Ana S Luis
email: ana.luis@medkem.gu.se.

Chunsheng Jin

Gabriel Vasconcelos Pereira

Robert W P Glowacki

Sadie Gugel

Shaleni Singh

Dominic P Byrne

Nicholas Pudlo

James A London

Arnaud Baslé

Mark Reihill

Stefan Oscarson

Patrick A Eyers

Mirjam

Czjzek

Gurvan Michel

Tristan Barbeyron

Edwin A Yates

Gunnar C Hansson

Niclas G Karlsson

Alan Cartmell
email: alan.cartmell@liverpool.ac.uk

Eric C Martens
email: emartens@umich.edu

A single

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The human gut microbiota (HGM) significantly impacts several aspects of intestinal health and disease, including inflammatory bowel disease (IBD) [START_REF] Arike | The Densely O-Glycosylated MUC2 Mucin Protects the Intestine and Provides Food for the Commensal Bacteria[END_REF] and colorectal cancer (CRC) [START_REF] Arike | Intestinal Muc2 mucin Oglycosylation is affected by microbiota and regulated by differential expression of glycosyltranferases[END_REF] . In the colon, secreted mucus creates a physical barrier that separates gut microbes from the intestinal epithelium [START_REF] Derrien | Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium[END_REF] preventing close contact that can lead to inflammation and eventual CRC if this barrier is either experimentally eliminated [START_REF] Pudlo | Symbiotic Human Gut Bacteria with Variable Metabolic Priorities for Host Mucosal Glycans[END_REF][START_REF] Barbeyron | Matching the Diversity of Sulfated Biomolecules: Creation of a Classification Database for Sulfatases Reflecting Their Substrate Specificity[END_REF] or has reduced glycosylation [START_REF] Hettle | The Molecular Basis of Polysaccharide Sulfatase Activity and a Nomenclature for Catalytic Subsites in this Class of Enzyme[END_REF][START_REF] Cartmell | How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans[END_REF][START_REF] Ndeh | HMSC073D09 HMPREF2955_03450 Human (lungs) A0A1M5DJ19 Bacteroides faecichinchillae SAMN05444349_12916 Human (feces) A0A1M6EIV9 Bacteroides stercorirosoris SAMN05444350_10912 1 Human (feces) A7LSB2 Bacteroides ovatus ATCC 8483 BACOVA_00697 Human B3C7H9 Bacteroides intestinalis DSM 17393 BACINT_00870 Human (feces) B5D3C1 Bacteroides plebeius DSM 17135 BACPLE_03515 Human (feces) B6VXI1 Bacteroides dorei DSM 17855 BACDOR_01990 Human (feces) C9KV90 Bacteroides finegoldii DSM 17565 BACFIN_06228 Human (feces) C9MNT2 Prevotella veroralis F0319 HMPREF0973_01270 Human (oral) C9PTV0 Prevotella sp. oral taxon 472 str. F0295 HMPREF6745_0389 Human (oral) D1VZ37 Prevotella timonensis CRIS 5C-B1 HMPREF9019_1482 Human (urogenital tract) D3I2J1 Prevotella melaninogenica D18 HMPREF0660_00136 Human (oral) D3IDU7 Prevotella sp. oral taxon 299 str. F0039 HMPREF0669_01601 Human (oral) D3IKS6 Prevotella sp. oral taxon 317 str. F0108 HMPREF0670_02095 Oral D9RR92 Prevotella melaninogenica ATCC 25845 HMPREF0659_A5130 Human (sputum) E6MSG7 Prevotella salivae DSM 15606 HMPREF9420_2435 Human (oral) E6SQJ1 Bacteroides helcogenes ATCC 35417 Bache_0950 Pig (Abscess) F3PXR8 Bacteroides fluxus YIT 12057 HMPREF9446_03566 Human (gut) F8N7E6 Prevotella multisaccharivorax DSM 17128 Premu_0918 Human (oral) G1VBK7 Prevotella sp. C561 HMPREF0666_00790 Human (Upper respiratory tract) G1WC24 Prevotella oulorum F0390 HMPREF9431_01375 Human G5GCE3 Alloprevotella rava F0323 HMPREF9332_01220 Human (oral) G6AUB5 Prevotella stercorea DSM 18206 HMPREF0673_00199 Human (feces) I0TBD8 Prevotella sp. oral taxon 306 str. F0472 HMPREF9969_1074 Human I4A3E0 Ornithobacterium rhinotracheale ATCC 51463 Ornrh_2345 Turkey (respiratory tract) I8UTS1 Bacteroides caccae[END_REF][9] . A major component of the colonic mucus is mucin 2 (MUC2), a glycoprotein that contains up to 80% glycans by mass and more than 100 different glycan structures that are O-linked to serine or threonine residues 10 . Mucin glycosylation is variable along the gastrointestinal (GI) tract with a marked increase in sulfation in the colon 11 . In mucins, O-linked sulfate may be attached to the 6-hydroxyl of N-acetyl-Dglucosamine (6S-GlcNAc) and non-reducing end D-galactose (Gal) sugars at hydroxyl positions 3-, 4-or 6-(3S-, 4S-and 6S-Gal, respectively) [11][12][START_REF] Robbe | Evidence of regio-specific glycosylation in human intestinal mucins -Presence of an acidic gradient along the intestinal tract[END_REF] (Fig. 1a). Sulfation often occurs as terminal caps that block enzymatic degradation of oligosaccharides. To degrade and utilize colonic mucin O-glycans, members of the HGM need to express appropriate carbohydrate sulfatases to remove these modifications. Bacteroides thetaiotaomicron (Bt) is a dominant member of the human gut microbiota that is able to utilize O-glycans as a sole nutrient source [START_REF] Martens | Mucosal Glycan Foraging Enhances Fitness and Transmission of a Saccharolytic Human Gut Bacterial Symbiont[END_REF] . Underscoring the importance of sulfatases, Bt requires active sulfatases for competitive colonization of the wild-type mouse gut [START_REF] Benjdia | Sulfatases and a radical S-adenosyl-L-methionine (AdoMet) enzyme are key for mucosal foraging and fitness of the prominent human gut symbiont, Bacteroides thetaiotaomicron[END_REF] and to induce inflammation in genetically-susceptible mice [START_REF] Hickey | Colitogenic Bacteroides thetaiotaomicron Antigens Access Host Immune Cells in a Sulfatase-Dependent Manner via Outer Membrane Vesicles[END_REF] . However, the specific sulfatases that mediate these effects remain unknown. Indeed, despite the critical roles of sulfatases in many biological processes, including several human diseases [START_REF] Diez-Roux | Sulfatases and human disease[END_REF] , a significant knowledge gap exists regarding the biochemical, structural and functional roles of these enzymes.

We hypothesized that specific Bt sulfatases play essential roles in initiation of Oglycan degradation. To test this, we measured the activities of 23 putative Bt sulfatases, determining that 12 enzymes are active on either model glycan substrates or purified colonic mucin O-glycans. Together with defining the specific activities of these enzymes, we determined the corresponding structures for 3 sulfatases, revealing the basis of substrate specificity. Using molecular genetics, we next assessed the contributions of these sulfatases to Bt fitness in vitro and in vivo, unexpectedly revealing that a single enzyme is essential for utilization of sulfated mucin O-glycans and plays a major role in competitive gut colonization. Identifying specific bacterial sulfatases that are critical for intestinal mucin degradation provides new potential targets, with a goal of blocking progression of diseases such as IBD and possibly other disorders that result from bacterial disruption of the mucus barrier.

Utilization of colonic mucins by HGM species

Several studies have identified HGM members that are able to utilize porcine gastric mucin O-glycans (gMO) as a sole carbon source [START_REF] Martens | Mucosal Glycan Foraging Enhances Fitness and Transmission of a Saccharolytic Human Gut Bacterial Symbiont[END_REF][START_REF] Wlodarska | Indoleacrylic Acid Produced by Commensal Peptostreptococcus Species Suppresses Inflammation[END_REF][START_REF] Tramontano | Nutritional preferences of human gut bacteria reveal their metabolic idiosyncrasies[END_REF] . However, this substrate does not adequately reflect the structural complexity of mucin O-glycans found in the colon, especially those with increased sulfation that are lacking in gMO 11 . To identify HGM species that utilize sulfated colonic mucins, we measured the growth of 19 Bacteroides type strains, plus Akkermansia muciniphila, on highly sulfated porcine colonic mucin oligosaccharides (cMO). We identified six Bacteroides strains that utilize cMO (Fig. 1b, Extended Data Fig. 1). Interestingly, two known mucin-degraders, A. muciniphila [START_REF] Derrien | Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium[END_REF] and B. massiliensis [START_REF] Pudlo | Symbiotic Human Gut Bacteria with Variable Metabolic Priorities for Host Mucosal Glycans[END_REF] , grew robustly on gMO but failed to utilize sulfated cMO as a substrate (Extended Data Fig. 1), highlighting the importance of employing colonic mucins as substrates to draw more physiologically-relevant conclusions about the metabolic targets of these organisms. Bt, the bacterium with the highest number of sulfatases (28), was one of the strains with the best growth on cMO (Fig. 1b), suggesting that some of these enzymes might play key roles in promoting this ability. Therefore, to understand the role of sulfatases in colonic mucin utilization by HGM bacteria we focused on the biochemical and genetic characterization of the Bt enzymes. See Supplementary Discussion 1 for additional details of GI mucins and bacterial growth kinetics.

Substrate specificity of Bt sulfatases

Sulfatases are classified into four main families (S1 to S4) in the SulfAtlas database according to sequence similarity, catalytic mechanism and fold [START_REF] Barbeyron | Matching the Diversity of Sulfated Biomolecules: Creation of a Classification Database for Sulfatases Reflecting Their Substrate Specificity[END_REF] . Family S1 is currently divided into 72 subfamilies (designated S1_X) and comprises the formylglycine sulfatases, which operate via a hydrolytic mechanism that utilizes a non-genetically coded formylglycine amino acid as its catalytic residue. In Bt and other anaerobic bacteria, this residue is introduced co-translationally by the anaerobic sulfatase maturating enzyme (anSME) [START_REF] Benjdia | Sulfatases and a radical S-adenosyl-L-methionine (AdoMet) enzyme are key for mucosal foraging and fitness of the prominent human gut symbiont, Bacteroides thetaiotaomicron[END_REF] , which converts a serine or cysteine, within the consensus sequence C/S-X-P/A/S-X-R, to formylglycine, which then serves as catalytic nucleophile [START_REF] Benjdia | Anaerobic sulfatase-maturating enzymes, first dual substrate radical S-adenosylmethionine enzymes[END_REF] . The Bt genome encodes 28 S1 sulfatases classified into twelve different subfamilies (Supplementary Table 1). Four Bt sulfatases have been previously characterized and all act on glycosaminoglycans (GAGs) that are components of extracellular matrix (Fig. 1c) [START_REF] Cartmell | How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans[END_REF][START_REF] Ndeh | Metabolism of multiple glycosaminoglycans by Bacteroides thetaiotaomicron is orchestrated by a versatile core genetic locus[END_REF] .

