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Pseudomonas aeruginosa , a common cause of morbidity in cystic fibrosis, chronically infects the patient's lungs by forming an alginate-containing biofilm. Alginate lyases are polysaccharide lyases that lyse alginate and are, therefore, potential biofilm-disruptive agents. However, cystic fibrosis sputum contains high levels of metals such as iron and zinc. The efficacy of alginate lyases under these conditions of elevated metal concentrations has not been categorically determined. Here, we have assessed the enzyme activity of two exolytic and five endolytic alginate lyases in the presence of metal ions (Fe 2+ , Zn 2+ , Mn 2+ , Mg 2+ , Ca 2+ , Ni 2+ , Cu 2+ ) elevated in the cystic fibrosis lung milieu. Several of these alginate lyases exhibited increased activity in the presence of Ca 2+ , and the polysaccharide lyase family 7 members studied here exhibited decreased activity in the presence of Zn 2+ . The enzyme activity of the PL7 alginate lyases from Cellulophaga algicola (CaAly/CaFLDAly) and Vibrio sp. (VspAlyVI) was not affected in the presence of a mix of all the above-mentioned metal ions at the elevated concentrations found in the cystic fibrosis lung milieu. Specific alginate lyases might, therefore, retain the ability to degrade the alginate-containing Pseudomonas biofilm in the presence of metal ions elevated in the cystic fibrosis lung milieu., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO)., (© 2024. The Author(s).)

Sialidases catalyze the removal of terminal sialic acids from sialylated biomolecules, and their substrate preference is frequently indicated in terms of the glycosidic linkages cleaved (α2-3, α2-6, and α2-8) without mention of the remaining sub-terminal reducing-end saccharide moieties. Many human gut commensal and pathogenic bacteria secrete sialidases to forage for sialic acids, which are then utilized as an energy source or assimilated into membrane/capsular structural components. Infant gut commensals similarly utilize sialylated human milk oligosaccharides containing different glycosidic linkages. Here, we have studied the preference of the bacterial sialidases, BbSia2 from Bifidobacterium bifidum , CpNanI from Clostridium perfringens , and HpNanH from Glaesserella parasuis, for the glycosidic linkages, Siaα2-3Gal, Siaα2-6Gal, and Siaα2-6GlcNAc, by employing 2-Aminobenzamide-labeled human milk oligosaccharides, 3'-Sialyllactose (3'-SL), 6'-Sialyllactose (6'-SL), and Sialyllacto-N-tetraose-b (LSTb), respectively, as proxies for these glycosidic linkages. BbSia2, Cp NanI, and HpNanH hydrolyzed these three oligosaccharides with the glycosidic linkage preferences, 3 ' -SL (Siaα2-3Gal) ≥ LSTb (Siaα2-6GlcNAc) ≥ 6 ' -SL (Siaα2-6Gal), 3 ' -SL (Siaα2-3Gal) ≥ 6 ' -SL (Siaα2-6Gal) > LSTb (Siaα2-6GlcNAc), and 3'-SL (Siaα2-3Gal) ≥ 6'-SL (Siaα2-6Gal) > LSTb (Siaα2-6GlcNAc), respectively. Our finding suggests that sub-terminal reducing-end saccharide moieties can profoundly influence the substrate preference of sialidases, and advocates for the characterization and indication of the substrate preference of sialidases in terms of both the glycosidic linkage and the sub-terminal reducing-end saccharide moiety., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Uncultured microbes represent a huge untapped biological resource of novel genes and gene products. Although recent genomic and metagenomic sequencing efforts have led to the identification of numerous genes that are homologous to existing annotated genes, there remains, yet, an enormous pool of unannotated genes that do not find significant sequence homology to existing annotated genes. Functional metagenomics offers a way to identify and annotate novel gene products. Here, we use functional metagenomics to mine novel carbohydrate binding domains that might aid human gut commensals in adherence, gut colonization, and metabolism of complex carbohydrates. We report the construction and functional screening of a metagenomic phage display library from healthy human fecal samples against dietary, microbial and host polysaccharides/glycoconjugates. We identify several protein sequences that do not find a hit to any known protein domain but are predicted to contain carbohydrate binding module-like folds. We heterologously express, purify and biochemically characterize some of these protein domains and demonstrate their carbohydrate-binding function. Our study reveals several previously unannotated carbohydrate-binding domains, including a levan binding domain and four complex N-glycan binding domains that might be useful for the labeling, visualization, and isolation of these glycans., (© 2023. The Author(s).)

