Glycan-Imprinted Nanoparticle as Artificial Neutralizing Antibody for Efficient HIV-1 Recognition and Inhibition.

The HIV-1 envelope is a heavily glycosylated class 1 trimeric fusion protein responsible for viral entry into CD4+ immune cells. Developing neutralizing antibodies against the specific envelope glycans is an alternative method for antiviral therapies. This work presents the first-ever development and characterization of artificial neutralizing antibodies using molecular imprinting technology to recognize and bind to the envelope protein of HIV-1. The prepared envelope glycan-imprinted nanoparticles (GINPs) can successfully prevent HIV-1 from infecting target cells by shielding the glycans on the envelope protein. In vitro experiments showed that GINPs have strong affinity toward HIV-1 (Kd = 36.7 ± 2.2 nM) and possess high anti-interference and specificity. GINPs demonstrate broad inhibition activity against both tier 1 and tier 2 HIV-1 strains with a pM-level IC50 and exhibit a significant inhibitory effect on long-term viral replication by more than 95%. The strategy provides a promising method for the inhibition and therapy of HIV-1 infection.

Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response.

Introduction: The pathogenic bacterium Helicobacter pylori has evolved glycan-mediated mechanisms to evade host immune defenses. This study tests the hypothesis that genetic disruption of H. pylori glycan biosynthesis alters immune recognition and response by human gastric epithelial cells and monocyte-derived dendritic cells. Methods: To test this hypothesis, human cell lines were challenged with wildtype H. pylori alongside an array of H. pylori glycosylation mutants. The relative levels of immune response were measured via immature dendritic cell maturation and cytokine secretion. Results: Our findings indicate that disruption of lipopolysaccharide biosynthesis diminishes gastric cytokine production, without disrupting dendritic cell recognition and activation. In contrast, variable immune responses were observed in protein glycosylation mutants which prompted us to test the hypothesis that phase variation plays a role in regulating bacterial cell surface glycosylation and subsequent immune recognition. Lewis antigen presentation does not correlate with extent of immune response, while the extent of lipopolysaccharide O-antigen elaboration does. Discussion: The outcomes of this study demonstrate that H. pylori glycans modulate the host immune response. This work provides a foundation to pursue immune-based tailoring of bacterial glycans towards modulating immunogenicity of microbial pathogens.

Probing altered receptor specificities of antigenically drifting human H3N2 viruses by chemoenzymatic synthesis, NMR, and modeling.

Prototypic receptors for human influenza viruses are N-glycans carrying α2,6-linked sialosides. Due to immune pressure, A/H3N2 influenza viruses have emerged with altered receptor specificities that bind α2,6-linked sialosides presented on extended N-acetyl-lactosamine (LacNAc) chains. Here, binding modes of such drifted hemagglutinin's (HAs) are examined by chemoenzymatic synthesis of N-glycans having 13C-labeled monosaccharides at strategic positions. The labeled glycans are employed in 2D STD-1H by 13C-HSQC NMR experiments to pinpoint which monosaccharides of the extended LacNAc chain engage with evolutionarily distinct HAs. The NMR data in combination with computation and mutagenesis demonstrate that mutations distal to the receptor binding domain of recent HAs create an extended binding site that accommodates with the extended LacNAc chain. A fluorine containing sialoside is used as NMR probe to derive relative binding affinities and confirms the contribution of the extended LacNAc chain for binding.

M1 macrophages polarized by crude polysaccharides isolated from Auricularia polytricha exhibit anti-tumor effect on human breast cancer cells.

