Sialidases from gut bacteria: a mini-review

Sialidases are a large group of enzymes, the majority of which catalyses the cleavage of terminal sialic acids from complex carbohydrates on glycoproteins or glycolipids. In the gastrointestinal (GI) tract, sialic acid residues are mostly found in terminal location of mucins via α2-3/6 glycosidic linkages. Many enteric commensal and pathogenic bacteria can utilize sialic acids as a nutrient source, but not all express the sialidases that are required to release free sialic acid. Sialidases encoded by gut bacteria vary in terms of their substrate specificity and their enzymatic reaction. Most are hydrolytic sialidases, which release free sialic acid from sialylated substrates. However, there are also examples with transglycosylation activities. Recently, a third class of sialidases, intramolecular trans-sialidase (IT-sialidase), has been discovered in gut microbiota, releasing (2,7-anhydro-Neu5Ac) 2,7-anydro-N-acetylneuraminic acid instead of sialic acid. Reaction specificity varies, with hydrolytic sialidases demonstrating broad activity against α2,3-, α2,6- and α2,8-linked substrates, whereas IT-sialidases tend to be specific for α2,3-linked substrates. In this mini-review, we summarize the current knowledge on the structural and biochemical properties of sialidases involved in the interaction between gut bacteria and epithelial surfaces.

The GI tract is heavily colonized with bacteria. Most species belong to the phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia. The microbiota composition varies longitudinally along the GI tract but also transversally from the mucosa to the lumen [9,10]. The terminal mucin O-glycans have been proposed to serve as metabolic substrates, providing a nutritional advantage to bacteria which have adapted to the GI mucosal environment [11,12]. The release of sialic acid from nonreducing ends by sialidases is an initial step in the sequential degradation of mucins, since the terminal location of sialic acid residues in the mucin oligosaccharide chains may prevent the action of other glycoside hydrolases (GHs). In bacteria, the genes involved in sialic acid metabolism are usually found clustered together forming what is denominated as a Nan cluster. The canonical nanA/K/E cluster was first described in Escherichia coli [13] and an alternative pathway defined by the nanLET cluster was later discovered in Bacteroides fragilis [14]. The majority of the bacteria that harbour a Nan cluster colonize mucus regions of the human body, such as the gut where sialic acid is highly abundant and can serve as a source of energy, carbon and nitrogen [15]. As described below, a number of gut bacteria employ sialidases in the release of host sialic acids, including Structures shown are representative and not exhaustive. These glycans are appended to the core mucin structures. In mice the fucose (Fuc) residues are more commonly linked to Gal rather GlcNAc, *structures common in human and rare in mice. multiple species of Clostridia [16], Bacteroides [17], certain subspecies/serovars/strains of Bifidobacterium longum [18], Vibrio cholerae [19], Ruminococcus gnavus and Akkermansia muciniphila [20]. However, some bacteria appear to have incomplete packages of enzymes for utilizing host sialic acids. For example, Bacteroides thetaiotaomicron VPI-5482 encodes a sialidase and can release free sialic acid, but lacks the Nan operon required to consume the liberated monosaccharide and does not appear capable of consuming free sialic acid [21]. On the other hand Clostridium difficile strain 630 encodes the Nan operon but lacks the sialidase [22] and thus relies on other sialidase-producing organisms to acquire this potential nutrient source from the mucosal environment [23,24]. In contrast, some bacteria appear to possess the complete pathway of sialic acid catabolism including a predicted sialidase gene e.g. B. fragilis strains [14,25]. A recent study reported that mice monoassociated with B. thetaiotaomicron exhibited a significantly higher concentration of free Neu5Ac compared with germ-free mice, consistent with the ability of B. thetaiotaomicron to liberate but not consume the monosaccharide, whereas colonization of mice with B. fragilis, which is able to catabolize Neu5Ac, did not result in increased free sialic acid [24]. In contrast with gut commensals, which appear to use sialidases primarily for nutrient acquisition, some pathogens of the GI tract such as V. cholerae or Clostridium perfringens strains also use sialidases to decrypt adhesin or toxin-binding sites [26]. All toxigenic strains of V. cholerae have a sialidase encoded within a pathogenicity island in their genomes [27]. However it is also worth noting that, within particular pathogenic or commensal species, the presence or absence of sialidase-encoding genes in bacterial genomes is often strain-specific. For example R. gnavus ATCC 29149 but not E1 expresses a sialidase [20,28], several strains of E. coli, e.g. enteropathogenic E. coli O127 strain (EPEC) [29] or probiotic strain Nissle 1917 [30] possess a sialidase-encoding gene whereas commensal E. coli strains such as E. coli strain EHV2 lack a sialidase [31]. However E. coli sialidases remain to be biochemically characterized. Similarly, not all Salmonella enterica strains encode a putative sialidase and only one sialidase has been functionally-characterized from S. enterica serovar typhimurium although it appears to have been acquired by horizontal transfer [32,33].

