A "terminal" case of glycan catabolism: Structural and enzymatic characterization of the sialidases of Clostridium perfringens.

Sialic acids are commonly found on the terminal ends of biologically important carbohydrates, including intestinal mucin O-linked glycans. Pathogens such as Clostridium perfringens, the causative agent of necrotic enteritis in poultry and humans, have the ability to degrade host mucins and colonize the mucus layer, which involves removal of the terminal sialic acid by carbohydrate-active enzymes (CAZymes). Here, we present the structural and biochemical characterization of the GH33 catalytic domains of the three sialidases of C. perfringens and probe their substrate specificity. The catalytically active domains, which we refer to as NanHGH33, NanJGH33, and NanIGH33, displayed differential activity on various naturally occurring forms of sialic acid. We report the X-ray crystal structures of these domains in complex with relevant sialic acid variants revealing the molecular basis of how each catalytic domain accommodates different sialic acids. NanHGH33 displays a distinct preference for α-2,3-linked sialic acid, but can process α-2,6-linked sialic acid. NanJGH33 and NanIGH33 both exhibit the ability to process α-2,3- and α-2,6-linked sialic acid without any significant apparent preference. All three enzymes were sensitive to generic and commercially available sialidase inhibitors, which impeded sialidase activity in cultures as well as the growth of C. perfringens on sialylated glycans. The knowledge gained in these studies can be applied to in vivo models for C. perfringens growth and metabolism of mucin O-glycans, with a view toward future mitigation of bacterial colonization and infection of intestinal tissues.

Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide.

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.

The pangenome of the wheat pathogen Pyrenophora tritici-repentis reveals novel transposons associated with necrotrophic effectors ToxA and ToxB.

BACKGROUND: In fungal plant pathogens, genome rearrangements followed by selection pressure for adaptive traits have facilitated the co-evolutionary arms race between hosts and their pathogens. Pyrenophora tritici-repentis (Ptr) has emerged recently as a foliar pathogen of wheat worldwide and its populations consist of isolates that vary in their ability to produce combinations of different necrotrophic effectors. These effectors play vital roles in disease development. Here, we sequenced the genomes of a global collection (40 isolates) of Ptr to gain insights into its gene content and genome rearrangements. RESULTS: A comparative genome analysis revealed an open pangenome, with an abundance of accessory genes (~ 57%) reflecting Ptr's adaptability. A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around effector coding genes, were detailed using long-read assemblies (PacBio RS II) generated in this work in addition to previously assembled genomes. We also discovered the involvement of large mobile elements associated with Ptr's effectors: ToxA, the gene encoding for the necrosis effector, was found as a single copy within a 143-kb 'Starship' transposon (dubbed 'Horizon') with a clearly defined target site and target site duplications. 'Horizon' was located on different chromosomes in different isolates, indicating mobility, and the previously described ToxhAT transposon (responsible for horizontal transfer of ToxA) was nested within this newly identified Starship. Additionally, ToxB, the gene encoding the chlorosis effector, was clustered as three copies on a 294-kb element, which is likely a different putative 'Starship' (dubbed 'Icarus') in a ToxB-producing isolate. ToxB and its putative transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and 'Icarus' were both present in a different non-coding isolate. This suggests that ToxB may have been mobile at some point during the evolution of the Ptr genome which is contradictory to the current assumption of ToxB vertical inheritance. Finally, the genome architecture of Ptr was defined as 'one-compartment' based on calculated gene distances and evolutionary rates. CONCLUSIONS: These findings together reflect on the highly plastic nature of the Ptr genome which has likely helped to drive its worldwide adaptation and has illuminated the involvement of giant transposons in facilitating the evolution of virulence in Ptr.

The glycoconjugate-degrading enzymes of Clostridium perfringens: Tailored catalysts for breaching the intestinal mucus barrier.

The gastrointestinal (GI) tract of humans and animals is lined with mucus that serves as a barrier between the gut microbiota and the epithelial layer of the intestine. As the proteins present in mucus are typically heavily glycosylated, such as the mucins, several enteric commensal and pathogenic bacterial species are well-adapted to this rich carbon source and their genomes are replete with carbohydrate-active enzymes targeted toward dismantling the glycans and proteins present in mucus. One such species is Clostridium perfringens, a Gram-positive opportunistic pathogen indigenous to the gut of humans and animals. The genome of C. perfringens encodes numerous carbohydrate-active enzymes that are predicted or known to target glycosidic linkages within or on the termini of mucus glycans. Through this enzymatic activity, the degradation of the mucosal layer by C. perfringens has been implicated in a number of GI diseases, the most severe of which is necrotic enteritis. In this review, we describe the wide array of extracellular glycoside hydrolases, and their accessory modules, that is possessed by C. perfringens, and examine the unique multimodularity of these proteins in the context of degrading the glycoconjugates in mucus as a potential component of disease.