Interestingly, several of the uncharacterized S1 sulfatases are encoded within polysaccharide utilization loci (PULs) that are known to be upregulated in vivo or during growth on gMO [START_REF] Martens | Mucosal Glycan Foraging Enhances Fitness and Transmission of a Saccharolytic Human Gut Bacterial Symbiont[END_REF] and encode other glycoside hydrolases enzymes potentially involved in degrading mucin O-glycans (Extended Data Fig. 2).

To understand the role of sulfatases in mucin metabolism, we cloned and expressed in soluble form 23 of the remaining 24 uncharacterized sulfatases. The recombinant proteins were tested for activity against a panel of commercially available sulfated saccharides (Supplementary Table 2). In this initial screen, we identified activities for twelve sulfatases (Fig. 1c, Extended Data Figs. 3,4 and Supplementary Table 3). Among these, 5 of the enzymes represent the first activities reported for their respective subfamilies: two S1_20 members (BT1636 and BT1622) were determined to target 3S-Gal, with BT1622 also preferentially cleaving 3S-N-acetyl-D-galactosamine (3S-GalNAc), two S1_16 enzymes (BT3796 and BT3057) cleave 4S-Gal/4S-GalNAc and one S1_46 enzyme (BT1918) cleaves 3S-GlcNAc, using the N-acetyl group as an absolute specificity determinant. This represents the first report of a bacterial sulfatase active on 3S-GalNAc, indicating that this sulfation could exist as a yet unidentified modification of host glycans. Subsequently, we refer to these enzymes by their gene/locus tag number with the corresponding activity in superscript (e.g., BT1636 3S-Gal ).

In addition to assigning new catalytic activities associated with three subfamilies that previously lacked any characterization, we also identified sulfatases displaying novel activities inside previously characterized subfamilies. These include three S1_15 enzymes (BT1624 6S-Gal/GalNAc , BT3109 6S-Gal/GalNAc and BT4631 6S-Gal/GalNAc ) that extend this family, previously only known to include 6S-GalNAc sulfatases, to those cleaving 6S-Gal.

Two members of S1_4 were active on 3S-Gal (BT4683 3S-Gal ) or 6S-Gal (BT3487 6S-Gal ), representing novel activities within this arylsulfatase subfamily. Finally, consistent with the activity previously described for S1_11 members, two enzymes were 6S-GlcNAc sulfatases (BT1628 6S-GlcNAc and BT3177 6S-GlcNAc ) (Fig. 1c and Extended Data Fig. 3).

Characterized sulfatases within the same subfamily, with the exception of two S1_4 members, cleaved the same sulfate ester linkages (Fig. 1c). However, despite these enzymes targeting the same linkages, their optimal activity depends on the surrounding glycan context. The activity of the 3S-Gal sulfatases is dependent on the linkage between Gal and GlcNAc with BT4683 3S-Gal showing a preference for 3'-sulfate-N-acetyl-D-lactosamine (3'S-LacNAc, 3'S-D-Gal-b1,4-D-GlcNAc). In contrast, BT1622 3S- Gal/GalNAc demonstrated enhanced activity towards 3'-sulfate-lacto-N-biose (3'S-LNB, 3'S-D-Gal-b1,3-D-GlcNAc) (Extended Data Fig. 4a and Supplementary Table 3).

Furthermore, additional affinity/activity studies revealed that BT1622 3S-Gal/GalNAc preferentially targets GalNAc and not Gal (Extended data Fig. 4b,c and Supplementary Table 3), suggesting that this sulfatase evolved to optimally target sulfate O3-linked to GalNAc. Bt sulfatase activity is also affected by the presence of terminal epitopes such as those that occur in Lewis antigens. Despite BT1636 3S-Gal being equally active on 3'S-LacNAc and 3'S-LNB, this protein has a lower affinity and it is 100-fold less active when L-fucose (Fuc) is linked to GlcNAc (3'S-Lewis-a/x) (Extended data Fig. 4a,b and Supplementary Table 3). While BT1622 3S-Gal/GalNAc is only weakly active on 3'S-Lewis-a antigen and not active at all on 3'S-Lewis-x, the reciprocal is true for BT4683 (Supplementary Table 3). The subfamily S1_15 enzyme BT1624 6S-Gal/GalNAc was only weakly active on 6S-Lewis-a/x antigens (Extended Data Fig. 4a), suggesting that these enzymes cannot accommodate Fuc linked to GlcNAc and that Fuc needs to be removed prior to sulfate cleavage. Additionally, affinity studies showed that BT3109 6S-Gal/GalNAc has a strong affinity for Gal, while the previously characterized GAG sulfatase BT3333 6S-GalNAc showed a preference for GalNAc, suggesting that optimal activity of S1_15 sulfatases likely depends on the glycan context and BT3333 6S-GalNAc evolved to target sulfated linkages in GAGs, a substrate that contains GalNAc but not Gal.

Bt sulfatase activity on colonic mucin oligosaccharides (cMO)

We next tested the activity of Bt sulfatases on custom purified cMO, which we determined to contain at least 131 different oligosaccharides (Supplementary Table 4).

Only 4 of the 6 sulfatases tested displayed activity on cMOs (Fig. 2a,b and Supplementary Table 4). BT1628 6S-GlcNAc and BT3177 6S-GlcNAc removed 6-O-sulfate from all GlcNAc structures but only when present at the non-reducing end of O-glycans confirming an exo-mode of action observed using commercial oligosaccharides (Fig. 2 and Extended Data Fig. 5a). After incubation with BT1636 3S-Gal , we were able to detect 14 new oligosaccharides and an overall increase of non-sulfated glycans (Fig. 2 and Extended Data Fig. 5b). Compared to the non-enzyme treated control, 36 oligosaccharides could no longer be detected after incubation with BT1636 3S-Gal (Fig. 2a and Supplementary Table 4). We determined the structures of 8 of these glycans and all present a terminal 3S-Gal (Fig. 2). This sulfatase was active on 3'S-Gal-b1,3-GalNAc (core 1) and more complex sulfated structures built around other common core structures (Fig. 2c), indicating that BT1636 3S-Gal evolved to accommodate the various linkages and substitutions found in mucin O-glycans. After incubation with BT4683 3S-Gal , 9 oligosaccharides disappeared, indicating that this enzyme is active on a smaller subset of structures (Fig. 2a and Supplementary Table 4). We were only able to determine the structure of one of those glycans and, unexpectedly, we found that this enzyme is endoactive on sialylated 3S-Gal (Fig. 2b), which is consistent with the protein structural data discussed below. Additionally, the strong activity of BT1636 3S-Gal on larger glycans with unknown structures (Supplementary Table 4) reveals that porcine cMOs are highly sulfated at the O3-position of galactose. Although previous studies have reported the presence of 3S-Gal in colonic mucins [26][27][28] , the analysis of such complex samples is technically challenging and, until now, no precise enzymatic tools were available to probe these linkages. Together, these results represent the first report of HGM sulfatases active on colonic mucin O-glycans and highlight the possibility to use gut bacterial sulfatases as analytical tools in structural characterization of mucin glycans (see Supplementary Discussion 2 for additional details of sulfatase activity on cMO).

Structural characterization of 3S-Gal/GalNAc sulfatases

To further understand the molecular details of carbohydrate recognition by S1 sulfatases, we determined the crystal structures of the three different 3S-Gal sulfatases that belong to two subfamilies. Consistent with previous structures of S1 sulfatases, all 3 enzymes display a a/b/a topology with a C-terminal sub-domain, and the active site residues interacting with the sulfate group are fully conserved (Extended data Fig. 6a,b).

The structure of BT1636 3S-Gal in complex with the product LacNAc revealed that His177 coordinates with O4 of Gal and mutation to alanine ablates enzyme activity, suggesting that this residue is the major specificity determinant for Gal (presenting an axial O4) over glucose (equatorial O4) (Fig. 3 and Supplementary Table 3). BT1636 3S-Gal also makes strong interactions with O2 via R353 and E334. The essential His177 in BT1636 3S-Gal is highly conserved (92%) within S1_20 sulfatases (Extended data Fig. 7). In BT1622 3S- Gal/GalNAc , mutation of the corresponding H176 to alanine causes a ~300-fold reduction in activity (Supplementary Table 3), further highlighting the importance of this residue in these enzymes have evolved to target sulfate groups in the different contexts in which they are found in complex host glycans. This is especially true for BT1636 3S-Gal which utilizes high affinity interactions with both O2 and O4 to drive enhanced activity to remove terminal 3S-Gal linkages in cMOs (see Supplementary Discussion 3 for additional details of sulfatase structural characterization and phylogeny).

Roles of sulfatases in B. thetaiotaomicron O-glycan utilization

Bt is able to utilize cMO as a sole carbon source (Fig. 1b), but the key enzymes involved in the degradation of these glycans remain unclear. Highlighting the importance of sulfatases to this symbiont's physiology, deletion of the gene encoding the only anaerobic sulfatase maturating enzyme (anSME) eliminates activation of all 28 S1 sulfatases [START_REF] Benjdia | Sulfatases and a radical S-adenosyl-L-methionine (AdoMet) enzyme are key for mucosal foraging and fitness of the prominent human gut symbiont, Bacteroides thetaiotaomicron[END_REF] and the ability of Bt to grow efficiently on cMOs (Fig. 4a). Based on this observation we generated a series of strains with compounded gene deletions in which one or several groups of sulfatases were eliminated based on their biochemical activity.

Deletion of all 3S-Gal/GalNAc sulfatases (Dbt1636 3S-Gal + Dbt1622 3S-Gal/GalNAc + Dbt4683 3S- Gal ) resulted in a growth phenotype similar to DanSME (Extended Data Fig. 8a).

Interestingly, we observed a similar growth defect when just BT1636 3S-Gal was deleted, but not the other 3S-Gal sulfatases (Fig. 4a and Extended Data Fig. 8a), consistent with the prominent activity of the recombinant form of this enzyme on cMOs. In contrast, a strain with compounded deletions of eight other sulfatases (Dbt1622 3S-Gal/GalNAc + Dbt4683 3S-Gal + Dbt1624 6S-Gal/GalNAc + Dbt3109 6S-Gal + Dbt4631 6S-Gal/GalNAc + Dbt1628 6S-GlcNAc + Dbt3177 6S-GlcNAc + Dbt3051 putative_6S-GlcNAc ) displayed a growth phenotype similar to wildtype (Fig. 4a), indicating that these enzymes are not essential for cMO utilization.

However, a D10X sulf mutant, which included the deletion of BT1636 3S-Gal and the two 4S-Gal/GalNAc sulfatases, showed a similar growth defect as DanSME and Dbt1636.

Complementation of this and other loss of function mutants with only bt1636 3S-Gal restored growth on cMO to levels similar to wild-type (Fig. 4a and Extended Data Fig. 8a). Cellular localization experiments revealed that BT1636 3S-Gal is located at the cell surface in Bt (Fig. 4b) and together these data suggest that this single 3S-Gal sulfatase is a critical cell surface enzyme involved in the utilization of sulfated O-glycans that are prominent in the colon.