Breast cancer has been reported to correlate with the infiltration of tumor-associated macrophages (TAMs) or M2-like macrophages in tumor microenvironment (TME) that could promote breast cancer progression. In contrast, M1-like macrophages displayed anti-tumor activity toward cancer. This study was focused on Auricularia polytricha (AP), a cloud ear mushroom, which has been reported for anti-tumor activity and immunomodulation. AP extracts were screened on differentiated THP-1 macrophages (M0). Results demonstrated that water extract (APW) and crude polysaccharides (APW-CP) could upregulate M1-related genes and cytokines production (IL-6, IL-1 ß and TNF-α) significantly. Moreover, APW and APW-CP showed a high expression of CD86 (M1 marker) compared to M0. The NF-κB signaling pathway is crucial for pro-inflammatory gene regulation. The APW and APW-CP treatment showed the induction of the NF-κB pathway in a dose-dependent manner, which related to the ß-glucan content in the extracts. Furthermore, APW-CP polarized macrophages were investigated for anti-tumor activity on human breast cancer cells (MCF-7 and MDA-MB-231). Results showed that APW-CP could inhibit the invasion of breast cancer cells and induce apoptosis. Therefore, M1 macrophages polarized by APW-CP showed anti-tumor activity against the breast cancer cells and ß-glucan may be the potential M1-phenotype inducer.

Pancreatic cancer-associated fibroblasts modulate macrophage differentiation via sialic acid-Siglec interactions.

Despite recent advances in cancer immunotherapy, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive due to an immunosuppressive tumor microenvironment, which is characterized by the abundance of cancer-associated fibroblasts (CAFs). Once identified, CAF-mediated immune inhibitory mechanisms could be exploited for cancer immunotherapy. Siglec receptors are increasingly recognized as immune checkpoints, and their ligands, sialic acids, are known to be overexpressed by cancer cells. Here, we unveil a previously unrecognized role of sialic acid-containing glycans on PDAC CAFs as crucial modulators of myeloid cells. Using multiplex immunohistochemistry and transcriptomics, we show that PDAC stroma is enriched in sialic acid-containing glycans compared to tumor cells and normal fibroblasts, and characterized by ST3GAL4 expression. We demonstrate that sialic acids on CAF cell lines serve as ligands for Siglec-7, -9, -10 and -15, distinct from the ligands on tumor cells, and that these receptors are found on myeloid cells in the stroma of PDAC biopsies. Furthermore, we show that CAFs drive the differentiation of monocytes to immunosuppressive tumor-associated macrophages in vitro, and that CAF sialylation plays a dominant role in this process compared to tumor cell sialylation. Collectively, our findings unravel sialic acids as a mechanism of CAF-mediated immunomodulation, which may provide targets for immunotherapy in PDAC.

Human brain glycoform coregulation network and glycan modification alterations in Alzheimer's disease.

Despite the importance of protein glycosylation to brain health, current knowledge of glycosylated proteoforms or glycoforms in human brain and their alterations in Alzheimer's disease (AD) is limited. Here, we report a proteome-wide glycoform profiling study of human AD and control brains using intact glycopeptide-based quantitative glycoproteomics coupled with systems biology. Our study identified more than 10,000 human brain N-glycoforms from nearly 1200 glycoproteins and uncovered disease signatures of altered glycoforms and glycan modifications, including reduced sialylation and N-glycan branching and elongation as well as elevated mannosylation and N-glycan truncation in AD. Network analyses revealed a higher-order organization of brain glycoproteome into networks of coregulated glycoforms and glycans and discovered glycoform and glycan modules associated with AD clinical phenotype, amyloid-ß accumulation, and tau pathology. Our findings provide valuable insights into disease pathogenesis and a rich resource of glycoform and glycan changes in AD and pave the way forward for developing glycosylation-based therapies and biomarkers for AD.

CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex.

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provide molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. IMPORTANCE: Cellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machinery. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