Allosteric inhibitors of calpains: Reevaluating inhibition by PD150606 and LSEAL.

BACKGROUND: The mercaptoacrylate calpain inhibitor, PD150606, has been shown by X-ray crystallography to bind to a hydrophobic groove in the enzyme's penta-EF-hand domains far away from the catalytic cleft and has been previously described as an uncompetitive inhibitor of calpains. The penta-peptide LSEAL has been reported to be an inhibitor of calpain and was predicted to bind in the same hydrophobic groove. The X-ray crystal structure of calpain-2 bound to its endogenous calpain inhibitor, calpastatin, shows that calpastatin also binds to the hydrophobic grooves in the two penta-EF-hand domains, but its inhibitory domain binds to the protease core domains and blocks the active site cleft directly. METHODS: The mechanisms of inhibition by PD150606 and LSEAL were investigated using steady-state kinetics of cleavage of a fluorogenic substrate by calpain-2 and the protease core of calpain1, as well as by examining the inhibition of casein hydrolysis by calpain and the autoproteolysis of calpain. RESULTS: PD150606 inhibits both full-length calpain-2 and the protease core of calpain-1 with an apparent noncompetitive kinetic model. The penta-peptide LSEAL failed to inhibit either whole calpain or its protease core in vitro. CONCLUSIONS: PD150606 cannot inhibit cleavage by calpain-2 of small substrates via binding to the penta-EF-hand domain. GENERAL SIGNIFICANCE: PD150606 is often described as a calpain-specific inhibitor due to its ability to target the penta-EF-hand domains of calpain, but we show that it must be acting at a site on the protease core domain instead.

Conversion of Phytochemicals by Lactobacilli: (Phospho)-ß-glucosidases Are Specific for Glucosylated Phytochemicals Rather than Disaccharides.

Food-fermenting lactobacilli convert glycosylated phytochemicals to glycosyl hydrolases and thereby alter their biological activity. This study aimed to investigate the microbial transformation of ß-glucosides of phytochemicals in comparison with utilization of cellobiose. Four homofermentative and four heterofermentative lactobacilli were selected to represent the metabolic diversity of Lactobacillaceae. The genomes of Lactobacillus crispatus, Companilactobacillus paralimentarius, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum encoded for 8 to 22 enzymes, predominantly phospho-ß-glucosidases, with predicted activity on ß-glucosides. Levilactobacillus hammesii and Furfurilactobacillus milii encoded for 3 ß-glucosidases, Furfurilactobacillus rossiae for one, and Fructilactobacillus sanfranciscensis for none. The hydrolysis of amygdalin, esculin, salicin, glucosides of quercetin and genistein, and ginsenosides demonstrated that several strains hydrolyzed ß-glucosides of phytochemicals but not cellobiose. Taken together, several of the carbohydrate-active enzymes of food-fermenting lactobacilli are specific for glycosides of phytochemicals.

In vitro and ex vivo metabolism of chemically diverse fructans by bovine rumen Bifidobacterium and Lactobacillus species.

BACKGROUND: Inulin and inulin-derived fructooligosaccharides (FOS) are well-known prebiotics for use in companion animals and livestock. The mechanisms by which FOS contribute to health has not been fully established. Further, the fine chemistry of fructan structures from diverse sources, such as graminan-type fructans found in cereal crops, has not been fully elucidated. New methods to study fructan structure and microbial responses to these complex carbohydrates will be key for evaluating the prebiotic potency of cereal fructans found in cattle feeds. As the rumen microbiome composition is closely associated with their metabolic traits, such as feed utilization and waste production, prebiotics and probiotics represent promising additives to shift the microbial community toward a more productive state. RESULTS: Within this study, inulin, levan, and graminan-type fructans from winter wheat, spring wheat, and barley were used to assess the capacity of rumen-derived Bifidobacterium boum, Bifidobacterium merycicum, and Lactobacillus vitulinus to metabolize diverse fructans. Graminan-type fructans were purified and structurally characterized from the stems and kernels of each plant. All three bacterial species grew on FOS, inulin, and cereal crop fructans in pure cultures. L. vitulinus was the only species that could metabolize levan, albeit its growth was delayed. Fluorescently labelled polysaccharides (FLAPS) were used to demonstrate interactions with Gram-positive bacteria and confirm fructan metabolism at the single-cell level; these results were in agreement with the individual growth profiles of each species. The prebiotic potential of inulin was further investigated within naïve rumen microbial communities, where increased relative abundance of Bifidobacterium and Lactobacillus species occurred in a dose-dependent and temporal-related manner. This was supported by in situ analysis of rumen microbiota from cattle fed inulin. FLAPS probe derived from inulin and fluorescent in situ hybridization using taxon-specific probes confirmed that inulin interacts with Bifidobacteria and Lactobacilli at the single-cell level. CONCLUSION: This research revealed that rumen-derived Bifidobacteria and Lactobacilli vary in their metabolism of structurally diverse fructans, and that inulin has limited prebiotic potential in the rumen. This knowledge establishes new methods for evaluating the prebiotic potential of fructans from diverse plant sources as prebiotic candidates for use in ruminants and other animals.