To further investigate the role of BT1636 3S-Gal in O-glycan utilization, we analyzed the oligosaccharides present in the culture supernatant of the wild-type and Dbt1636 3S-Gal strains after growth on cMO. Consistent with a robust ability of Bt to degrade diverse colonic O-glycans, no oligosaccharides were detected in wild-type supernatant (Supplementary Table 5). Compared to the cMO used as substrate (control), the supernatant of Dbt1636 3S-Gal showed a 20-fold accumulation of terminal 3S-Gal capped glycans, suggesting that these could not be degraded. Indeed, the three most common 3S-Gal structures detected in the Dbt1636 3S-Gal supernatant accounted for 50% of the total oligosaccharides detected in this sample (Fig. 4c). These results reveal that deletion of BT1636 3S-Gal results in loss of the ability to utilize 3S-Gal O-glycans. Interestingly, 49 of the 72 glycans detected in Dbt1636 3S-Gal supernatant were not present in the control sample (Supplementary Table 5), suggesting that although the mutant does not grow well on cMO it is able to modify these oligosaccharides to generate new glycans (Supplementary discussion 5). These data, combined with the cell surface location of BT1636 3S-Gal , suggest that this sulfatase is required early in O-glycan catabolism likely by cleaving 3S-Gal from O-glycans prior to importing them into the periplasm where these oligosaccharides will be sequentially degraded by additional enzymes and also serve as cues for activating transcription of the O-glycan PULs (Extended Data Fig. 2). Although Bt does encode two additional 3S-Gal/GalNAc sulfatases, the low activity of these additional sulfatases on cMOs (Fig. 2) and their likely periplasmic location, suggests why these enzymes cannot compensate for loss of BT1636 3S-Gal . Interestingly, all of the Bacteroides species able to utilize cMO (Fig. 1a) have homologues of BT1636 3S-Gal , suggesting that this activity plays a key role in mucin utilization by other HGM members (Supplementary Table 6).

Finally, to investigate the requirement for specific sulfatases in vivo, we utilized gnotobiotic mice in which we competed individual mutants against the wild-type strain to evaluate their colonization fitness. It has been reported that mouse colonic Muc2 prominently displays 6S-GlcNAc modifications 29 . However, mutants lacking either just the two active 6S-GlcNAc sulfatases (D6S-GlcNAc double mutant), or these two enzymes, another putative 6S-GlcNAc sulfatase and all three 6S-Gal/GalNAc sulfatases (D6S-GlcNAc + D6S-Gal/GalNAc hexa mutant), competed equally with wild-type (Extended Data Fig. 8c), suggesting that neither of these two sulfatase activities are essential determinants in vivo. A significant defect was observed with a mutant lacking all 3S-Gal/GalNAc sulfatases (Fig. 4d). The fitness defect was exacerbated by eliminating 3S-Gal/GalNAc and 6S-GlcNAc sulfatase activities together (D3S-Gal/GalNAc + D6S-GlcNAc) (Fig. 4d), suggesting that they synergize in vivo. Consistent with its prominent role in cMO utilization in vitro, a mutant lacking just BT1636 Gal-3Sulf displayed a significant defect that was similar or slightly more severe compared to the D3S-Gal/GalNAc mutant when competed with the wild-type strain (Fig. 4d), further suggesting that this enzyme plays an essential role in gut colonization by allowing Bt to access 3S-Gal O-glycans (see Supplementary Discussion 4-5 for additional details regarding growth and competition of mutants in vitro and in vivo).

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Conclusion

To degrade the complex O-glycans found in mucins some HGM bacteria have evolved complex arsenals of degradative enzymes which include diverse sulfatases. Disarming all of the sulfatases in Bt via anSME deletion results in drastically reduced competitive colonization (Extended Data Fig. 8c) [START_REF] Benjdia | Sulfatases and a radical S-adenosyl-L-methionine (AdoMet) enzyme are key for mucosal foraging and fitness of the prominent human gut symbiont, Bacteroides thetaiotaomicron[END_REF] and an inability to elicit colitis in an animal model of IBD [START_REF] Hickey | Colitogenic Bacteroides thetaiotaomicron Antigens Access Host Immune Cells in a Sulfatase-Dependent Manner via Outer Membrane Vesicles[END_REF] . While these findings support a critical role for active sulfatases in both fitness and promoting inflammation, they provide no insight into the complexity of catalytic modifications carried out by these enzymes. In this study, we reveal that Bt has a robust ability to grow on highly sulfated mucin oligosaccharides from colonic tissue and that it possesses active sulfatases capable of removing sulfate groups in all contexts in which sulfation is known to occur in mucin, including novel specificities. Surprisingly, we found that a single key sulfatase is essential for growth on colonic mucin O-glycans. This cell surface enzyme removes 3-sulfate capping Gal allowing the degradation of these glycans by additional enzymes. This critical role of BT1636 3S-Gal supports the conclusion that keystone steps exist in the complex pathway of mucin degradation. Further delineation of these critical steps, along with identification of the corresponding enzymes(s), are a prerequisite to modulating such events and potentially inhibiting mucindegrading activities in bacteria that contribute to disease.

Methods

Recombinant Protein Production

Genes were amplified by PCR using the appropriate primers and the amplified DNA cloned in pET28b using NheI/XhoI restriction sites or pETite (Expresso TM T7 cloning and expression system, Lucigen) generating constructs with either N-or C-terminal His6 tags (Supplementary Table 7). The catalytic serine was mutated to cysteine since Escherichia coli in only able to convert cysteine to formylglycine. Recombinant genes were expressed in Escherichia coli strains BL21 (DE3) or TUNER (Novagen), containing the appropriate recombinant plasmid, and cultured to mid-exponential phase before induction with 1 mM (BL21(DE3)) or 0.2 mM (TUNER) of isopropyl β-D-1-thiogalactopyranoside; cells were cultured for another 16 h at 16°C and 180 rpm.

Recombinant protein were purified to >90% electrophoretic purity by immobilized metal ion affinity chromatography using a cobalt-based matrix (Talon, Clontech) and eluted with imidazole as described previously [START_REF] Cartmell | How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans[END_REF] . For the proteins selected for structural studies, another step of size exclusion chromatography was performed using a Superdex 16/60 S200 column (GE Healthcare), with 10 mM HEPES, pH 7.5, and 150 mM NaCl as the eluent, and they were judged to be ≥95% pure by SDS-PAGE. Protein concentrations were determined by measuring absorbance at 280 nm using the respective molar extinction coefficient. When necessary, proteins were then concentrated by centrifugaton using a molecular mass cutoff of 30 kDa.

Site-Directed Mutagenesis

Site-directed mutagenesis was conducted using the PCR-based QuikChange kit (Stratagene) and conducted according to the manufacturer's instructions, using the appropriate plasmid as the template and primers (Supplementary Table 8). All mutations were confirmed by DNA sequencing.

Sources of purified carbohydrates

All carbohydrates were from Sigma, Carbosynth or Dextra Laboratories. All other chemical reagents were purchased from Sigma. The 3S-GalNAc was chemically synthesized as previously described 30 .

Mucin purification

Gastric mucin oligosaccharides (gMO) were purified from commercial available porcine gastric mucins (type III, Sigma) as previously described [START_REF] Martens | Mucosal Glycan Foraging Enhances Fitness and Transmission of a Saccharolytic Human Gut Bacterial Symbiont[END_REF] . Colonic mucins oligosaccharides (cMO) were purified from pig distal pig colons and rectum. Briefly, the tissue was open and the fecal contents were carefully removed. The mucosa was scrapped off and mucus was extracted by homogenizing the tissue in at least 5 times volume of extraction buffer (6 M guanidine chloride, 5 mM EDTA, 10 mM NaH2PO4, pH 6.5) and slow stirring at 4ºC for 16 h. The solution was spun down at 15,000 rpm and 10ºC for 30 min and supernatant was discharged. The pellets were resuspended in extraction buffer and the process was repeated until the supernatant was clear for at least two extractions. After the extraction the mucins were solubilized by reducing the disulfide bonds. The pellets were resuspended in fresh reduction buffer (6 M guanidine chloride, 0.1 M Tris, 5 mM EDTA, pH 8.0) containing 25 mM of 1,4-dithiothreitol and slowly stirred at 37ºC for 5 h. After this incubation, 62.5 mM of iodoacetamide were added and the solution was stirred slowly in the dark at room temperature for 16 h. The solution was centrifuged at 10,000 rpm at 4ºC for 30 min and the supernatant containing the solubilized mucins was extensively dialysed into water. Samples were dissolved into 100 mM Tris-HCl pH 8.0 containing 1 mg/ml of trypsin and incubated slowly stirring at 37ºC for 16 h.

The glycans were b-eliminated by adding 0.1 M NaOH and 1 M NaBH4 and incubate the solution at 65ºC for 18 h. After cooling the solution to room temperature, the pH was adjusted to 7.0 with concentrated HCl and extensively dialysed in water. The released porcine colonic mucin glycans were recovered by lyophilization the solution until completely dry and used in further experiments.

HPLC and TLC sulfatase enzymatic assays

The sulfatase activity screen against commercially available sulfated oligosaccharides (Supplementary Table 2) was performed with 1 µM of recombinant enzyme and 1 mM of substrate in 10 mM MES pH6.5 with 5 mM CaCl2 for 16h at 37ºC. Sulfated N-acetyl-D-lactosamine and lacto-N-biose were generated by incubating the respective sulfated Lewis antigens with 1 μM of a-1,3/1,4-fucosidase BT1625 31 in the same conditions.

Reactions were analysed by thin layer chromatography (TLC). Briefly, 2 μL of each sample was spotted onto silica plates and resolved in butanol:acetic acid:water (2:1:1) running buffer. The TLC plates were dried, and the sugars were visualized using diphenylamine stain (1 ml of 37.5% HCl, 2 ml of aniline, 10 ml of 85% H3PO3, 100 ml of ethyl acetate and 2 g diphenylamine) and heated at 100°C for 20 min. When relevant, the enzymatic activity was confirmed by high-performance anionic exchange chromatography (HPAEC) with pulsed amperometric detection using standard methodology. The sugars (reaction substrate/products) were bound to a Dionex CarboPac P100 column and eluted with an initial isocratic flow of 10 mM NaOH during 20 min then a gradient of 10-100 mM of NaOH for 20 min at a flow rate of 1.0 ml min -1 . The reaction products were identified using the appropriated standards. All experiments were performed in triplicate.