Chronic mucoid Pseudomonas aeruginosa infections are a major scourge in cystic fibrosis patients. Mucoid P. aeruginosa displays structured alginate-rich biofilms that are resistant to antibiotics. Here, we have assessed the efficacy of a panel of alginate lyases in combating mucoid P. aeruginosa biofilms in cystic fibrosis. Albeit we could not demonstrate alginate degradation by alginate lyases in sputum, we demonstrate that the endotypic alginate lyases, Ca Aly (from Cellulophaga algicola ) and Vsp AlyVI (from Vibrio sp. QY101) and the exotypic alginate lyases, Fsp AlyFRB (from Falsirhodobacterium sp. alg1), and SA1-IV (from Sphingomonas sp. A1), indeed inhibit biofilm formation by a mucoid P. aeruginosa strain isolated from the sputum of a cystic fibrosis patient with comparative effect to that of the glycoside hydrolase PslG, a promising candidate for biofilm treatment. We believe that these enzymes should be explored for in vivo efficacy in future studies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Some bacterial flagellins are O-glycosylated on surface-exposed serine/threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called motility-associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, serine/threonine-O-linked N-acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and serine/threonine-O-linked 3-deoxy-D-manno-octulosonic acid or keto-deoxyoctulosonate (Ser/Thr-O-KDO), both catalyzed by Geobacillus kaustophilus Maf and Clostridium botulinum Maf. We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were coexpressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. Our finding that both G. kaustophilus Maf (putative flagellin sialyltransferase) and C. botulinum Maf (putative flagellin legionaminic acid transferase) catalyzed Neu5Ac and KDO transfer on to flagellin indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Alginate lyases are potential agents for disrupting alginate-rich Pseudomonas biofilms in the infected lungs of cystic fibrosis patients but there is as yet no clinically approved alginate lyase that can be used as a therapeutic. We report here the endolytic alginate lyase activity of a recombinant Cellulophaga algicola alginate lyase domain (CaAly) encoded by a gene that also codes for an N-terminal carbohydrate-binding module, CBM6, and a central F-type lectin domain (CaFLD). CaAly degraded both polyM and polyG alginates with optimal temperature and pH of 37°C and pH 7, respectively, with greater preference for polyG. Recombinant CaFLD bound to fucosylated glycans with a preference for H-type 2 glycan motif, and did not have any apparent effect on the enzyme activity of the co-associated alginate lyase domain in the recombinant protein construct, CaFLD_Aly. We assessed the potential of CaAly and other alginate lyases previously reported in published literature to inhibit biofilm formation by a clinical strain, Pseudomonas aeruginosa MCC 2081. Of all the alginate lyases tested, CaAly displayed most inhibition of in vitro biofilm formation on plastic surfaces. We also assessed its inhibitory ability against P. aeruginosa 2081 biofilms formed over a monolayer of A549 lung epithelial cells. Our study indicated that CaAly is efficacious in inhibition of biofilm formation even on A549 lung epithelial cell line monolayers., (© 2020 International Union of Biochemistry and Molecular Biology.)

F-type lectins are typically L-fucose binding proteins with characteristic L-fucose-binding and calcium-binding sequence motifs, and an F-type lectin fold. An exception is Ranaspumin-4, an F-type lectin of the Tungra frog, Engystomops pustulosus. Ranaspumin-4 is D-galactose specific and does not bind to L-fucose although it has the conserved L-fucose binding sequence motif and shares overall sequence similarity with other F-type lectins. Here, we report the detailed glycan-binding profile of wild-type Ranaspumin-4 using hemagglutination inhibition assays, flow cytometry assays and enzyme-linked lectin assays, and identify residues important for D-galactose recognition using rational site-directed mutagenesis. We demonstrate that Ranaspumin-4 binds to terminal D-galactose in α or β linkage with preference for α1-3, α1-4, β1-3, and β1-4 linkages. Further, we find that a methionine residue (M31) in Ranaspumin-4 that occurs in place of a conserved Gln residue (in other F-type lectins), supports D-galactose recognition. Resides Q42 and F156 also likely aid in D-galactose recognition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Various factors including diet, age, geography, culture and socio-economic status have a role in determining the composition of the human gut microbiota. The human gut microbial composition is known to be altered in disease conditions. Considering the important role of the gut microbiome in maintaining homeostasis and overall health, it is important to understand the microbial diversity and the functional metagenome of the healthy gut. Here, we characterized the microbiota of 31 fecal samples from healthy individuals of Indian ethnic tribes from Ladakh, Jaisalmer and Khargone by shotgun metagenomic sequencing. Sequence analysis revealed that Bifidobacterium and Prevotella were the key microbes contributing to the differences among Jaisalmer, Khargone and Ladakh samples at the genus level. Our correlation network study identified carbohydrate-active enzymes and carbohydrate binding proteins that are associated with specific genera in the different Indian geographical regions studied. Network analysis of carbohydrate-active enzymes and genus abundance revealed that the presence of different carbohydrate-active enzymes is driven by differential abundance of genera. The correlation networks were different in the different geographical regions, and these interactions suggest the role of less abundant genera in shaping the gut environment. We compared our data with samples from different countries and found significant differences in taxonomic composition and abundance of carbohydrate-active enzymes in the gut microbiota as compared to the other countries., Competing Interests: The authors have declared that no competing interests exist.