SACCHARIS v2: Streamlining Prediction of Carbohydrate-Active Enzyme Specificities Within Large Datasets.

Carbohydrates are chemically and structurally diverse, composed of a wide array of monosaccharides, stereochemical linkages, substituent groups, and intermolecular associations with other biological molecules. A large repertoire of carbohydrate-active enzymes (CAZymes) and enzymatic activities are required to form, dismantle, and metabolize these complex molecules. The software SACCHARIS (Sequence Analysis and Clustering of CarboHydrate Active enzymes for Rapid Informed prediction of Specificity) provides a rapid, easy-to-use pipeline for the prediction of potential CAZyme function in new datasets. We have updated SACCHARIS to (i) simplify its installation by re-writing in Python and packaging for Conda; (ii) enhance its usability through a new (optional) interactive GUI; and (iii) enable semi-automated annotation of phylogenetic tree output via a new R package or the commonly-used webserver iTOL. Significantly, SACCHARIS v2 has been developed with high-throughput omics in mind, with pipeline automation geared toward complex (meta)genome and (meta)transcriptome datasets to reveal the total CAZyme content ("CAZome") of an organism or community. Here, we outline the development and use of SACCHARIS v2 to discover and annotate CAZymes and provide insight into complex carbohydrate metabolisms in individual organisms and communities.

Structural, Biochemical, and Phylogenetic Analysis of Bacterial and Fungal Carbohydrate Esterase Family 15 Glucuronoyl Esterases in the Rumen.

Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/ß hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome.

Carbohydrate flow through agricultural ecosystems: Implications for synthesis and microbial conversion of carbohydrates.

Carbohydrates are chemically and structurally diverse biomolecules, serving numerous and varied roles in agricultural ecosystems. Crops and horticulture products are inherent sources of carbohydrates that are consumed by humans and non-human animals alike; however carbohydrates are also present in other agricultural materials, such as soil and compost, human and animal tissues, milk and dairy products, and honey. The biosynthesis, modification, and flow of carbohydrates within and between agricultural ecosystems is intimately related with microbial communities that colonize and thrive within these environments. Recent advances in -omics techniques have ushered in a new era for microbial ecology by illuminating the functional potential for carbohydrate metabolism encoded within microbial genomes, while agricultural glycomics is providing fresh perspective on carbohydrate-microbe interactions and how they influence the flow of functionalized carbon. Indeed, carbohydrates and carbohydrate-active enzymes are interventions with unrealized potential for improving carbon sequestration, soil fertility and stability, developing alternatives to antimicrobials, and circular production systems. In this manner, glycomics represents a new frontier for carbohydrate-based biotechnological solutions for agricultural systems facing escalating challenges, such as the changing climate.

Development of α-helical calpain probes by mimicking a natural protein-protein interaction.

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein-protein interactions.

Structure of the AlgKX modification and secretion complex required for alginate production and biofilm attachment in Pseudomonas aeruginosa.

Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.

Mechanistic insights into the digestion of complex dietary fibre by the rumen microbiota using combinatorial high-resolution glycomics and transcriptomic analyses.

There is a knowledge gap regarding the factors that impede the ruminal digestion of plant cell walls or if rumen microbiota possess the functional activities to overcome these constraints. Innovative experimental methods were adopted to provide a high-resolution understanding of plant cell wall chemistries, identify higher-order structures that resist microbial digestion, and determine how they interact with the functional activities of the rumen microbiota. We characterized the total tract indigestible residue (TTIR) from cattle fed a low-quality straw diet using two comparative glycomic approaches: ELISA-based glycome profiling and total cell wall glycosidic linkage analysis. We successfully detected numerous and diverse cell wall glycan epitopes in barley straw (BS) and TTIR and determined their relative abundance pre- and post-total tract digestion. Of these, xyloglucans and heteroxylans were of higher abundance in TTIR. To determine if the rumen microbiota can further saccharify the residual plant polysaccharides within TTIR, rumen microbiota from cattle fed a diet containing BS were incubated with BS and TTIR ex vivo in batch cultures. Transcripts coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences encoded CAZymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan. This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can be extended to other feed-host systems and inform next-generation solutions to improve the performance of ruminants fed low-quality forages.

