for these glycan-binding domains and providing opportunities to engineer proteases targeting specific glycoproteins for both biomedical and industrial applications

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

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 yet uncharacterized GAG-inducible genes in its genome and that 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 or Bt) and 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 Δ4,5UA-GlcNAc, Δ4,5UA-GlcNAc-GlcA-GlcNac, Δ4,5UA-[GlcNAc-GlcA]2-GlcNac and Δ4,5UA-[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 homologues 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

Structural and functional analyses of glycoside hydrolase 138 enzymes targeting chain A galacturonic acid in the complex pectin rhamnogalacturonan II

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiome. The selection pressures in this environment have spurred the evolution of a complex reservoir of microbial genes encoding carbohydrate-active enzymes (CAZymes). Previously, we have shown that the human gut bacterium Bacteroides thetaiotaomicron (Bt) can depolymerize the most structurally complex glycan, the plant pectin rhamnogalacturonan II (RGII), commonly found in the human diet. Previous investigation of the RGII-degrading apparatus in Bt identified BT0997 as a new CAZyme family, classified as glycoside hydrolase 138 (GH138). The mechanism of substrate recognition by GH138, however, remains unclear. Here, using synthetic substrates and biochemical assays, we show that BT0997 targets the D-galacturonic acid-α-1,2-L-rhamnose linkage in chain A of RGII and that it absolutely requires the presence of a second D-galacturonic acid side chain (linked β-1,3 to L-rhamnose) for activity. NMR analysis revealed that BT0997 operates through a double displacement retaining mechanism. We also report the crystal structure of a BT0997 homolog, BPA0997 from Bacteroides paurosaccharolyticus, in complex with ligands at 1.6 Å resolution. The structure disclosed that the enzyme comprises four domains, including a catalytic TIM (α/β)8 barrel. Characterization of several BT0997 variants identified Glu-294 and Glu-361 as the catalytic acid/base and nucleophile, respectively, and we observed a chloride ion close to the active site. The three-dimensional structure and bioinformatic analysis revealed that two arginines, Arg-332 and Arg-521, are key specificity determinants of BT0997 in targeting D-galacturonic acid residues. In summary, our study reports the first structural and mechanistic analyses of GH138 enzymes

Single cell fluorescence imaging of glycan uptake by intestinal bacteria

Microbes in the intestines of mammals degrade dietary glycans for energy and growth. The pathways required for polysaccharide utilization are functionally diverse; moreover, they are unequally dispersed between bacterial genomes. Hence, assigning metabolic phenotypes to genotypes remains a challenge in microbiome research. Here we demonstrate that glycan uptake in gut bacteria can be visualized with fluorescent glycan conjugates (FGCs) using epifluorescence microscopy. Yeast α-mannan and rhamnogalacturonan-II, two structurally distinct glycans from the cell walls of yeast and plants, respectively, were fluorescently labeled and fed to Bacteroides thetaiotaomicron VPI-5482. Wild-type cells rapidly consumed the FGCs and became fluorescent; whereas, strains that had deleted pathways for glycan degradation and transport were non-fluorescent. Uptake of FGCs, therefore, is direct evidence of genetic function and provides a direct method to assess specific glycan metabolism in intestinal bacteria at the single cell level.</p

Ascertaining the biochemical function of an essential pectin methylesterase in the gut microbe Bacteroides thetaiotaomicron

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II–derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/β-hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed β-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella. Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut

How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans

The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for Bacteroides thetaiotaomicron, a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the B. thetaiotaomicron Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS, and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut Bacteroides, indicating that the model developed is of generic relevance to this important microbial community

Sulfation of arabinogalactan proteins confers privileged nutrient status to Bacteroides plebeius

The human gut microbiota (HGM) contributes to the physiology and health of its host. The health benefits provided by dietary manipulation of the HGM requires knowledge of how glycans, the major nutrients available to this ecosystem, are metabolized. Arabinogalactan proteins (AGPs) are a ubiquitous feature of plant polysaccharides available to the HGM. Although the galactan backbone and galactooligosaccharide side chains of AGPs are conserved, the decorations of these structures are highly variable. Here we tested the hypothesis that these variations in arabinogalactan decoration provides a selection mechanism for specific Bacteroides species within the HGM. The data showed that only a single bacterium, B. plebeius, grew on red wine (Wi-AGP) and seaweed (SW-AGP) AGP in mono- or mixed culture. Wi-AGP thus acts as a privileged nutrient for a Bacteroides species within the HGM that utilizes marine and terrestrial plant glycans. The B. plebeius polysaccharide utilization loci (PULs) upregulated by AGPs encoded a polysaccharide lyase, located in the enzyme family GH145, which hydrolysed Rha-Glc linkages in Wi-AGP. Further analysis of GH145 identified an enzyme with two active sites that displayed glycoside hydrolase and lyase activities, respectively, which conferred substrate flexibility for different AGPs. The AGP degrading apparatus of B. plebeius also contained a sulfatase, BACPLE00986, active on SW-AGP and Wi-AGP, which played a pivotal role in the utilization of these glycans by the bacterium. BACPLE00986 enabled other Bacteroides species to access the sulfated AGPs, providing a route to introducing privileged nutrient utilization into probiotic and commensal organisms that could improve human health

Exploring the sequence-function space of microbial fucosidases

Microbial α-l-fucosidases catalyse the hydrolysis of terminal α-l-fucosidic linkages with diverse substrate/linkage specificities and can be used in transglycosylation reactions to synthesise oligosaccharides. Based on sequence identity, α-l-fucosidases have been classified in distinct glycoside hydrolases (GHs) families in the carbohydrate-active enzymes (CAZy) database. Here, we explored the sequence-function space of fucosidases from GH29 family. Based on sequence similarity network (SSN) analyses, 16 GH29 α-l-fucosidases were selected for functional characterisation. Using activity assays combined with HPAEC-PAD and LC-FD-MS/MS analyses, we determined the substrate and linkage specificities of these enzymes against a range of defined oligosaccharides and glycoconjugates, revealing a range of specificities for α1,2, α1,3, α1,4 and α1,6 linked fucosylated ligands. The structural basis for the substrate specificity of GH29 fucosidase from Bifidobacterium asteroides towards α1-6 linkages and FA2G2 N-glycan was further determined by X-ray crystallography and saturation transfer difference NMR. TLC combined with electrospray ionization – MS and NMR confirmed the capacity of this enzyme to carry out transfucosylation reactions with GlcNAc and Fuc1,3GlcNAc as acceptors. Taken together, these experimental data validate the use of SSN as a reliable bioinformatics approach to predict the substrate specificity and transfucosylation activity of GH29 fucosidases.<br/

Fucosidases from the human gut symbiont Ruminococcus gnavus

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers