In Silico Study on a Binding Mechanism of ssDNA Aptamers Targeting Glycosidic Bond-Containing Small Molecules.

Aptamer-based detection targeting glycoconjugates has attracted significant attention for its remarkable potential in identifying structural changes in saccharides in different stages of various diseases. However, the challenges in screening aptamers for small carbohydrates or glycoconjugates, which contain highly flexible and diverse glycosidic bonds, have hindered their application and commercialization. In this study, we investigated the binding conformations between three glycosidic bond-containing small molecules (GlySMs; glucose, N-acetylneuraminic acid, and neomycin) and their corresponding aptamers in silico, and analyzed factors contributing to their binding affinities. Based on the findings, a novel binding mechanism was proposed, highlighting the central role of the stem structure of the aptamer in binding and recognizing GlySMs and the auxiliary role of the mismatched bases in the adjacent loop. Guided by this binding mechanism, an aptamer with a higher 6'-sialyllactose binding affinity was designed, achieving a KD value of 4.54 ± 0.64 µM in vitro through a single shear and one mutation. The binding mechanism offers crucial guidance for designing high-affinity aptamers, enhancing the virtual screening efficiency for GlySMs. This streamlined workflow filters out ineffective binding sites, accelerating aptamer development and providing novel insights into glycan-nucleic acid interactions.

In Silico Selection and Validation of High-Affinity ssDNA Aptamers Targeting Paromomycin.

Glycans are promising for disease diagnosis since glycan biosynthesis is significantly affected by disease states, and glycosylation changes are probably more pronounced than protein expression during the transformation to the diseased condition. Glycan-specific aptamers can be developed for challenging applications such as cancer targeting; however, the high flexibility of glycosidic bonds and scarcity of studies on glycan-aptamer binding mechanisms increased the difficulty of screening. In this work, the model of interactions between glycans and ssDNA aptamers synthesized based on the sequence of rRNA genes was developed. Our simulation-based approach revealed that paromomycin as a representative example of glycans is preferred to bind base-restricted stem structures of aptamers because they are more critical in stabilizing the flexible structures of glycans. Combined experiments and simulations have identified two optimal mutant aptamers. Our work would provide a potential strategy that the glycan-binding rRNA genes could act as the initial aptamer pools to accelerate aptamer screening. In addition, this in silico workflow would be potentially applied in the more extensive in vitro development and application of RNA-templated ssDNA aptamers targeting glycans.

Recent Research and Application Prospect of Functional Oligosaccharides on Intestinal Disease Treatment.

The intestinal tract is an essential digestive organ of the human body, and damage to the intestinal barrier will lead to various diseases. Functional oligosaccharides are carbohydrates with a low degree of polymerization and exhibit beneficial effects on human intestinal health. Laboratory experiments and clinical studies indicate that functional oligosaccharides repair the damaged intestinal tract and maintain intestinal homeostasis by regulating intestinal barrier function, immune response, and intestinal microbial composition. Functional oligosaccharides treat intestinal disease such as inflammatory bowel disease (IBD) and colorectal cancer (CRC) and have excellent prospects for therapeutic application. Here, we present an overview of the recent research into the effects of functional oligosaccharides on intestinal health.

Exploring Effects of Chitosan Oligosaccharides on the DSS-Induced Intestinal Barrier Impairment In Vitro and In Vivo.

Intestinal barrier dysfunction is an essential pathological change in inflammatory bowel disease (IBD). The mucus layer and the intestinal epithelial tight junction act together to maintain barrier integrity. Studies showed that chitosan oligosaccharide (COS) had a positive effect on gut health, effectively protecting the intestinal barrier in IBD. However, these studies usually focused on its impact on the intestinal epithelial tight junction. The influence of COS on the intestinal mucus layer is still poorly understood. In this study, we explored the effect of COS on intestinal mucus in vitro using human colonic mucus-secreted HT-29 cells. COS relieved DSS (dextran sulfate sodium)-induced mucus defects. Additionally, the structural characteristics of COS greatly influenced this activity. Finally, we evaluated the protective effect of COS on intestinal barrier function in mice with DSS-induced colitis. The results indicated that COS could manipulate intestinal mucus production, which likely contributed to its intestinal protective effects.

Blood-Brain Barrier Permeable Chitosan Oligosaccharides Interfere with ß-Amyloid Aggregation and Alleviate ß-Amyloid Protein Mediated Neurotoxicity and Neuroinflammation in a Dose- and Degree of Polymerization-Dependent Manner.