Combined whole cell wall analysis and streamlined in silico carbohydrate-active enzyme discovery to improve biocatalytic conversion of agricultural crop residues.

The production of biofuels as an efficient source of renewable energy has received considerable attention due to increasing energy demands and regulatory incentives to reduce greenhouse gas emissions. Second-generation biofuel feedstocks, including agricultural crop residues generated on-farm during annual harvests, are abundant, inexpensive, and sustainable. Unlike first-generation feedstocks, which are enriched in easily fermentable carbohydrates, crop residue cell walls are highly resistant to saccharification, fermentation, and valorization. Crop residues contain recalcitrant polysaccharides, including cellulose, hemicelluloses, pectins, and lignin and lignin-carbohydrate complexes. In addition, their cell walls can vary in linkage structure and monosaccharide composition between plant sources. Characterization of total cell wall structure, including high-resolution analyses of saccharide composition, linkage, and complex structures using chromatography-based methods, nuclear magnetic resonance, -omics, and antibody glycome profiling, provides critical insight into the fine chemistry of feedstock cell walls. Furthermore, improving both the catalytic potential of microbial communities that populate biodigester reactors and the efficiency of pre-treatments used in bioethanol production may improve bioconversion rates and yields. Toward this end, knowledge and characterization of carbohydrate-active enzymes (CAZymes) involved in dynamic biomass deconstruction is pivotal. Here we overview the use of common "-omics"-based methods for the study of lignocellulose-metabolizing communities and microorganisms, as well as methods for annotation and discovery of CAZymes, and accurate prediction of CAZyme function. Emerging approaches for analysis of large datasets, including metagenome-assembled genomes, are also discussed. Using complementary glycomic and meta-omic methods to characterize agricultural residues and the microbial communities that digest them provides promising streams of research to maximize value and energy extraction from crop waste streams.

Approaches to Investigate Selective Dietary Polysaccharide Utilization by Human Gut Microbiota at a Functional Level.

The human diet is temporally and spatially dynamic, and influenced by culture, regional food systems, socioeconomics, and consumer preference. Such factors result in enormous structural diversity of ingested glycans that are refractory to digestion by human enzymes. To convert these glycans into metabolizable nutrients and energy, humans rely upon the catalytic potential encoded within the gut microbiome, a rich collective of microorganisms residing in the gastrointestinal tract. The development of high-throughput sequencing methods has enabled microbial communities to be studied with more coverage and depth, and as a result, cataloging the taxonomic structure of the gut microbiome has become routine. Efforts to unravel the microbial processes governing glycan digestion by the gut microbiome, however, are still in their infancy and will benefit by retooling our approaches to study glycan structure at high resolution and adopting next-generation functional methods. Also, new bioinformatic tools specialized for annotating carbohydrate-active enzymes and predicting their functions with high accuracy will be required for deciphering the catalytic potential of sequence datasets. Furthermore, physiological approaches to enable genotype-phenotype assignments within the gut microbiome, such as fluorescent polysaccharides, has enabled rapid identification of carbohydrate interactions at the single cell level. In this review, we summarize the current state-of-knowledge of these methods and discuss how their continued development will advance our understanding of gut microbiome function.

Combinatorial Glycomic Analyses to Direct CAZyme Discovery for the Tailored Degradation of Canola Meal Non-Starch Dietary Polysaccharides.

Canola meal (CM), the protein-rich by-product of canola oil extraction, has shown promise as an alternative feedstuff and protein supplement in poultry diets, yet its use has been limited due to the abundance of plant cell wall fibre, specifically non-starch polysaccharides (NSP) and lignin. The addition of exogenous enzymes to promote the digestion of CM NSP in chickens has potential to increase the metabolizable energy of CM. We isolated chicken cecal bacteria from a continuous-flow mini-bioreactor system and selected for those with the ability to metabolize CM NSP. Of 100 isolates identified, Bacteroides spp. and Enterococcus spp. were the most common species with these capabilities. To identify enzymes specifically for the digestion of CM NSP, we used a combination of glycomics techniques, including enzyme-linked immunosorbent assay characterization of the plant cell wall fractions, glycosidic linkage analysis (methylation-GC-MS analysis) of CM NSP and their fractions, bacterial growth profiles using minimal media supplemented with CM NSP, and the sequencing and de novo annotation of bacterial genomes of high-efficiency CM NSP utilizing bacteria. The SACCHARIS pipeline was used to select plant cell wall active enzymes for recombinant production and characterization. This approach represents a multidisciplinary innovation platform to bioprospect endogenous CAZymes from the intestinal microbiota of herbivorous and omnivorous animals which is adaptable to a variety of applications and dietary polysaccharides.