Rauwolfia polysaccharide can inhibit the progress of ulcerative colitis through NOS2-mediated JAK2/STAT3 pathway.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory disease of the digestive tract. Rauwolfia polysaccharide (Rau) has therapeutic effects on colitis in mice, but its mechanism of action needs to be further clarified. In the study, we explored the effect of Rau on the UC cell model induced by Lipopolysaccharide (LPS). METHODS: We constructed a UC cell model by stimulating HT-29 cells with LPS. Dextran sodium sulfate (DSS) was used to induce mice to construct an animal model of UC. Subsequently, we performed Rau administration on the UC cell model. Then, the therapeutic effect of Rau on UC cell model and was validated through methods such as Cell Counting Kit-8 (CCK8), Muse, Quantitative real­time polymerase chain reaction (RT-qPCR), Western blotting, and Enzyme-linked immunosorbent assay (ELISA). RESULTS: The results showed that Rau can promote the proliferation and inhibit the apoptosis of the HT-29 cells-induced by LPS. Moreover, we observed that Rau can inhibit the expression of NOS2/JAK2/STAT3 in LPS-induced HT-29 cells. To further explore the role of NOS2 in UC progression, we used siRNA technology to knock down NOS2 and search for its mechanism in UC. The results illustrated that NOS2 knockdown can promote proliferation and inhibit the apoptosis of LPS-induced HT-29 cells by JAK2/STAT3 pathway. In addition, in vitro and in vivo experiments, we observed that the activation of the JAK2/STAT3 pathway can inhibit the effect of Rau on DSS-induced UC model. CONCLUSION: In short, Rauwolfia polysaccharide can inhibit the progress of ulcerative colitis through NOS2-mediated JAK2/STAT3 pathway. This study provides a theoretical clue for the treatment of UC by Rau.

Exposing the molecular heterogeneity of glycosylated biotherapeutics.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

Multifaceted roles of plant glycosyl hydrolases during pathogen infections: more to discover.

MAIN CONCLUSION: Carbohydrates are hydrolyzed by a family of carbohydrate-active enzymes (CAZymes) called glycosidases or glycosyl hydrolases. Here, we have summarized the roles of various plant defense glycosidases that possess different substrate specificities. We have also highlighted the open questions in this research field. Glycosidases or glycosyl hydrolases (GHs) are a family of carbohydrate-active enzymes (CAZymes) that hydrolyze glycosidic bonds in carbohydrates and glycoconjugates. Compared to those of all other sequenced organisms, plant genomes contain a remarkable diversity of glycosidases. Plant glycosidases exhibit activities on various substrates and have been shown to play important roles during pathogen infections. Plant glycosidases from different GH families have been shown to act upon pathogen components, host cell walls, host apoplastic sugars, host secondary metabolites, and host N-glycans to mediate immunity against invading pathogens. We could classify the activities of these plant defense GHs under eleven different mechanisms through which they operate during pathogen infections. Here, we have provided comprehensive information on the catalytic activities, GH family classification, subcellular localization, domain structure, functional roles, and microbial strategies to regulate the activities of defense-related plant GHs. We have also emphasized the research gaps and potential investigations needed to advance this topic of research.

Distinct CD16a features on human NK cells observed by flow cytometry correlate with increased ADCC.

Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N-glycan processing pathway. Kifunensine treatment also increased the antibody-binding affinity of Fc γ receptor IIIa/CD16a. Here we demonstrate that inhibiting NK cell N-glycan processing increased ADCC. We reduced N-glycan processing with the CRIPSR-CAS9 knockdown of MGAT1, another early-stage N-glycan processing enzyme, and showed that these cells likewise increased antibody binding affinity and ADCC. These experiments led to the observation that NK cells with diminished N-glycan processing capability also revealed a clear phenotype in flow cytometry experiments using the B73.1 and 3G8 antibodies binding two distinct CD16a epitopes. We evaluated this "affinity profiling" approach using primary NK cells and identified a distinct shift and differentiated populations by flow cytometry that correlated with increased ADCC.

Anti-Aging in Caenorhabditis elegans of Polysaccharides from Polygonatum cyrtonema Hua.