The typical F-type lectin domain (FLD) has an L-fucose-binding motif [HX(26)RXDX(4)R/K] with conserved basic residues that mediate hydrogen bonding with alpha-L-fucose. About one-third of the nonredundant FLD sequences in the publicly available databases are "atypical"; they have motifs with substitutions of these critical residues and/or variations in motif length. We addressed the question if atypical FLDs with substitutions of the critical residues retain lectin activity by performing site-directed mutagenesis and assessing the glycan-binding functions of typical and atypical FLDs. Site directed mutagenesis of an L-fucose-binding FLD from Streptosporangium roseum indicated that the critical His residue could be replaced by Ser and the second Arg by Lys without complete loss of lectin activity. Mutagenesis of His to other naturally substituting residues and mutagenesis of the first Arg to the naturally substituting residues, Lys, Ile, Ser, or Cys, resulted in loss of lectin activity. Glycan binding analysis and site-directed mutagenesis of atypical FLDs from Actinomyces turicensis, and Saccharomonospora cyanea confirmed that Ser and Thr can assume the L-fucose-binding role of the critical His, and further suggested that the residue in this position is dispensable in certain FLDs. We identified, by sequence and structural analysis of atypical FLDs, a Glu residue in the complementarity determining region, CDR5 that compensates for a lack of the critical His or other appropriate polar residue in this position. We propose that FLDs lacking a typical FLD sequence motif might nevertheless retain lectin activity through the recruitment of other strategically positioned polar residues in the CDR loops. © 2018 IUBMB Life, 71(3):385-397, 2019., (© 2018 International Union of Biochemistry and Molecular Biology.)

Individual lectin-carbohydrate interactions are usually of low affinity. However, high avidity is frequently attained by the multivalent presentation of glycans on biological surfaces coupled with the occurrence of high order lectin oligomers or tandem repeats of lectin domains in the polypeptide. F-type lectins are l-fucose binding lectins with a typical sequence motif, HX(26)RXDX(4)R/K, whose residues participate in l-fucose binding. We previously reported the presence of a few eukaryotic F-type lectin domains with partial sequence duplication that results in the presence of two l-fucose-binding sequence motifs. We hypothesized that such partial sequence duplication would result in greater avidity of lectin-ligand interactions. Inspired by this example from Nature, we attempted to engineer a bacterial F-type lectin domain from Streptosporangium roseum to attain avid binding by mimicking partial duplication. The engineered lectin demonstrated 12-fold greater binding strength than the wild-type lectin to multivalent fucosylated glycoconjugates. However, the affinity to the monosaccharide l-fucose in solution was similar and partial sequence duplication did not result in an additional functional l-fucose binding site. We also cloned, expressed and purified a Branchiostoma floridae F-type lectin domain with naturally occurring partial sequence duplication and confirmed that the duplicated region with the F-type lectin sequence motif did not participate in l-fucose binding. We found that the greater binding strength of the engineered lectin from S. roseum was instead due to increased oligomerization. We believe that this Nature-inspired strategy might be useful for engineering lectins to improve binding strength in various applications.

F-type lectins are phylogenetically widespread but selectively distributed fucose-binding lectins with L-fucose- and calcium-binding sequence motifs and an F-type lectin fold. Bacterial F-type lectin domains frequently occur in tandem with various protein domains in diverse architectures, indicating a possible role in directing enzyme activities or other biological functions to distinct fucosylated niches. Here, we report the biochemical characterization of a Streptosporangium roseum protein containing an F-type lectin domain in tandem with an NPCBM-associated domain and a family GH 29A alpha-l-fucosidase domain. We show that the F-type lectin domain of this protein recognizes fucosylated glycans in both α and β linkages but has high affinity for a Fuc-α-1,2-Gal motif and that the alpha-l-fucosidase domain displays hydrolytic activity on glycan substrates with α1-2 and α1-4 linked fucose. We also show that the F-type lectin domain does not have any effect on the activity of the cis-positioned alpha-l-fucosidase domain with the synthetic substrate, 4-Methylumbelliferyl-alpha-l-fucopyranoside or on inhibition of this activity by l-fucose or deoxyfuconojirimycin hydrochloride. However, the F-type lectin domain together with the NPCBM-associated domain enhances the activity of the cis-positioned alpha-l-fucosidase domain for soluble fucosylated oligosaccharide substrates. While there are many reports of glycoside hydrolase activity towards insoluble and soluble polysaccharides being enhanced by cis-positioned carbohydrate binding modules on the polypeptide, this is the first report, to our knowledge, of enhancement of activity towards aqueous, freely diffusible, small oligosaccharides. We propose a model involving structural stabilization and a bind-and-jump action mediated by the F-type lectin domain to rationalize our findings.