Limb-girdle muscular dystrophy type 2A can result from accelerated autoproteolytic inactivation of calpain 3.

Loss-of-function mutations in calpain 3 have been shown to cause limb-girdle muscular dystrophy type 2A (LGMD2A), an autosomal recessive disorder that results in gradual wasting of the muscles of the hip and shoulder areas. Due to the inherent instability of calpain 3, recombinant expression of the full-length enzyme has not been possible, making in vitro analysis of specific LGMD2A-causing mutations difficult. However, because calpain 3 is highly similar in amino acid sequence to calpain 2, the recently solved crystal structure of full-length, Ca2+-bound, calpastatin-inhibited rat calpain 2 has allowed us to model calpain 3 as a Ca2+-bound homodimer. The model revealed three distinct areas of the enzyme that undergo a large conformational change upon Ca2+ binding. Located in these areas are several residues that undergo mutation to cause LGMD2A. We investigated the in vitro effects of six of these mutations by making the corresponding mutations in rat calpain 2. All six mutations examined in this study resulted in a decrease in enzyme activity. All but one of the mutations caused an increased rate of autoproteolytic degradation of the enzyme as witnessed by SDS-PAGE, indicating the decrease in enzyme activity is caused, at least in part, by an increase in the rate of autoproteolytic degradation. The putative in vivo effects of these mutations on calpain 3 activity are discussed with respect to their ability to cause LGMD2A.

Structural analysis of broiler chicken small intestinal mucin O-glycan modification by Clostridium perfringens.

Clostridium perfringens is a Gram-positive opportunistic pathogen that is the principal etiological agent of necrotic enteritis (NE) in poultry. The ability of C. perfringens to incite NE depends upon its ability to penetrate the protective mucus barrier within the small intestine, which is largely composed of heavily glycosylated proteins called mucins. Mucins are decorated by N- and O-linked glycans that serve both as a formidable gel-like barrier against invading pathogens and as a rich carbon source for mucolytic bacteria. The composition of avian O-linked glycans is markedly different from mucins in other vertebrates, being enriched in sulfated monosaccharides and N-acetyl-d-neuraminic acid (Neu5Ac, sialic acid). These modifications increase the overall negative charge of mucins and are believed to impede colonization by enteric pathogens. The mechanism by which C. perfringens penetrates the poultry intestinal mucus layer during NE is still unknown. However, the CAZome (i.e., the total collection of proteins encoded within a genome active on carbohydrates) of C. perfringens strain CP1 encodes several putative and known enzymes with activities consistent with the modification of mucin. To further investigate this relationship, O-glycans from Gallus gallus domesticus mucus were extracted from the small intestine and characterized using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Chicken mucin monosaccharides included l-fucose (Fuc), d-mannose (Man), d-galactose (Gal), N-acetyl-d-galactosamine (GalNAc), N-acetyl-d-glucosamine (GlcNAc), and Neu5Ac (sialic acid). Using these monosaccharides as sole carbon sources, we showed that C. perfringens CP1 grew on Neu5Ac, Man, Gal, and GlcNAc but not on Fuc and GalNAc. We also demonstrated C. perfringens grew on different native-state preparations of intestinal mucins and mucus including porcine mucins, chicken mucus, and chicken mucins. Finally, anaerobic incubation of chicken mucin O-glycans with C. perfringens and subsequent analysis of the glycans revealed that there was preferential removal of Neu5Ac. These observations are discussed in the context of the predicted metabolic potential of C. perfringens CP1 and the mucolytic enzymes encoded within its CAZome.

Gram-negative synthase-dependent exopolysaccharide biosynthetic machines.

Bacteria predominantly exist as matrix embedded communities of cells called biofilms. The biofilm matrix is made up of a variety of self-produced extracellular components including DNA, proteins, and exopolysaccharides. Bacterial exopolysaccharides have been implicated in surface adhesion, resistance to antibiotics, and protection from host immune systems. Herein we review the structure and function of the proteins involved in the production of the Gram-negative synthase-dependent exopolysaccharides: alginate, poly-ß(1,6)-N-acetyl-d-glucosamine (PNAG), cellulose, and the Pel polysaccharide. We highlight the similarities and differences that exist at the molecular level in these synthase systems.