Polygonatum cyrtonema Hua, the dried rhizome of Polygonum multiflorum from the Liliaceae family, is a widely used medicinal herb with a long history of application. Its main active ingredients are polysaccharides, which have been demonstrated in contemporary studies to effectively delay the aging process. In the present study, homogeneous polysaccharide (PCP-1) was obtained after the purification and isolation of polysaccharides from Polygonatum cyrtonema Hua (PCP). The anti-aging activities of both were compared, and the possible mechanism of action for exerting anti-aging activity was explored using Caenorhabditis elegans (C. elegans). Research has indicated that PCP and PCP-1 exhibit potent anti-oxidant and anti-aging properties. Of particular note is that PCP-1 acts better than PCP. The two were able to prolong the lifespan of nematodes, improve the stress resistance of nematodes, reduce the accumulation of lipofuscin in the intestine, decrease the content of ROS and MDA in the body, increase the activity of the antioxidant enzymes SOD and CAT, promote the nuclear translocation of DAF-16, down-regulate the mRNA levels of the age-1 and daf-2 genes of the IIS pathway in nematodes, and up-regulate the expression of the daf-16, skn-1, sod-3, and hsp-16.2 genes. Based on the aforementioned findings, it is possible that the mechanism by which PCP and PCP-1 exert anti-aging effects may be through negative regulation of the IIS pathway, activation of the transcription factor DAF-16/FOXO, and enhancement of oxidative defenses and stress resistance in nematodes. Overall, the present study illustrated the great potential of polysaccharides from Polygonatum cyrtonema Hua in anti-aging and antioxidant activities. Specifically, PCP-1 demonstrated superior characteristics, which provides a reference for the future development of Polygonatum cyrtonema Hua polysaccharides.

Study of the Structure and Bioactivity of Polysaccharides from Different Parts of Stemona tuberosa Lour.

The polysaccharides from Stemona tuberosa Lour, a kind of plant used in Chinese herbal medicine, have various pharmacological activities, such as anti-inflammatory and antioxidant properties. However, the effects of the extraction methods and the activity of polysaccharides from different parts are still unknown. Therefore, this study aimed to evaluate the effects of different extraction methods on the yields, chemical compositions, and bioactivity of polysaccharides extracted from different parts of Stemona tuberosa Lour. Six polysaccharides were extracted from the leaves, roots, and stems of Stemona tuberosa Lour through the use of hot water (i.e., SPS-L1, SPS-R1, and SPS-S1) and an ultrasound-assisted method (i.e., SPS-L2, SPS-R2, and SPS-S2). The results showed that the physicochemical properties, structural properties, and biological activity of the polysaccharides varied with the extraction methods and parts. SPS-R1 and SPS-R2 had higher extraction yields and total sugar contents than those of the other SPSs (SPS-L1, SPS-L2, SPS-S1, and SPS-S2). SPS-L1 had favorable antioxidant activity and the ability to downregulate MUC5AC expression. An investigation of the anti-inflammatory properties showed that SPS-R1 and SPS-R2 had greater anti-inflammatory activities, while SPS-R2 demonstrated the strongest anti-inflammatory potential. The results of this study indicated that SPS-L1 and SPS-L2, which were extracted from non-medicinal parts, may serve as potent natural antioxidants, but further study is necessary to explore their potential applications in the treatment of diseases. The positive anti-inflammatory effects of SPS-R1 and SPS-R2 in the roots may be further exploited in drugs for the treatment of inflammation.

Complex N-glycans are important for interspecies transmission of H7 influenza A viruses.

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.

A polyethylene glycol-grafted pullulan polysaccharide adhesive improves drug loading capacity and release efficiency.

In this study, polyethylene glycol was grafted onto pullulan polysaccharides, resulting in the development of a novel adhesive termed PLUPE, offering superior drug loading capacity and rapid release efficiency. The efficacy of PLUPE was rigorously evaluated through various tests, including the tack test, shear strength test, 180° peel strength test, and human skin adhesion test. The results demonstrated that PLUPE exhibited a static shear strength that was 4.6 to 9.3 times higher than conventional PSAs, ensuring secure adhesion for over 3 days on human skin. A comprehensive analysis, encompassing electrical potential evaluation, calculation of interaction parameters, and FT-IR spectra, elucidated why improved the miscibility between the drug and PSAs, that the significant enhancement of intermolecular hydrogen bonding in the PLUPE structure. ATR-FTIR, rheological, and thermodynamic analyses further revealed that the hydrogen bonding network in PLUPE primarily interacted with polar groups in the skin. This interaction augmented the fluidity and free volume of PSA molecules, thereby promoting efficient drug release. The results confirmed the safety profile of PLUPE through skin irritation tests and MTT assays, bolstering its viability for application in TDDS patches. In conclusion, PLUPE represented a groundbreaking adhesive solution for TDDS patches, successfully overcoming longstanding challenges associated with PSAs.