Liquid Chromatograph-Electrospray Ionization Tandem Mass Spectrometry

Enzymatic reactions of sulfatases in colonic mucin oligosaccharides and culture supernatant were cleaned up with graphitized carbon 32 . Reactions with sulfated defined saccharides were reduced and desalted. Briefly, reactions were dried in Speed vac, reconstituted in 20 µL of 50 mM NaOH and 500 mM NaBH4 and incubated at 50ºC for 3 h. Reactions were cool down on ice, neutralized with 1 µL of glacial acetic acid and desalted using a cation exchange column containing AG ® 50W-X8 resin. All cleaned and desalted reactions were reconstituted in water before analysis by liquid chromatographelectrospray ionization tandem mass spectrometry (LC-ESI/MS). The oligosaccharides were separated on a column (10 cm × 250 µm) packed in-house with 5 µm porous graphite particles (Hypercarb, Thermo-Hypersil, Runcorn, UK). The oligosaccharides were injected on to the column and eluted with a 0-40 % acetonitrile gradient in 10 mM ammonium bicarbonate over 46 min at a flow rate of 10 µl/min.. A 40 cm × 50 µm i.d. fused silica capillary was used as transfer line to the ion source. Samples were analyzed in negative ion mode on a LTQ linear ion trap mass spectrometer (Thermo Electron, San José, CA), with an IonMax standard ESI source equipped with a stainless steel needle kept at -3.5 kV. Compressed air was used as nebulizer gas. The heated capillary was kept at 300°C, and the capillary voltage was -33 kV. Full scan (m/z 380-2,000, two microscan, maximum 100 ms, target value of 30,000) was performed, followed by datadependent MS 2 scans (two microscans, maximum 100 ms, target value of 10,000) with normalized collision energy of 35%, isolation window of 2.5 units, activation q=0.25 and activation time 30 ms). The threshold for MS 2 was set to 300 counts. Data acquisition and processing were conducted with Xcalibur software (Version 2.0.7). Glycans were identified from their MS/MS spectra by manual annotation and validated by available structures stored in Unicarb-DB database (2020-01 version) (Supplementary Fig. 1) 33 .

O-Glycan structural characterization was based on diagnostic fragment ions 34 .The schematic glycosidic or cross-ring cleavages were assigned according to the Domon and Costello nomenclacture 35 . For comparison of glycan abundance between samples, the individual glycan structures were quantified relative to the total content by integration of the extracted ion chromatogram peak area using Progenesis QI. The area under the curve (AUC) of each structure was normalized to the total AUC and expressed as a percentage.

Microfuidic-based enzymatic desulfation assays

Sulfated carbohydrates were labelled at their reducing end with BODIPY which has a maximal emission absorbance of ~503 nm, which can be detected by the EZ Reader via LED-induced fluorescence. Non-radioactive mobility shift carbohydrate sulfation assays were optimised in solution with a 12-sipper chip coated with CR8 reagent using a PerkinElmer EZ Reader II system using EDTA-based separation buffer. This approach allows real-time kinetic evaluation of substrate de-sulfation 36 . Pressure and voltage settings were adjusted manually (1.8 psi, upstream voltage:2250 V, downstream voltage: 500 V) to afford optimal separation of the sulfated product and unsulfated substrate with a sample (sip) time of 0.2 s, and total assay times appropriate for the experiment. Individual de-sulfation assays were carried out at 28°C after assembly in a 384-well plate in a final volume of 80 μl in the presence of substrate concentrations between 0.5 and 20 µM with 100 mM Bis-Tris-Propane, 150 mM NaCl, 0.02% (v/v) Brij-35 and 5 mM CaCl2.

The degree of de-sulfation was calculated by peak integration using EZ Reader software, which measures the sulfated carbohydrate : unsulfated carbohydrate ratio at each individua time-point. The activity of sulfatase enzymes was quantified in 'kinetic mode' by monitoring the amount of unsulfated glycan generated over the assay time, relative to control assay with no enzyme; with sulfate loss limited to ∼20% to prevent of substrate and to ensure assay linearity. kcat/KM values, using the equation V0=(Vmax/KM)/S, were determined by linear regression analysis with GraphPad Prism software. Substrate concentrations were varied to ensure assay linearity, and substrate concentrations present were significantly <KM.

NMR desulfation assays

NMR experiments, monitoring the de-sulfation of 6S-D-galactose and 6S-N-acetyl-Dgalactosamine, were conducted in D2O with 50 mM sodium phosphate, pH 7.0, supplemented with 150 mM NaCl at 25°C on a 800MHz Bruker Avance III spectrometer equipped with a TCI CryoProbe and a 600MHz Bruker Avance II+ spectrometer, also fitted with a TCI CryoProbe. 1D and 2D proton and TOCSY spectra (mixing time 80 ms)

were measured using standard pulse sequences provided by the manufacturer. Spectra were processed and analysed using TopSpin 3.4A and TopSpin 4.0 software (Bruker).

Galactose integrals were recorded directly for the C(6)H2-OH peak within the region 3.694 to 3.721ppm, referenced to the combined C(2) peaks of D-galactose and 6S-D-galactose with in the region 3.415 to 3.475ppm. Similarly, 6S-N-acetyl-D-galactosamine integrals were recorded directly for the C(6)H2-OH peak within the region 3.674 to 3.747ppm, referenced to the combined C(4) peaks for N-acetyl-D-galactosamine and 6S-N-acetyl-Dgalactosamine in the region 3.925 to 3.968ppm.

Differential scanning fluorimetry

Thermal shift/stability assays (TSAs) were performed using a StepOnePlus Real-Time PCR machine (LifeTechnologies) and SYPRO-Orange dye (emission maximum 570 nm, Invitrogen) as previously described 37 with thermal ramping between 20 and 95°C in 0.3°C step intervals per data point to induce denaturation in the presence or absence of various carbohydrates as appropriate to the sulfatase being analysed. The melting temperature (Tm) corresponding to the midpoint for the protein unfolding transition was calculated by fitting the sigmoidal melt curve to the Boltzmann equation using GraphPad Prism, with R 2 values of >0.99. Data points after the fluorescence intensity maximum were excluded from the fitting. Changes in the unfolding transition temperature compared with the control curve (ΔTm) were calculated for each ligand. A positive ΔTm value indicates that the ligand stabilises the protein from thermal denaturation, and confirms binding to the protein. All TSA experiments were conducted using a final protein concentration of 5μM in 100 mM Bis-Tris-Propane (BTP), pH 7.0, and 150 mM NaCl supplemented with the appropriate ligand. When BT1622 3S-Gal/GalNAc and BT1636 3S-Gal were assessed against 3'S-LacNAc and 3'S-LNB 100 mM Hepes (pH 7.0) was employed instead of BTP, although no difference in the Tm value of the proteins was observed. Three independent assays were performed for each protein and protein ligand combination.

Glycan labelling

Sulfated saccharide samples were labelled according to a modification of the method previously described reporting the formation of N-glycosyl amines for 4,6-O-benzilidene protected D-gluopyranose monosaccharides with aromatic amines 38 . Briefly, the lyophilised sugar (1 mg) was dissolved in 0.50 ml anhydrous methanol in a 1.5 ml screwtop PTFE microcentrifuge tube. 0.1 mg, BODIPY-FL hydrazide (4,4-difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid hydrazide, lex./em. 505/513, extinction coefficient 80,000 M -1 cm -1 ) was added and the mixture vortexed (1 min), then incubated in darkness at 65ºC for 24 h. The products were then cooled and a portion purified by TLC on silica coated aluminium plates and developed with methanol or 1:1 v/v ethyl acetate/methanol to provide Rf values suitable to allow separation of unreacted label from labelled glycan product. The unreacted BODIPY-FL label (orange on the TLC plate) was identified by reference to a lane containing the starting material (BODIPY-FL hydrazide) on the TLC plate, allowing differentiation from the putative labelled product (also orange). This latter band was scraped from the plates and extracted in fresh methanol (2 x 0.5 ml), spun for 3 min at 13,000 x g and the supernatant was recovered and dried (rotary evaporator) to recover the fluorescent-coloured product (bright green when dissolved in aqueous solution), which was then employed in subsequent experiments.

Anaerobic bacterial culture and genetic manipulation

All strains were anaerobic grown at 37 °C in a chamber (10% H2, 5% CO2, and 85% N2;

Coy Manufacturing, Grass Lake, MI). Bacteroides type strains were culture in either tryptone-yeast extract-glucose medium (TYG), brain heart infusion medium or minimal medium (MM) containing an appropriate carbon source. Bacteroides massilliensis and Akkermansia muciniphila were culture as described before [START_REF] Pudlo | Symbiotic Human Gut Bacteria with Variable Metabolic Priorities for Host Mucosal Glycans[END_REF]39 . Bt strains containing specific gene deletions or inactivated versions of enzymes (BT1636 3S-Gal S77A) were made by counterselectable allelic exchange as previously described [START_REF] Koropatkin | Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices[END_REF] . Complemention of deletion strains was performed using pNBU2 vector as previously described [START_REF] Koropatkin | Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices[END_REF] , containing a constitutive promotor used previously [START_REF] Degnan | Human gut microbes use multiple transporters to distinguish vitamin B(1)(2) analogs and compete in the gut[END_REF] . All primers used to generate the mutants and complementation are listed in Supplementary Table 9. Growth of the WT and mutants was measured on an automated plate reader by increase in absorbance at 600 nm in 96-well plates containing 200 µl of minimal media mixed with the respective filter-sterilised (monosaccharide and gMO) or autoclave-sterilised cMO as described before 39 . To achieve consistent growth, all carbon sources were used at 5 mg/ml with exception of gMO that was added in a final concentration of 10 mg/ml. All growth curves presented are averages and s.e.m of three technical replicates.

Crystallization of carbohydrate sulfatases

After purification, all proteins were carried forward in the same eluent as used for the size exclusion chromatography (see Recombinant Protein Production). Sparse matrix screens were set up in 96-well sitting drop TTP Labtech plates (400-nL drops). Initial hits crystals for all proteins were obtained between 20 and 35 mg/ml. For BT1622 3S-Gal/GalNAc and BT1636 3S-Gal wildtype Bt variants were used, having a Ser at the catalytic formylglycine position, whilst for for BT4683 3S-Gal the S73C mutant was used. BT1622 3S-Gal/GalNAc with 20 mM LNB crystallised in 20% PEG 3350 and 0.2 M sodium citrate tribasic dihydrate.

BT1636 3S-Gal with 20 mM LacNAc crystallised in 40% MPD and 0.2 M sodium cacodylate pH 6.5. for BT4683 3S-Gal with 20 mM LacNAc crystallised in 20% PEG 3350, 0.2 M sodium iodide and BTP pH 8.5. All crystals were cryoprotected with the addition of the ligand they were crystallised with plus 20% PEG 400 and 20% glycerol was used as the cryoprotectant for BT4683 3S-Gal and BT1622 3S-Gal/GalNAc , respectively. No cryoprotectant was added to BT1636 3S-Gal crystals. Data were collected at Diamond Light Source (Oxford) on beamlines I03, I04, I04-1 and I24 at 100 K. The data were integrated with XDS 42 , or Xia2 43 3di or 3dii and scaled with Aimless [START_REF] Evans | How good are my data and what is the resolution?[END_REF] . Five percent of observations were randomly selected for the Rfree set. The phase problem was solved by molecular replacement using the automated molecular replacement server Balbes 45 for all proteins except BT1622 3S-Gal/GalNAc . The phase problem for BT1622 3S-Gal/GalNAc was initially solved using Molrep [START_REF] Vagin | Molecular replacement with MOLREP[END_REF] and BT1636 3S-Gal as the search model. This gave a partial solution, which could not be fully solved due to twinning. An acceptable model of BT1622 3S-Gal/GalNAc was constructed to be used to better solve the phase problem and the molecular replacement was re-performed. Models underwent recursive cycles of model building in Coot [START_REF] Emsley | Features and development of Coot[END_REF] and refinement cycles in Refmac5 [START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF] . Bespoke ligands were generated using JLigand [START_REF] Lebedev | JLigand: a graphical tool for the CCP4 template-restraint library[END_REF] . The models were validated using Coot 47 and MolProbity [START_REF] Chen | MolProbity: all-atom structure validation for macromolecular crystallography[END_REF] . Structural figures were made using Pymol and all other programs used were from the CCP4 [START_REF]Collaborative computational project, n. The CCP4 suite: Programs for protein crystallography[END_REF] and CCP4i2 suite [START_REF] Potterton | CCP4i2: the new graphical user interface to the CCP4 program suite[END_REF] . The data processing and refinement statistics are reported in Supplementary Table 10.