This communication probes ligand binding by human Intelectin-1 with several saccharides. Human Intelectin-1 was previously reported to bind to microbial glycans via ribofuranoside or galactofuranoside residues, whereas subsequently, a crystal structure of ligand bound hITLN1 indicated that hITLN1 does not bind to ribofuranoside but distinguishes between microbial and human glycans through a glycan motif - a terminal, acyclic 1,2-diol, which is present on galactofuranose and other microbial saccharides. Here, we demonstrate that besides glycerol and glycerol derivatives (which have an acyclic 1,2-diol), and 2-deoxy-d-galactose, d-ribose and 2-deoxy-d-ribose, which have been previously reported as human Intelectin-1 ligands, 2-C-hydroxymethyl-d-ribose, d-talose, d-idose, d-altrose and sorbitol also elute human Intelectin-1 from Sepharose CL-6B. Interestingly, Sepharose, 2-deoxy-d-galactose (in its pyranose form), 2-C-hydroxymethyl-d-ribose, d-ribose and 2-deoxy d-ribose lack a terminal, acyclic 1,2-diol. We discuss the implications of these observations and rationalize the discrepancies in the apparent affinity of saccharide ligands for hITLN1 with different assay formats. We also report the distinct saccharide binding profiles of the hITLN1 homologues, HaloITLN and XL35ITLN, and demonstrate that hITLN1 binding to a saccharide ligand may modulate binding to its protein ligand, lactoferrin and vice versa., (Copyright © 2017 Elsevier B.V. All rights reserved.)

F-type lectins are phylogenetically widespread albeit selectively distributed lectins with an L-fucose-binding sequence motif and an F-type lectin fold. Several F-type lectins from fishes have been extensively studied, and structural information is available for F-type lectin domains from fish and bacterial proteins. F-type lectins have been demonstrated to be involved in self-/nonself-recognition and therefore have an important role in pathogen defense in many metazoan animals. F-type lectin domains also have been implicated in functions related to fertilization, protoplast regeneration, and bacterial virulence. We have recently analyzed and reported the taxonomic spread, phylogenetic distribution, architectural contexts, and sequence characteristics of prokaryotic and eukaryotic F-type lectin domains. Interestingly, while eukaryotic F-type lectin domains were frequently present as stand-alone domains, bacterial F-type lectin domains were mostly found co-occurring with enzymatic or nonenzymatic domains in diverse domain architectures, suggesting that the F-type lectin domain might be involved in targeting enzyme activities or directing other biological functions to distinct glycosylated niches in bacteria. We and others have probed the fine oligosaccharide-binding specificity of several F-type lectin domains. The currently available wealth of sequence, structural, and biochemical information about F-type lectin domains provides opportunities for the generation of designer lectins with improved binding strength and altered binding specificities. We discuss the prevalence, provenance, properties, peculiarities, and potential of F-type lectin domains for future applications in this review.

F-type lectins are fucose binding lectins with characteristic fucose binding and calcium binding motifs. Although they occur with a selective distribution in viruses, prokaryotes and eukaryotes, most biochemical studies have focused on vertebrate F-type lectins. Recently, using sensitive bioinformatics search techniques on the non-redundant database, we had identified many microbial F-type lectin domains with diverse domain organizations. We report here the biochemical characterization of F-type lectin domains from Cyanobium sp. PCC 7001, Myxococcus hansupus and Leucothrix mucor. We demonstrate that while all these three microbial F-type lectin domains bind to the blood group H antigen epitope on fucosylated glycans, there are fine differences in their glycan binding specificity. Cyanobium sp. PCC 7001 F-type lectin domain binds exclusively to extended H type-2 motif, Myxococcus hansupus F-type lectin domain binds to B, H type-1 and Lewis b motifs, and Leucothrix mucor F-type lectin domain binds to a wide range of fucosylated glycans, including A, B, H and Lewis antigens. We believe that these microbial lectins will be useful additions to the glycobiologist's toolbox for labeling, isolating and visualizing glycans., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The yeast Saccharomyces boulardii is used worldwide as a probiotic to alleviate the effects of several gastrointestinal diseases and control antibiotics-associated diarrhea. While many studies report the probiotic effects of S. boulardii, no genome information for this yeast is currently available in the public domain., Results: We report the 11.4 Mbp draft genome of this probiotic yeast. The draft genome was obtained by assembling Roche 454 FLX + shotgun data into 194 contigs with an N50 of 251 Kbp. We compare our draft genome with all other Saccharomyces cerevisiae genomes., Conclusions: Our analysis confirms the close similarity of S. boulardii to S. cerevisiae strains and provides a framework to understand the probiotic effects of this yeast, which exhibits unique physiological and metabolic properties.