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Sialic acid catabolism in the gut is important as increased free sialic acid levels in the intestinal mucosal compartment, e.g. post-antibiotic treatment, will favour outgrowth of some bacterial pathogenic strains of S. Typhimurium and C. difficile [24] or the outgrowth of E. coli during inflammation [31]. Such cross-feeding activity has also been reported between commensal bacteria, e.g. Bifidobacterium breve UCC2003 (containing a functional Nan cluster for sialic utilization) can utilize sialic acid released by the sialidase activity of Bifidobacterium bifidum PRL201048 [34]. The gut symbiont, R. gnavus ATCC 29149, is different from the above as it possesses the complete Nan cluster and an intramolecular trans-sialidase (IT-sialidase), thus producing (2,7-anhydro-Neu5Ac) 2,7-anydro-N-acetylneuraminic acid instead of free Neu5Ac, suggesting a novel mechanism of adaptation to the mucosal environment [20]. The biological role of bacterial sialidases produced by human gut commensal and pathogenic bacteria has been reviewed previously [15,26,35]. Here we focus on the structural and biochemical properties Mechanism of action hydrolytic/trans/IT-sialidases sialidases act via a two-step double-displacement mechanism so that the α-configuration of the glycosidic bond is retained The glycosylation step is the same for all three classes of sialidase, but for the deglycosylation step the incoming molecule can be water, another sugar or the internal oxygen atom, as indicated. Figure adapted from www.cazypedia.org.
of characterized sialidases involved in the interaction between gut bacteria and epithelial surfaces.

General features
Sialidases (also commonly referred to as neuraminidases) are a large group of enzymes, the majority of which are exo-sialidases catalysing the cleavage of terminal sialic acids from complex carbohydrates on glycoproteins or glycolipids. Based on amino acid sequence similarities, bacterial exosialidases are classified in the GH family 33 (GH33) of the CAZy classification (www.cazy.org) [36]. Hydrolysis occurs via an acid/base-catalysed double-displacement mechanism involving a covalent sialyl-enzyme intermediate, resulting in overall retention of configuration at the anomeric centre [37,38] ( Figure 2). Unusually the catalytic nucleophile is a tyrosine residue activated by a proximal glutamic acid, due to the charge on sialic acid itself as shown biochemically [39] and confirmed structurally [40] (Figure 2). Sialidases from the retaining sialidase families GH34 and GH83 are both restricted to viruses and examples of GH58 inverting endosialidases are found in some E. coli strains (www.cazy.org).
The GH33 catalytic domains adopt a six-bladed βpropeller fold (Figures 3A and 3B). GH33 catalytic domains are often associated with additional domains [46] including membrane-binding domains [47] and carbohydrate-binding modules (CBMs) such as sialic acid-specific CBM40 [19,20] and broadly specific CBM32 [48] as classified in CAZy (www.cazy.org). CBMs are believed to mediate adherence of the enzyme to cognate carbohydrate substrates and enhance the hydrolase activity of the catalytic domains by increasing enzyme substrate proximity [49]. Both exoand trans-sialidases share a set of active site residues and cleave the terminal α-linked sialic acid residue by the same catalytic mechanism. This conserved active site includes a glutamic acid-tyrosine charge relay with the tyrosine acting as the catalytic nucleophile [40] and an aspartate residue as the general acid/base (Figure 2). The incoming sialic acid residue is orientated in the active site via a trio of arginines, which co-ordinates the sialic acid carboxylate moiety and a hydrophobic pocket which accommodates the ligand Nacetyl group ( Figure 3C). Aspartic acid-boxes are motifs commonly found at the termini of sialidase β-propeller blades, they may stabilize the protein fold by providing interblade contacts [50,51].

Hydrolytic exo-sialidases
Among GI commensals, Bacteroidetes species are found at high abundance and many of them express sialidases in culture [17]. B. thetaiotaomicron spp. encode the sialidases required to cleave and release terminal sialic acid from the mucosal glycoconjugates, but do not encode the Nan cluster required to consume the liberated monosaccharide [21]. The purified sialidase from B. thetaiotaomicron VPI-5482 has been shown to hydrolyse sialylglycoconjugates including fetuin and transferrin [52] (Table 1). Presumably, the release of sialic acids allows B. thetaiotaomicron to access highly coveted underlying carbohydrates in the mucus. Recently, a sialidase from Bacteroides vulgatus BVU 4143 has been shown to be active against 4MU-Neu5Ac and PNP-Neu5Ac and inhibited by N-acetyl-2,3-didehydro-2deoxyneuraminic acid (Neu5Ac2en) inhibitor [31] (Table 1). B. fragilis strains are among those bacteria that have been shown to possess the complete pathway of sialic acid catabolism including the sialidase. Several sialidases from Can produce Neu5Ac2en + [19] *P/E refers to whether the characterization is carried out with purified (P) enzymes (including recombinant enzyme) or with bacterial extract (E). † This column indicates whether the enzyme is active ( + ) or not (-) against the substrates tested, + + is used to denote more activity than + , where relative activity is indicated. These strains are 'flesh-eating' strains isolated from gangrene rather than gut bacteria but are included because more biochemical data are available. ‡ Details of enzymes are not currently in CAZy 'characterized' page.