Immunolabelling of BT1636 in Bt cell surface

For the fluorescence microscopy, Bt cells (Wild type (Dtdk) and Dbt1636 3S-Gal ) were grown to early exponential phase (Abs600nm 0.25-0.35) in rich TYG medium. One ml of the cultures was collected, centrifuged at 13,000 x g, and subsequently washed three times in MM with no carbon source. Bt cells incubated with cMO for four hours and fixed in 4.5% formalin overnight at 4ºC with gentle rocking. Cells were stained with a polyclonal antibody raised in rabbit against purified recombinant BT1636 (BT1636 Ab , Cocalico Biologicals) and detected with an Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes). Images were taken with Zeiss Apotome using the same exposure time between samples.

Gnotobiotic Mouse Experiments

All experiments involving animals, including euthanasia via carbon dioxide asphyxiation, were approved by the University Committee on Use and Care of Animals at the University of Michigan (NIH Office of Laboratory Animal Welfare number A3114-01) and overseen by a veterinarian. Groups of 3 to 5, 6-8 week old germfree Swiss Webster mice were randomly assigned to each experiment. 7 days prior gavage the animals diet was switched to a fiber-free diet (Envigo-Teklad TD 130343) that was maintained through all the experiment. At day 0, mice were gavage with equal amount of Bt WT strain and mutant and fecal samples were collected at day 2 and every 5 days until day 42. At the end-point of the experiment distal small intestine and cecal contents were also collected. The bacteria gDNA extraction and quantification by qPCR of the relative abundance of each strain on the various samples was carried out as described previously 39 .

Phylogenetic analysis

To maximise sequence coverage, and avoid repetition, we selected 800 and 920 representative sequences of subfamily S1_20 (composed of 1356 sequences) and S1_4

(composed of 1895 sequences), respectively. The sequences were aligned by MAFFT v.7 53 using L-INS-i algorithm. The multiple sequence alignment was visualized by Jalview software v.11.0 [START_REF] Clamp | The Jalview Java alignment editor[END_REF] and non-aligned regions were removed. 404 and 364 positions were used for the S1_4 and S1_20 phylogeny, respectively. Phylogeny was made using RAxML v. 8.2.4 55 . The phylogenetic tree was build with the Maximum Likelihood method [START_REF] Felsenstein | Evolutionary trees from DNA sequences: a maximum likelihood approach[END_REF] and the LG matrix as evolutive model 57 using a discrete Gamma distribution to model evolutionary rate differences among sites (4 categories). The rate variation model allowed for some sites to be evolutionarily invariable. The reliability of the trees was tested by bootstrap analysis using 1,000 resamplings of the dataset [START_REF] Felsenstein | Confidence Limits on Phylogenies: An Approach Using the Bootstrap[END_REF] . All the final global phylogenetic trees were obtained with MEGA v.7 [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets[END_REF] . Fifteen S1_0 sequences from the sulfAtlas database were used as an outgroup.

Quantification and statistical analysis

For in vivo competitions, when three or more fecal samples were collected, Student's t tests (one-tailed, paired) were performed for each time point in Excel. When necessary, the statistical analysis for remaining samples in stated in the respective figure legend. Maximum absorbance (600 nm)

B . f r a g i l i s B . t h e t a i o t a o m i c r o n B . c a c c a e B . d o r e i B . v u l g a t u s B . s a l y e r s i a e B . n o r d i i B . f i n e g o l d i i B . c e l l u l o s i l y t i c u s B . i n t e s t i n a l i s B . o v a t u s B . x y l a n i s o l v e n s B . u n i f o r m i s B . o l e i c i p l e n u s B . c l a r u s B . e g g e r t h i i B . m a s s i l l i e n s i s A . m u c i n i p h i l a B . f l u x u s B . s t e r c o r i

Bf Bt Bv

Time (h)

Bm

3S-Gal/GalNAc [ bt1636(S77A) + bt1622 + bt4683]

3S-Gal/GalNAc + 6S-GlcNAc [ bt1636(S77A) + bt1622 + bt4683 + bt1628 + bt3177 + bt3051]

A. muciniphila O-glycans utilizer

Gastric O-glycans utilizer Lewis-x 6S-Lewis-

GH2 GH20 GH95 GH18 GH29 GH2 GH20 GH36 GH29 BT3172 S1_11 S1_15 S1_11 S1_20 S1_16 S1_27 GH* BT3173 BT3174 BT3175 BT3176 BT3177 BT3178 BT3179 BT3180 BT1623 BT1624 BT1625 BT1626 BT1627 BT1628 BT1629 BT1630 BT1631 BT1632 BT1634 BT1633 BT1635 BT1636 BT3796 BT3797 BT3798 BT3799 BT3780 GH20 S1_20 BT1617 BT1618 BT1619 BT1620 BT1621 BT1622 TM GH* GH2 GH20 GH95 S1_4 BT4681 BT4682 BT4683 BT4684 BT4631 BT4632 BT4633 BT4634
GH2 BT3092 GH43 S1_4 BT3093 BT3094 S1_67 BT3095 GH51 BT3096 BT3097 BT3098BT3099 BT3100 S1_8 BT3101 BT3102 BT3103 BT3104 BT3105 S1_4 BT3106 GH43 BT3107 BT3108 S1_15 BT3109 S1_4 GH43 BT2912 GH88 BT2913 S1_7 BT2914 BT2915 BT2916 BT2917 BT2918 BT2919 BT2920 BT2921 GH2 BT2922 BT2923 BT0754 BT0755 S1_7 BT0756 GH2 BT0757 BT0752 BT0753 BT3483 BT3484 BT3481 BT3482 BT3485 S1_22 BT3486 S1_4 BT3487 S1_4 BT3488 S1_31 BT3489 BT3490 BT3491 BT3492 BT3493 BT3494 BT3495 BT3496 BT3498 BT3499 BT3500 GH20 BT3501 BT3502 BT3503 BT3504 BT3505 BT3506
R E X R E W(Y) R E X R(K) E X R(M/V/I/L) E X R(M/V/I/L) E X R(A) E X R E W R(A) E X R(K) E X R E X S1_4 R(M/V/I/L) E W(Y/F/H) R(K) E W R E X Predicted activity 3S-Gal 3S-Gal/GalNAc E Q H E R E Q H E R E Q H E R E Q H E R E Q(H) H E R E Q(H) H E R E Q H E R E Q H E R E Q H E R E Q H E R E Q H E R E Q H E R E Q H E R E Q H E R X Q H E R

Utilization of different mucin O-glycans sources by HGM

Mucin composition varies throughout the gastrointestinal (GI) tract, with the stomach having mainly MUC5AC and the colon mainly MUC2 1 . The glycosylation of these respective mucins also varies along the GI tract with higher levels of sulfated and sialylated structures observed in the distal colon compared to the upper GI tract 2 .

Among the 20 bacterial strains tested for growth, 12 failed to grow on gastric mucin O-glycans (gMO) or colonic mucin O-glycans (cMOs) (Fig. 1b). Only 1b and Extended Data Fig. 1). The differences observed in the growth profiles were reproducible in two different batches of purified cMOs (Fig. 1b) Indeed, it is likely that different HGM members have evolved to target different (or only a subset) of the available O-glycans and this fine-tuning of host glycan utilization may have important implications in gut colonization and symbiosis. Additionally, B. massiliensis and Akkermansia mucinipila grew on gMO but failed to utilize cMO (Fig. 1b and Extended Data Fig. 1). Both strains were able to grow on N-acetylglucosamine (GlcNAc) and Akkermansia mucinipila grew on GlcNAc in the presence of cMO suggesting that these O-glycans do not inhibit the growth of this bacterium. Previous studies have determined that B. massiliensis and Akkermansia mucinipila are mucin-degraders by demonstrating growth on gastric mucins [START_REF] Derrien | Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium[END_REF][START_REF] Pudlo | Symbiotic Human Gut Bacteria with Variable Metabolic Priorities for Host Mucosal Glycans[END_REF] . However, the lack of growth in colonic O-glycans suggests that these bacteria are not able to initiate the degradation of more complex, sulfated colonic glycans. This finding highlights the importance of taking into account O-glycosylation differences along the GI tract and the need to utilize colonic mucins to draw conclusions regarding the full mucin-degrading potential of the colonic HGM.

Sulfatase activity in cMO

Despite all 12 sulfatases being active on defined oligosaccharides, of those tested on cMO, BT1622 3S-Gal/GalNAc (S1_20 subfamily) and BT1624 6S-Gal/GalNAc (S1_15 subfamily) did not show any activity on this complex substrate (Fig. 2, Extended Data Fig. 5 and Supplementary Table 4). These findings are consistent with the results observed in defined commercial oligosaccharides where BT1622 3S-Gal/GalNAc showed a preference for sulfated GalNAc over Gal glycans (Extended Data Fig. 4) and BT1624 6S-Gal/GalNAc activity is blocked by the presence of additional substitutions (such as Lewis antigens) (Extended Data Fig. 4a). Additionally, 2 of 6 detected 6S-Gal structures contained a capping sialic acid and a terminal blood group H type 2 [Fuc-a1,2-(6S)Gal-b1,4-GlcNAc-] (Fig. 2). The lack of activity of BT1624 6S-Gal/GalNAc towards such structures confirms an exo-mode of action that we describe for this sulfatase using commercial substrates.

Overall, when compared to the non-enzyme treated control, we detected an increase of non-sulfated structures and decrease of sulfated oligosaccharides in all samples with the active enzymes (BT4683 3S-Gal , BT1636 3S-Gal , BT1628 6S-GlcNAc and BT3177 6S-GlcNAc ) (Extended Data Fig. 5b). BT1636 3S-Gal (S1_20) was active towards all detected 3S-Gal structures with the exception of glycan 1055_1 that is a doubly sulfated 3S-Gal/6S-GlcNAc fucosylated structure (Extended Data Fig. 5a). As we observed using commercial substrates, the presence of Lewis-a/x epitopes leads to a decrease in the activity of this sulfatase (Extended Data Fig. 4a and Supplementary Table 4) and the presence of a second sulfate group might exacerbate this negative effect leading to the lack of activity towards this complex sulfated O-glycan. The incubation of the 6S-GlcNAc sulfatases BT1628 6S-GlcNAc and BT3177 6S-GlcNAc with cMO suggests that these enzymes are redundant, but because they are encoded in different PULs they could be expressed in response to different activating cues (Fig. 2 and Supplementary Table 4). Compared to the non-enzyme treated control, 16 glycans

were not detected after incubation with these sulfatases, 14 of these structures have a terminal 6S-GlcNAc (Fig. 2 and Supplementary Table 4). BT1628 6S-GlcNAc and BT3177 6S-GlcNAc were active in 6S-GlcNAc core 3 (GlcNAc-b1,3-GalNAc) and core 4 (GlcNAc-b1,6-GalNAc) structures (Extended Data Fig. 5a), suggesting that these sulfatases are well suited to accommodate the variations in linkages/sugars found in mucin O-glycans. Additionally, we also detect 7 new glycans that are likely to be reaction products of BT1628 6S-GlcNAc and BT3177 6S-GlcNAc (Fig. 2 and Supplementary Table 4).