The S1 spike protein of SARS-CoV-2 upregulates the ERK/MAPK signaling pathway in DC-SIGN-expressing THP-1 cells.

Dendritic cells, macrophages, neutrophils, and other antigen-presenting cells express various C-type lectin receptors that function to recognize the glycans associated with pathogens. The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds various pathogens such as HIV glycoprotein 120, the Ebola glycoprotein, hemagglutinin, and the dengue virus glycoprotein in addition to the SARS-CoV-2 spike protein, and also triggers antigen-presenting cell endocytosis and immune escape from systemic infections. Many studies on the binding of SARS-CoV-2 spike protein with glycans have been published, but the underlying mechanism by which intracellular signaling occurs remains unclear. In this study, we report that the S1 spike protein of SARS-CoV-2 induces the phosphorylation of extracellular signal-regulated kinases (ERKs) in THP-1 cells, a DC-SIGN-expressing human monocytic leukemic cell line. On the other hand, the phosphorylation level of NF-κB remained unchanged under the same conditions. These data suggest that the major cell signaling pathway regulated by the S1 spike protein is the ERK pathway, which is superior to the NF-κB pathway in these DC-SIGN-expressing THP-1 cells and may contribute to immune hyperactivation in SARS-CoV-2 infections. Additionally, several glycans such as mannans, mannosylated bovine serum albumin, the serum amyloid beta protein, and intracellular adhesion molecule 3 suppressed ERK phosphorylation, suggesting that these molecules are target molecules for SARS-CoV-2 infection by suppressing immune hyperactivation that occurs in the ERK signaling pathway.

Genomic characterization and expression profiling of the lytic polysaccharide monooxygenases AA9 family in thermophilic fungi Thermothelomyces fergusii in response to carbon source media.

Enhancing enzyme activity and stability in biomass degradation can improve substrate saccharification and, increases biorefinery efficiency. For the first time, we identified 20 lytic polysaccharide monooxygenases (LPMOs) AA9 genes in the genome of Thermothelomyces fergusii. Our results showed that TfAA9 was categorized into LPMOs1, LPMOs2, and LPMOs3 subgroups based on protein diversity. Protein- 3D structure analysis showed strong interactions between Myceliophthora thermophila AA9 proteins and 17 TfAA9 proteins. Gene ontology analysis indicated a high enrichment of cellulase activity in TfAA9 genes. KEGG pathways analysis revealed the role of TfAA9 proteins in the endohydrolysis of 1,4-beta-D-glucosidic linkages in cellulose. Numerous TfAA9s gene transcripts were up-regulated on avicel, cellobiose, and glucose, with a higher proportion on avicel. Protein concentration, endoglucanase, and cellulase activity were also boosted on avicel. However, limited fungal biomass was observed on avicel, despite the abundance of AA9 LPMOs in the T. fergusii genome. These findings expand our understanding of fungal AA9 genes and their role in lignocellulolytic degradation. The disparity between biomass and enzymatic activity suggests screening TfAA9 genes for highly active enzymes and redundant genes via heterologous expression. In short, functional characterization of these genes could contribute to improving the saccharification process of industrial raw materials.

Functional Characterization of Endo- and Exo-Hydrolase Genes in Arabinan Degradation Gene Cluster of Bifidobacterium longum subsp. suis.