Trans-sialidases and IT-sialidases
Most trans-sialidases have been characterized from trypanosome species [77]. Trans-sialidases have not been reported in the gut microbiota. However a few examples of exosialidases from gut bacteria have been reported to perform trans-glycosylation reactions under certain experimental conditions. These include the aforementioned SiaBb2 from B. bifidum and NanI from C. perfringens [69] (Table 1).
IT-sialidases are unique in that they catalyse an intramolecular reaction in which the O7-hydroxy group of the bound sialic acid glycerol group attacks the positively charged C2 atom of the oxocarbenium intermediate [44,78].
The altered reaction pathway leads to release of 2,7-anhydro-Neu5Ac instead of Neu5Ac ( Figure 2). The first example of this enzyme class was described in NanL [79,80], which is purported to be from the leech Macrobdella decora, but may be from a bacterial source in the leech GI tract, as previously suggested [81]. Three IT-sialidases have been biochemically and structurally described: NanL [44,78], NanB from Streptococcus pneumonia [45] and RgNanH from R. gnavus [20] ( Figure 3B). The active site of ITsialidases is characterized by a conserved threonine residue which sterically hinders the substrate glycerol group, forcing it into an axial position whence it can attack at the anomeric C2 carbon and form the intramolecular linkage [44,78] (Figure 3C). An additional characteristic feature of these enzymes is a hydrophobic rim close to the arginine triad, formed by tryptophan-tyrosine stack [20,44,45]. This feature provides strict specificity for α2-3-linked substrates and may also provide an important contribution to the reaction mechanism by providing a desolvated, hydrophobic environment. This allows the intramolecular reaction to proceed, as the O7 hydroxy of the glycerol group must outrun any incoming water molecules that would otherwise attack the C2 carbon and produce Neu5Ac ( Figure 3C).
RgNanH from R. gnavus ATCC 29149 is the first example of an IT-sialidase functionally characterized in gut bacteria [20,28]. The enzyme produces 2,7-anhydro-Neu5Ac with strict specificity towards α2-3 glycosidic substrate linkages. RgNanH is a three-domain modular protein with an N-terminal lectin-like domain (L-domain) classified as a CBM40, a GH33 catalytic domain (N-domain) and a domain inserted into the catalytic domain (I-domain). The crystal structure of the RgNanH catalytic domain has been solved and demonstrates the six-bladed β-propeller fold characteristic of sialidases [20]. A domain of unknown function protrudes from between two blades of the βpropeller (I-domain). Crystal structures in complex with 2,7-anhydro-Neu5Ac and known inhibitors of hydrolytic sialidases, allowed interrogation of the active site. Of particular importance is the conservation of the active site threonine (Thr 557 ), which is proposed to sterically force the substrate glycerol group into a position from where it can attack the C2 atom [20] (Figure 3C). This residue also impacts on the response to sialidase inhibitors, as shown by poor inhibition by Neu5Ac2en and micromolar inhibition by siastatin B [20].
Bioinformatics analyses revealed that the presence of ITsialidases is shared by other members of the gut microbiota, in particular Blautia hansenii, Ruminococcus torques, all 10 strains of Clostridium perfringens with available genome data, C. sp. 7 2 43 FAA, C. celatum, C. nexile, C. spiroforme, three unclassified Lachnospiraceae, more than 100 strains of Streptococcus agalactiae and three of the genome-sequenced publicly available Lactobacillus salivarius strains. The detection of IT-sialidase homologues in at least 11 % of gut metagenomes of a population of diseased and healthy humans confirmed that this enzyme is widespread across gut bacteria, especially in Firmicutes. This analysis also revealed a greater abundance of IT-sialidase encoding species in patients with inflammatory bowel diseases (IBD) as compared with healthy individuals [20]. The specific niche colonization of these bacteria may reflect their adaptation to particular mucin glycosylation profiles associated with intestinal inflammation and/or infection [82,83].

Conclusions and perspectives
Bacterial sialidases and their sialoglycan targets contribute to host-microbe interactions at the mucosal surface. An imbalance in the proportion of gut commensals able to modulate mucosal sialic acid levels or a change in host mucin sialylation is often associated with enteric infection or intestinal inflammation. Maintaining a balance in the ability of gut commensals to produce and/or consume sialic acid in the mucosal compartment is therefore essential to gut homoeostasis.
Further investigations of bacterial sialidases should clarify the type of sialylated structures that are accessible to the gut bacteria and the specificity of sialidases towards sialic acids with different modifications and in different linkages. These include gaining structural insights into the diversity of sialic acid derivatives that can be produced and/or taken up by commensal and pathogenic bacteria. Thus, for therapeutic purposes, modulation of sialidase expression might be effectively achieved by appropriate use of specific inhibitors or pro/prebiotic approaches targeting specific bacterial strains.

Funding
The work was supported by the Biotechnology and Biological Sciences Research Council [grant number BB/J004529/1].