The identification and characterization of the first sulfatases active on mucin Oglycans creates the opportunity to improve our understanding of O-glycan structures by using these enzymes as analytical tools. After the treatment with BT1636 3S-Gal several oligosaccharides predicted to contain a terminal sulfate linked to Gal were not detected. Although we could not determine the specific sulfate linkage by mass spectrometry, the activity of the 3S-sulfatase suggests that these oligos contain a terminal 3S-Gal (Extended Data Fig. 5a and Supplementary Table 4). The specificity of the 6S-GlcNAc sulfatases for non-fucosylated O-glycans also illuminates their potential use as tools to characterize the structure of these complex structures since it allows the differentiation of different isomers. For example, we detect two oligosaccharides with mass 1096, however, after incubation with BT1628 6S-GlcNAc or BT3177 6S-GlcNAc , only the isomer 1096_2 was detected, indicating that the isomer 1096_1 contain a terminal 6S-GlcNAc (Extended Data Fig. 5a and Supplementary Table 4).

Conserved structural features of the S1 formylglycine family

Protein fold and subsites nomenclature (Extended data Fig. 6a). The subsite nomenclature for carbohydrate sulfatases is such that the invariant sulfate binding site is denoted as the S site. The S site sulfate is appended to the 0 subsite sugar. Subsites then increase in number (i.e. +1, +2, +3)

as the sugar moves toward the reducing end (free O1) and decreases in number as the sugar chain moves towards the non-reducing end (i.e. -1, -2, -3) [START_REF] Hettle | The Molecular Basis of Polysaccharide Sulfatase Activity and a Nomenclature for Catalytic Subsites in this Class of Enzyme[END_REF] .

S1 formylglycine active site conservation

The sulfate binding site (S site) is invariant across the S1 family and comprises the catalytic residues (nucleophile and catalytic acid) and a calcium binding site (Extended data Fig. 6b). An invariant histidine is likely the potential catalytic acid but a lysine has also been suggested to possibly fulfil this role [START_REF] Cartmell | How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans[END_REF] . The pKa of His is ~6.0, whilst Lys has a pKa of >10, making it more chemically feasible that His performs the role of the catalytic acid. Homologues of these residues (H252 and K352 in BT1636 3S- Gal ) make hydrogen bonds to the scissile sulfoester linkage (Extended data Fig. 6b).

Previously published work with BT1596 and BT4656, which are 2S-Uronic acid and 6S-GlcNAc sulfatases, respectively, showed that the mutation of either residue to alanine causes inactivation [START_REF] Cartmell | How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans[END_REF] . Consistent with this work, a BT4683 3S-Gal H219A mutant was inactive. However, in BT1622 3S-Gal/GalNAc , the mutation of H255 to Ala caused only a ~30-fold decrease in activity (Supplementary Table 3). Thus, it is possible that in BT1622 3S-Gal/GalNAc the loss of H255 is compensated by the invariant residue K356 and interestingly BT1622 3S-Gal/GalNAc has a pH optimum ~2 units higher than most sulfatases assayed (Supplementary Fig. 4).

The calcium binding site is located at the base of the S site interacting with the sulfate group. This calcium ion is an essential component of the catalytic mechanism helping to stabilise negative charges that occur during the catalysis. All three of the solved structures had occupation for calcium. In BT1636 3S-Gal D328 and the sulfate group of the substrate coordinate above and below the calcium with D37, D38, N329

and the formylglycine binding in a plane completing an octahedral coordination (Extended Data Fig. 6b). These three Asp and the Asn coordinated with calcium are structurally conserved in all 3S-Gal/GalNAc sulfatases structures (Extended data Fig. 6b).

The solved structures of BT1636 3S-Gal and BT1622 3S-Gal/GalNAc were native Bt proteins having a Ser at the formylglycine position. However, the structure of BT4683 3S-Gal was obtained with the active protein where S73 was mutated to Cys (as E. coli can only convert Cys, not Ser, to formylglycine). The analysis of BT4683 3S-Gal reveals that the crystallized protein still has the Cys and not formyglycine indicating poor installation of the formylglycine. This observation means the kinetic data, (although the rates are significant and readily measurable) may be an underestimation of true catalytic performance. This will affect the kcat component of the kcat/KM measurement and thus the kcat/KM reported in Supplementary Table 3 is an underestimate of the true activity.

Additional 3S-Gal/GalNAc specificity determinants based on structures BT1636 3S-Gal was solved in complex with the product LacNAc; the Gal at 0 subsite is well ordered and makes extensive interactions, whilst the +1 GlcNAc is highly disordered and appears to make no interactions with the protein (Fig. 3 and Extended Data Fig. 6c). O2 of the Gal hydrogen bonds with Oe1 of E334 and NH2 of R353. Mutation of these residues to Ala causes ~300 and ~60-fold reductions in kcat/KM, respectively. The O6 group of Gal potentially coordinates with Oe2 of E100 and Ne2 of Q173 and mutations of these residues to Ala cause ~80 and ~50-fold decreases in kcat/KM, respectively (Fig. 3 and Supplementary Table 3). Comparison of the BT1622 3S-Gal/GaNAc structure with BT1636 3S-Gal shows that E98 and Q172 (which correspond to E100 and Q173 in BT1636 3S-Gal ) are conserved (Fig. 3) and mutating E98 to Ala caused only a 15-fold decrease in kcat/KM (Supplementary Table 3).

Additionally, in BT1622 3S-Gal/GaNAc the hydrophobic interactions with the N-acetyl group,

and the more open pocket, may offset the effects H176A (300-fold loss in activity) when compared to H177A (complete loss in activity) in BT1636 3S-Gal (Fig. 3 and Supplementary Table 3).

BT4683 Gal-3S also displayed the same 3S-Gal activity as the S1_20 enzymes but showed a preference for 3'S-LacNAc, reciprocal to BT1622 3S-Gal . BT4683 3S-Gal bound the O2 Gal of LacNAc via Oe2 of E335 (equivalent to E334 in BT1636 3S-Gal ) and through either Ne or NH1 of R72 (Fig. 3). Although R72 is sequentially distal to R353 in BT1636 3S-Gal it is spatially similar and likely contributes in a similar capacity (Fig. 3).

Despite the mutations R72A and E335A resulting in loss of activity, the Glu and Arg are only conserved in 62 % and 19 % of S1_4 sequences, respectively, suggesting there is a significant but not absolute selection for an equatorial O2 in this subfamily (Extended Data Fig. 7 and Supplementary Fig. 2). Uniquely among the 3S-Gal sulfatases identified, BT4683 3S-Gal utilises a hydrophobic stacking interaction through W505 to provide a platform for the +1 GlcNAc and partially the 0 Gal. Mutation of W505 to Ala almost completely abolishes activity on 3'S-LacNAc (Supplementary Table 3) but surprisingly this residue is not conserved in our phylogenetic analyses of S1_4 being present in only 8 other sequences (Extended Data 7 and Supplementary Fig. 2). It is important to note, that W505 is not well conserved; potential equivalent aromatic residues can be found in some additional clades, which are coloured light brown (or bronze), pink or dark red, but it is not evident from the alignment that these are functional equivalents. Future structural work is needed to confirm if other aromatic residues take equivalent positions in those sulfatases. Additionally, the BT4683 3S-Gal activity against defined sulfated saccharides was suggestive of an exo-acting enzyme that cleaves terminal 3S-Gal (Extended Data Fig. 4). However, a close analysis of this sulfatase structure shows that the active site is located in an open cleft characteristic of an endo-active enzyme [START_REF] Hettle | The Molecular Basis of Polysaccharide Sulfatase Activity and a Nomenclature for Catalytic Subsites in this Class of Enzyme[END_REF] . This more open cleft of BT4683 3S-Gal may allow additional sugars/sulfates to be accommodated on the O6 of both the 0 Gal and +1 GlcNAc. Indeed, the activity determined in cMO shows that this sulfatase can act on sialylated O-glycan (Fig. 2). Further modelling of different O-glycan structures (using the crystallographically solved LacNAc as an 'anchor') indicate that this enzyme can accommodate complex O-glycans with internal sulfation (Fig. 3 and Extended Data 6d). Together, these results suggest that BT4683 Gal-3S , and its close homologues, could be endo 3S sulfatases where the 0 subsite specificity for Gal is driven by glycan context and/or distal subsites such as -1 and +2, rather than an axial O4 as in S1_20.

Additionally, it is unclear why BT1636 Gal-3S acts better on LacNAc substrates than BT1622 3S-Gal/GalNAc . It is interesting to note, however, that both BT1636 3S-Gal and BT4683 3S-Gal perform well on LacNAc configured substrates and utilise an Arg and Glu to coordinate O2 whilst BT1622 3S-Gal lacks these residues (Fig. 3). These residues may lead to the enhanced activity on LacNAc (b1,4 glycan) vs. LNB (b1,3 substrate).

Another thing to note is that a b1,4 vs b1,3 linkage will rotate the GlcNAc ~60° but switch the position of the Fuc residue from being on the 'N-acetyl side' of the glycosidic bond to the 'O6 side' of the glycosidic bond, and this may also be the cause of the differential activities on b1,4 vs b1,3 linked substrates.

Phylogenetic analyses of S1_20 specificity determinants

The essential His that acts as a key specificity determinant of galacto-over gluco-substrates (H177 and H176 in BT1636 3S-Gal and BT1622 3S-Gal/GalNAc , respectively) is highly conserved (92% of S1_20 sequences) (Extended data Fig. 7 and Supplementary Fig. 3). The Gln (Q173 and Q172 in BT1636 3S-Gal and BT1622 3S- Gal/GalNAc , respectively) is only conserved in 66% of sequences and in 25% of the cases is substituted with a histidine, a residue that can also fulfill the same role of Gln interacting with Gal O6. Indeed, these conserved residues are located in a highly a Q173 may function indirectly to select for an axial O4 and thus these residues may operate as a selectivity 'dyad' for Gal with S1_20. Additionally, the residues implicated in recognition of Gal over GalNAc, E335 and R353 in BT1636 3S-Gal are conserved in 64 and 74% of S1_20 sequences, whilst the residue that allows the accommodation of O2 N-acetyl and activity in GalNAc (N334 in BT1622 3S-Gal/GalNAc ) is only found in 8% of members of this family (Extended Data Fig. 7 and Supplementary Fig. 3). This observation suggests that the majority of the S1_20 sulfatases evolved to target sulfated Gal and only a subset of this subfamily's members can actually also be active on GalNAc. Interestingly, all of the close homologs of BT1636 3S-Gal and BT1622 3S- Gal/GalNAc that share the critical specificity determinants of these proteins (Supplementary Tables 12 and13) were isolated from mammals at body regions rich in mucins, highlighting the role of these sulfatases in accessing sulfated host glycans.