Bifidobacteria are probiotic microorganisms commonly found in the gastrointestinal tract, some of which are known to utilize linear arabino-oligosaccharides (AOS) as prebiotic carbohydrates. In general, the synergistic actions of exo-type α-l-arabinofuranosidases (ABFs) and endo-α-1,5-l-arabinanases (ABNs) are required for efficient arabinan degradation. In this study, the putative gene cluster for arabinan degradation was discovered in the genome of Bifidobacterium longum subsp. suis. It consists of a variety of genes encoding exo- and endo-hydrolases, sugar-binding proteins, ABC-binding cassettes, and transcriptional regulators. Among them, two endo-ABNs GH43 (BflsABN43A and BflsABN43B), two exo-ABFs GH43 (BflsABF43A and BflsABF43B), and an exo-ABF GH51 (BflsABF51) were predicted to be the key hydrolases for arabinan degradation. These hydrolase genes were functionally expressed in Escherichia coli, and their enzymatic properties were characterized. Their synergism in arabinan degradation has been proposed from the detailed modes of action. Extracellular endo-BflsABN43A hydrolyzes sugar beet and debranched arabinans into the short-chain branched and linear AOS. Intracellularly, AOS can be further degraded into l-arabinose via the cooperative actions of endo-BflsABN43B, exo-BflsABF43A with debranching activity, α-1,5-linkage-specific exo-BflsABF43B, and exo-BflsABF51 with dual activities. The resulting l-arabinose is expected to be metabolized into energy through the pentose phosphate pathway by three enzymes expressed from the ara operon of bifidobacteria. It is anticipated that uncovering arabinan utilization gene clusters and their detailed functions in the genomes of diverse microorganisms will facilitate the development of customized synbiotics.

Degradation and fermentation of hyaluronic acid by Bacteroides spp. from the human gut microbiota.

Bacteroides spp. are prominent members of the human gut microbiota that play critical roles in the metabolism of complex carbohydrates from the daily diet. Hyaluronic acid (HA) is a multifunctional polysaccharide which has been extensively used in the food and biomedical industry. However, how HA is degraded and fermented by Bacteroides spp. has not been fully characterized. Here, we comprehensively investigated the detailed degradation profiles and fermentation characteristics of four different HAs with discrete molecular weight (Mw) by fourteen distinctive Bacteroides spp. from the human gut microbiota. Our results indicated that high-Mw HAs were more degradable and fermentable than low-Mw HAs. Interestingly, B. salyersiae showed the best degrading capability for both high-Mw and low-Mw HAs, making it a keystone species for HA degradation among Bacteroides spp.. Specifically, HA degradation by B. salyersiae produced significant amounts of unsaturated tetrasaccharide (udp4). Co-culture experiments indicated that the produced udp4 could be further fermented and utilized by non-proficient HA-degraders, suggesting a possible cross-feeding interaction in the utilization of HA within the Bacteroides spp.. Altogether, our study provides novel insights into the metabolism of HA by the human gut microbiota, which has considerable implications for the development of new HA-based nutraceuticals and medicines.

Bioconversion of α-Chitin by a Lytic Polysaccharide Monooxygenase OsLPMO10A Coupled with Chitinases and the Synergistic Mechanism Analysis.

The whole enzymatic conversion of chitin is a green and promising alternative to current strategies, which are based on lytic polysaccharide monooxygenases (LPMOs) and chitinases. However, the lack of LPMOs with high activity toward α-chitin limits the efficient bioconversion of α-chitin. Herein, we characterized a high chitin-active LPMO from Oceanobacillus sp. J11TS1 (OsLPMO10A), which could promote the decrystallization of the α-chitin surface. Furthermore, when coupled with OsLPMO10A, the conversion rate of α-chitin to N-acetyl chitobiose [(GlcNAc)2] by three chitinases (Serratia marcescens, ChiA, -B, and -C) reached 30.86%, which was 2.03-folds that without the addition of OsLPMO10A. Moreover, the results of synergistic reactions indicated that OsLPMO10A and chitinases promoted the degradation of α-chitin each other mainly on the surface. To the best of our knowledge, this study achieved the highest yield of N-acetyl chitooligosaccharides (N-acetyl COSs) among reported LPMOs-driven bioconversion systems, which could be regarded as a promising candidate for α-chitin bioconversion.