Growth of sulfatase mutants on O-glycans

The deletion strain lacking 4S-Gal/GalNAc sulfatases (Dbt3057 + Dbt3796) did not show any phenotype in cMO (Extended Data Fig. 8a), a result that is consistent with the lack of these sulfated linkages in colonic mucins (Supplementary Table 4).

Unexpectedly, the deletion strains lacking the identified 6S-Gal/GalNAc sulfatases (Dbt1624 + Dbt3109 + Dbt4631) and 6S-GlcNAc sulfatases (Dbt1628 + Dbt3051 + Dbt3177) also did not show any growth defect on cMOs (Extended Data Fig. 8a).

Analysis of cMO by mass spectrometry showed that this substrate contains a low abundance of 6S-Gal but a relatively high abundance of 6S-GlcNAc, especially in shorter structures (Supplementary Table 4). Although the low abundance of O6sulfated Gal could explain the lack of phenotype of the 6S-Gal/GalNAc sulfatase deficient strain, the lack of effect in the D6S-GlcNAc mutant in cMO was unexpected (Extended Data Fig. 8a). Due to the limitations of the mass spectrometry technique it is not possible to analyse sulfation in longer oligos, making the real complexity of glycans found in colonic mucins unclear. Indeed, the lack of phenotype of D6S-GlcNAc mutant in cMO suggests that 6S-GlcNAc might not be a major terminal epitope in colonic mucins. It is also important to note that the mutant D6S-GlcNAc is the deletion of two characterized 6S-GlcNAc sulfatases active on cMO (BT1628 6S-GlcNAc and BT3177 6S-GlcNAc ) and a third closely related S1_11 sulfatase (BT3051 putative_6S-GlcNAc ) for which no activity was found. This putative 6S-GlcNAc sulfatase was deleted to avoid possible compensation of function after loss of BT1628 6S-GlcNAc and BT3177 6S-GlcNAc activities.

The deletion of previously characterized GAG-specific sulfatases 8 (Dbt1596 + Dbt3333 + Dbt3349 + Dbt4656) did not result in any observable phenotype in cMO (Extended Data Fig. 8a), indicating that this substrate was not contaminated with additional endogenous host glycans. Additionally, despite some mutants exhibiting growth defects on sulfated cMO, all of the mutants grew well on gMO and glucose (Fig. 4a and Extended Data Fig. 8a), suggesting that the phenotypes observed are dependent on the mucin source (colon) and cannot be observed utilizing mucins from other regions of the gastrointestinal tract. Together these results highlight the contribution of sulfatases in utilization of colonic mucins by the HGM.

Analysis of Dbt1636 3S-Gal culture supernatant by MS

The analysis of the oligosaccharides present in Dbt1636 3S-Gal culture supernatant after 96h incubation revealed that the detected glycans are different from the cMO profile in the starting material (Fig. 4c, Extended Data Fig. 8b and Supplementary Table 5). We detected 114 glycans in the cMO sample, of which 39 were sulfated and fucosylated (44% total) (Extended Data Fig. 8b) and the three most common structures (12% total) were 6S-GlcNAc oligosaccharides (Fig. 4c and Supplementary Table 5). In the control sample, the levels of sulfation, sialylation and fucosylation were 92%, 40% and 77%, respectively (Supplementary Table 5). In the Dbt1636 3S- Gal culture supernatant, we detected 72 glycans, of which 41 were substituted only with O-sulfate (84% total) (Extended Data Fig. 8b). In the mutant supernatant the levels of sulfation (95%) were similar to cMO, however the levels of sialylation (11%) and fucosylation (5%) decreased substantially (Supplementary Table 5), suggesting that this mutant is not able to utilize sulfated structures and these accumulate in culture media.

Additionally, a total of 98 of the 114 structures present in cMO were not detected in Dbt1636 3S-Gal culture supernatant whereas in mutant supernatant, we detected 49 glycans that were not detected in the initial substrate (Supplementary Table 5). This suggests that some of the oligosaccharides present in cMO can support the limited growth of Dbt1636

Supplementary Table 7

Primers designed to clone Bt sulfatases Suplementary Fig. 2. Phylogenetic tree of representative sulfatases from subfamily S1_20 The annotations concern the presence or absence of conservation of the following residues E100, Q173 H177, E334, R353, which are crucial in substrate recognition by BT1636 (acc-code Q8A789; i.e. E100 means an equivalent glutamate is present, and for example E334A means that the E-equivalent position is replaced by an alanine); in a certain number of sequences no obvious ortologous residue can be deduced from the alignment and this is indicated (red text); the branches have the same colour if a pattern is in common; the colour codes are the same and as defined in Extended Fig. 7 . Red filled circles designate sequences of S1_20 sulfatases from B. thetaiotaomicron.

Figure 1 .

 1 Figure 1. Bacterial growth on colonic mucin and Bt sulfatase activities. a, Schematic representation of mucin O-glycans and relevant terminal epitopes (dashed boxes). Sugars are shown according to the Symbol Nomenclature for Glycan system 60 . b, Growth of Bacteroides type strains and Akkermansia muciniphila on colonic mucin Oglycans (cMO) and number of respective encoded S1 sulfatases. The bars represent the average of two independent experiments with different batches of cMO. Bacterial species able to utilize gastric mucin glycans are highlighted in blue. Maximum absorbance is the difference of the maximum absorbance value (Abs600nm) for each culture and the initial absorbance at time 0 (T0h). Graphic shows the example of growth curves for B. fragilis (Bf), B. thetaiotaomicron (Bt), B. vulgatus (Bv) and B. massilliensis (Bm). c, Phylogeny of Bt sulfatases showing the 28 S1 sulfatases and their respective substrates where known, including this study. Enzymes are color coded according the respective subfamilies with sulfatases characterized in this study highlighted in bold. * indicates sulfatase activity previously characterized and arrows point the substrate preferentially targeted by the respective enzyme. Sulfatases on a shared branch that share more than 86% and 39-58% of sequence identity are highlighted in blue and green background, respectively. Data from biological replicates n = 3 to 6 and error bars denote s.e.m..

Figure 2 .

 2 Figure 2. Activity of Bt sulfatases on colonic mucin O-glycans. a, Representation of O-glycans detected by mass spectrometry in cMO (control) and after sulfatase treatment from the lower (top) to the higher (bottom) mass range. b, Relative abundance and putative structures for the specific m/z shown in panel a. Remaining structures are shown in Extended figure 5a. c, Schematic representation of the putative structures that were not detected after treatment with BT1636 3S-Gal .

Figure 3 .

 3 Figure 3. Crystal structures of 3S-Gal/GalNAc sulfatases. a, Schematicrepresentation of the residues interacting with targeted sugars, including the putative catalytic residues (in dark red), the calcium ion (grey sphere) and subsites S, 0 and +1 highlighted in red. BT1636 3S-Gal and BT4683 3S-Gal in complex with LacNAc (D-Gal-b1,4-

Figure 4 .Extended Data 8 .

 48 Figure 4. BT1636 3S-Gal activity is required for the utilization of cMO and competitive fitness in vivo. a, Growth of Bt wild-type Dtdk (WT), different sulfatase gene-deletion mutants (named "DbtXXXX") and strains complemented with bt1636 3S-Gal on colonic or gastric mucin O-glycans (cMO and gMO, respectively) (line represents the average of biological replicates (n = 3) and error bars denote s.e.m.) b, Immunofluorescent and differential interference contrast (DIC) microscopy of Bt WT and sulfatase mutant staining with polyclonal antibody (Ab) against BT1636 3S-Gal (green) and DNA staining with DAPI (blue). c, Relative abundance of different O-glycans detected by mass spectrometry in Dbt1636 3S-Gal culture supernatant or cMO in minimal media without bacteria (control), after 96h in anaerobic conditions. The mass and associated structure of the 3 more abundant glycans in both samples are shown. d, in vivo competitions in gnotobiotic mice (n = 5-9 separate mice from two separate experiments, except competition of the D6S-GlcNAcmutant that showed no defect in one experiment) fed fiber-free diet and inoculated with WT and mutants. The fecal relative abundance of each strain was determined along the time course and in small intestine and cecum at day 42 (experimental endpoint). The relative abundance in each mouse is represented in the respective light colour. The error bars denote s.e.m. Significant differences between wild-type and mutant strain were compared at each time point using student's t-test (paired, one tail) and * indicates sample days in which the mutant was significantly different (p < 0.01) from the wild-type.

  Fig 1.

  Fig 2.

  Fig 4.

  -D-galactosamine (GalNAc) N-Acetyl-D-glucosamine (GlcNAc)

  a l p e l l e t 6S-GlcNAc ( bt1628 + bt3177) 6S-GlcNAc + 6S-Gal/GalNAc ( bt1628 + bt3177 + bt3051 + bt1624 + bt3109 + bt4631)

S1 sulfatases comprise the

  most common and largest family of sulfatases, currently encompass 36,816 members in sulfAtlas and are found in all domains of life 5 . S1 sulfatases are part of the alkaline phosphatase superfamily and adopt an alkaline phosphatase-like fold. This is an N-terminal a/b/a domain with S1 sulfatases also possessing a smaller C-terminal 'sub domain'. The active site is located in the Nterminal domain that has a large mixed b-sheet composed of ~10 b strands, sandwiched between a helices above and below. The C-terminal 'sub-domain' is composed of a 4 stranded antiparallel b-sheet and a single amphipathic terminal helix. This C-terminal domain abuts the N-terminal domain through the antiparallel b-sheet with loops from the b strands sometimes contributing to the active site architecture

  conserved domain with the consensus sequence [CDNS]-[QH]-[RVF]-[QHLD]-[AG]-H-[NRST]-[YHF]-[YF]-P (Prosite syntax). With H177 targeting the axial O4 of Gal directly,

Fig. 4 )

 4 , supplemented with 150 mM NaCl, 5 mM CaCl2 and 0.02 % Brijj. 6S-Gal/GalNAc activity was analysed in 50 mM sodium phosphate with 150 mM NaCl at 25°C by NMR. (WT) wild-type sulfatases with catalytic Ser mutated to Cys. (NQ) Active but not possible to quantify, (NA) Not active; (LacNAc) N-acetyl-Dlactosamine; (LNB) lacto-N-biose; (GlcNAc) N-acetyl-D-glucosamine; (Gal) D-galactose; (GalNAc) N-acetyl-D-galactosamine; (GlcNAc) N-acetyl-D-glucosamine. All values were performed in triplicate and represent averaged data with the standard error of the mean, unless otherwise stated. * These values are duplicates of specific activities at 500 µM substrate and are in units of h -1 .

2 Suplementary Fig. 2 .

 22 Phylogenetic tree of representative sulfatases from subfamily S1_4. The annotations concern the presence or absence of conservation of the following residues R72, E335 and W505, which are crucial in substrate recognition by BT4683 (acc-code Q89YP8; i.e. R72 means an equivalent arginine is present, and for example R72I means that the R-equivalent position is replaced by an isoleucine); the branches have the same colour if a pattern is in common; the colour codes are the same and as defined in Extended Fig 7.Red filled circles designate sequences of S1_4 sulfatases from B. thetaiotaomicron.

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  6 bacteria were able to utilize both O-glycans substrates but growth was variable. In both O-glycan

substrates, Bacteroides thetaiotaomicron (Bt), B. caccae, B. fragilis and B. fluxus grew better than B. dorei and B. vulgatus (Fig.

  3S-Gal and, although this mutant is not able to utilize many sulfated cMO structures, it can still modify the glycans to create novel structures. It remains unclear which enzymes are encoded by the mutant to modify the O-glycans, but the presence of a cell surface sialidase 9 can explain the decrease of sialylation levels in structures found in Dbt1636 3S-Gal supernatant. Additionally, the presence of surface endo-acting glycoside hydrolases able to cleave O-glycans into shorter oligosaccharides 10 can also contribute to new glycan structures in the mutant culture supernatant. Together these results show that Dbt1636 3S-Gal is not able to utilize most sulfated O-glycans explaining the limited growth of this mutant in cMO. Briliute, J. et al. Complex N-glycan breakdown by gut Bacteroides involves an_SI. Nat Microbiol 4, 1571-1581, doi:10.1038/s41564-019-0466-x (2019). 10 Crouch, L. I. et al. Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown. Nat Commun 11, 4017, doi:10.1038/s41467-020-17847-5 (2020).Reactions performed at 28°C and in 100 mM Bis-Tris-Propane, at the appropriate pH optimum (Supplementary

				kcat/KM (min -1 M -1 )
		6S-Gal*	6S-GalNAc*	6S-GlcNAc	4S-Gal	4S-GalNAc
	WT BT1624 6S-Gal/GalNAc (S79C)	3.9 ± 0.55	1.95 ± 0.6	NA	NA	NA
	WT BT3109 6S-Gal/GalNAc (S79C)	2.73 ± 0.74	NQ	NA	NA	NA
	WT BT3333 6S-Gal/GalNAc (S79C)	NQ	3.1 ± 0.02	NA	NA	NA
	WT BT4631 6S-Gal/GalNAc (S79C)	NQ	NQ	NA	NA	NA
	WT BT3177 6S-GlcNAc (S79C)	NA	NA	(2.6 ± 0.2) x 10 4	NA	NA
	WT BT3057 4S-Gal/GalNAc (S88C)	NA	NA	NA	NQ	(5.2 ± 0.1) x 10 2
	WT BT3796 4S-Gal/GalNAc (S88C)	NA	NA	NA	NQ	(4.3 ± 0.09) x 10 3

Table 8

 8 Primers designed to generate the site-directed mutants of Bt sulfatases Primers designed to generate the in-frame gene deletions and complementations of Bt sulfatases catalytic residue was mutated to alanine to knockdown the sulfatase activity of this protein; # complementation of bt1636 deletion; Restriction sites highlighted in bold.

	Gene Vector	Primer name	Primer sequence (3' to 5')
	BT0756 pETite_Ntag	BT0756_Fw BT0756_Rv	catcatcaccaccatcacgagaacctgtacttccagggc cagcacaatgtctccgatgtttcc gtggcggccgctctatta ttatttttttaatgatgatttcttcac
	BT1622	pETite_Ntag pET28b _Ntag	catcatcaccaccatcacgagaacctgtacttccagggc caaacccaacccgcatctccc gtggcggccgctctatta aaagtccggaccgtttctagc BT1622_Fw_NheI ggaatagctagc caaacccaacccgcatctcccaac BT1622_Fw BT1622_Rv BT1622_Rv_XhoI gagatactcgag ttaaaagtccggaccgtttctagc
	BT1624	pETite_Ctag pET28b _Ntag	gaaggagatatacatatg caggagaaacaaacccagcgtcc gtgatggtggtgatgatg tagtggcagaccgtagcgtc BT1624_Fw_NheI ggaatagctagc aaacaaacccagcgtccgaatgtt BT1624_Fw BT1624_Rv BT1624_Rv_XhoI gagatactcgag ttatagtggcagaccgtagcgtcc
	BT1628 pETite_Ctag	BT1628_Fw BT1628_Rv	gaaggagatatacatatg acaaacaagcagaaaacgacagaacaaaagccg gtgatggtggtgatgatg ttccttgtccctttccggaga
	BT1636	pETite_Ctag pET28b _Ntag	gaaggagatatacatatg cagaaaaacaacacaaagccgaatatccctc gtgatggtggtgatgatg tttcttctccggtagtgtaacgg BT1636_Fw_NheI ggaatagctagc cagaaaaacaacacaaagccgaat BT1636_Fw BT1636_Rv BT1636_Rv_XhoI gagatactcgag ttatttcttctccggtagtgtaac
	BT1918 pETite_Ctag	BT1918_Fw BT1918_Rv	gaaggagatatacatatg caaaaacaaccgcacattatcttaattatg gtgatggtggtgatgatg tcgcttttctttcggatagttag
	BT2914 pET28a_Ntag	BT2914_Fw_NheI ggaatagctagc cagaataataagatgaatgttttg BT2914_Rv_XhoI gagatactcgag ctataattgagatttccggatctt
			BT3051_Fw	gaaggagatatacatatg caacctgccggacaacgcccc
	BT3051 pETite_Ctag	BT3051_Rv	gtgatggtggtgatgatg cttattatagtatttttcaagcagtttctgc
	BT3057	pETite_Ctag	BT3057_Fw BT3057_Rv	gaaggagatatacatatg caggaacgcccgaatattattgtcttcc gtgatggtggtgatgatg aaagtaaacaaaaggcttcaactcatttcc
		pET28a_Ntag	BT3057_Fw_NheI ggaatagctagc caggaacgcccgaatattattgtct BT3057_Rv_XhoI gagatactcgag ttaaaagtaaacaaaaggcttcaa
	BT3093 pETite_Ctag	BT3093_Fw BT3093_Rv	gaaggagatatacatatg caagaaaagcctaatatcattgttatattggcgg gtgatggtggtgatgatg cttatctctatattctttaaagtccttccgg
	BT3095 pETite_Ctag	BT3095_Fw BT3095_Rv	gaaggagatatacatatg cagcgttttgtattacgggattggc gtgatggtggtgatgatg ttgttcgggttggagataattataagg

*

* * * * * * * * * * * * * * * * * *

Acknowledgements

This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement N o 748336. This work was supported by National Institutes of Health grants (DK118024 and DK125445 awarded to ECM, U01AI095473 awarded to GCH), the European Research Council ERC (694181), The Knut and Alice Wallenberg Foundation (2017.0028), Swedish Research Council (2017-00958) and the academy of medical sciences/Wellcome Trust through the springboard grant SBF005\1065 163470 awarded to AC. The authors acknowledge access to the SOLEIL and Diamond Light sources via both the University of Liverpool and Newcastle university BAGs (proposals mx21970 and mx18598, respectively). We thank the staff of DIAMOND, SOLEIL, and members of the Liverpool's Molecular biophysics group for assistance with data collection. Mass spectrometry of glycans was performed in the Swedish infrastructure for biologic mass spectrometry (BioMS) supported by the Swedish Research Council. We are also grateful for Dr. Erwan Corre's help regarding bioinformatics analyses (ABIMS platform, Station Biologique de Roscoff, France).

Data availability statement

Source Data for all experiments, along with corresponding statistical test values, where appropriate, are provided within the paper and in Supplementary information. The crystal structure dataset generates have been deposit in the in the Protein Data Bank (PDB) under the following accession numbers: 7ANB, 7ANA, 7AN1 and 7ALL. The LC-MS/MS raw files and annotated structures are submitted to the Glycopost (https://glycopost.glycosmos.org/preview/12430260615f9d5733a1a5d, code 1955) and Unicarb-DB database, respectively. Code availability statement: No new codes were developed or compiled in this study . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 21, 2020. ; https://doi.org/10.1101/2020.11.20.392076 doi: bioRxiv preprint

Competing interests statement

The authors declare no competing interests.

Author contributions

ASL, AC and ECM designed experiments and wrote the manuscript. ASL and AC cloned, generated proteins mutants, expressed and purified sulfatases. ASL and AC performed enzyme assay, binding and kinetic analyses with assistance of DPB and PAE. MR and SO synthesized the O3-sulfated N-acetylgalactosamine. EAY labelled glycan substrates. AC grew, solved, collected and analysed crystallographic data with assistance from AB.

AC and JAL carried out NMR kinetic analyses. CJ, ASL, GCH and NGK performed and interpreted analytical glycobiology experiments. ASL, GP, RWPG, SG, SS and NAP performed bacterial growth experiments and analysed in vivo competition data. MC, GM and TB performed sulfatase phylogenetic analyses. All authors read and approved the manuscript.

Supplementary Table 1

Family S1 sulfatase subfamiles encoded in the genomes of different Bacteroides type strains and Akkermansia

Supplementary Table 2

List of sulfated saccharides used in the initial sulfatase activity screen

Supplementary Table 3

Sulfatase kinetics for WT and mutants against different saccharides kcat/KM (min -1 M -1 )

3'S-Lewis-a 3'S-Lewis-x 3'S-LacNAc 3'S-LNB 3S-GalNAc 3S,6S-GlcNAc

WT BT1622 3S-Gal/GalNAc (S75C) (1.8 ± 0.04) x 10 2 NQ (1.9 ± 0.04) x 10 3 (3.6 ± 0.13) x 10 4 Supplementary Table 4 LC-MS analysis of colonic mucin oligosaccharides (cMO)

Provided as a separate multi-tab Excel file Supplementary Table 5 LC-MS analysis of O-glycans in culture supernatant of Dbt1636 3S-Gal mutant by LC-MS/MS Provided as a separate multi-tab Excel file

Supplementary Table 6

Conservation of S1_20 3S-Gal/GalNAc specificity residues in Bacteroides type strains and Akkermansia muciniphila * Protein without critical calcium binding residues (predicted to be inactive); Proteins

Supplementary Table 10

Values in parenthesis are for the highest resolution shell. Rfree was calculated using a set (5%) of randomly selected reflections that were excluded from refinement.

BT1622 3S-Gal/GalNAc BT1622 3S-Gal/GalNAc with GalNAc BT1636 3S-Gal with LacNAc BT4683

Supplementary Table 11

Melting temperatures of galactose targeting sulfatases with and without ligands.

Melting temperature (°C)

Supplementary Table 12

S1_20 homologues of BT1636

Supplementary Figure 2

Phylogenetic analysis of S1_4 subfamily showing the conservation of BT4683 3S-Gal specificity residues Provided as separate PDF file due to size Supplementary Figure 3 Phylogenetic analysis of S1_20 subfamily showing the conservation of BT1636 3S-Gal specificity residues Provided as separate PDF file due to size Supplementary Figure 4 pH profile of different sulfatases

Graphics showing the pH optimum for BT1636 3S-Gal and BT3057 4S-Gal/GalNAc is 6; BT4683 3S-Gal had a pH optimum of 6.5; BT19183S-GlcNAc had a pH optimum of 7.0; BT1622 S-Gal and BT3177 6S-GlcNAc had a pH optimum of 8, whilst BT3796 4S-Gal/GalNAc had a pH optimum of 8.5. All reactions were performed using 100 mM of the appropriate buffer supplemented with 150 mM NaCl and 5 mM CaCl2. A substrate concentration of 1 µM was used and an enzyme concentration of between 0.1 -2 µM was deployed depending on the enzyme.