It is proven that ß-amyloid (Aß) aggregates containing cross-ß-sheet structures led to oxidative stress, neuroinflammation, and neuronal loss via multiple pathways. Therefore, reduction of Aß neurotoxicity via inhibiting aggregation of Aß or dissociating toxic Aß aggregates into nontoxic forms might be effective therapeutic methods for Alzheimer's disease (AD) treatment. This study was designed to explore interference of chitosan oligosaccharides (COS) on ß-(1-42)-amyloid protein (Aß42) aggregation and Aß42-induced cytotoxicity. Here it was demonstrated that COS showed good blood-brain barrier (BBB) penetration ability in vitro and in vivo. The experimental results showed that COS efficiently interfered with Aß42 aggregation in dose- and degree of polymerization (DP)-dependent manners, and COS monomer with DP6 showed the best effect on preventing conformational transition into ß-sheet-rich structures. Based on the binding affinity analysis by microscale thermophoresis (MST), it was confirmed that COS could directly bind with Aß42 in a DP-dependent manner. Our findings demonstrated that different performance of COS monomers with different DPs against Aß42 assembly was, to some extent, attributable to their different binding capacities with Aß42. As a result, COS significantly ameliorated Aß42-induced cytotoxicity. Taken together, our studies would point towards a potential role of COS in treatment of AD.

Chitosan Oligosaccharides Coupling Inhibits Bacterial Biofilm-Related Antibiotic Resistance against Florfenicol.

The formation of bacterial biofilms has increased the resistance of bacteria to various environmental factors and is tightly associated with many persistent and chronic bacterial infections. Herein we design a strategy conjugating florfenicol, an antibiotic commonly used in the treatment of streptococcus, with the antimicrobial biomaterial, chitosan oligosaccharides. The results demonstrated that the florfenicol-COS conjugate (F-COS) efficiently eradicated the mature Streptococcus hyovaginalis biofilm, apparently inhibiting drug resistance to florfenicol. A quantity of 250 µg/mL F-COS showed effective inhibitory activity against planktonic cells and biofilm of the bacteria, and a 4-fold improvement of the F-COS compared to unmodified florfenicol was observed. Furthermore, the conjugate showed a broad-spectrum activity against both Gram-positive and Gram-negative bacteria. It suggested that F-COS might have a potential for application in the treatment of biofilm-related infections.

Inhibition of Liver Tumor Cell Metastasis by Partially Acetylated Chitosan Oligosaccharide on A Tumor-Vessel Microsystem.

Chitooligosaccharides (COS), the only cationic oligosaccharide in nature, have been demonstrated to have anti-tumor activity. However, the inhibitory effects of COS on different stages of tumor metastasis are still unknown, and it is not clear what stage(s) of tumor metastasis COS targeted. To study the inhibitory effects of a new partially acetylated chitooligosaccharide (paCOS) with fraction of acetylation (FA) 0.46 on each phase of liver cancer cell metastasis, a dynamic tumor-vessel microsystem undergoing physiological flow was leveraged. paCOS (FA = 0.46) significantly inhibited proliferation of HepG2 cells through vascular absorption on the chip, and inhibited migration of HepG2 cells by inhibiting the formation of pseudopod in liver tumor cells. It was also found that paCOS at 10 µg/mL had a stronger inhibitory effect on liver tumor cells invading blood vessels than that of paCOS at 100 µg/mL, and paCOS at 100 µg/mL, which had a significant destructive effect on tumor vascular growth and barrier function. Moreover, paCOS reduced the number of liver tumor cells adhering onto the surface of HUVECs layer after 3 h of treatment. Therefore, the results revealed that paCOS had considerable potential as drugs for anti-tumor metastasis.

Synthesis and Evaluation of a Chitosan Oligosaccharide-Streptomycin Conjugate against Pseudomonas aeruginosa Biofilms.

Microbial biofilms are considerably more resistant to antibiotics than planktonic cells. It has been reported that chitosan coupling with the aminoglycoside antibiotic streptomycin dramatically disrupted biofilms of several Gram-positive bacteria. This finding suggested the application of the covalent conjugate of antimicrobial natural polysaccharides and antibiotics on anti-infection therapy. However, the underlying molecular mechanism of the chitosan-streptomycin conjugate (CS-Strep) remains unclear and the poor water-solubility of the conjugate might restrict its applications for anti-infection therapy. In this study, we conjugated streptomycin with water-soluble chitosan oligosaccharides (COS). Unlike CS-Strep, the COS-streptomycin conjugate (COS-Strep) barely affected biofilms of tested Gram-positive bacteria. However, COS-Strep efficiently eradicated established biofilms of the Gram-negative pathogen Pseudomonas aeruginosa. This activity of COS-Strep was influenced by the degree of polymerization of chitosan oligosaccharide. The increased susceptibility of P. aeruginosa biofilms to antibiotics after conjugating might be related to the following: Suppression of the activation of MexX-MexY drug efflux pump system induced by streptomycin treatment; and down-regulation of the biosynthesis of biofilm exopolysaccharides. Thus, this work indicated that covalently linking antibiotics to chitosan oligosaccharides was a possible approach for the development of antimicrobial drugs against biofilm-related infections.

Unraveling synthesis of the cryptococcal cell wall and capsule.

Fungal pathogens cause devastating infections in millions of individuals each year, representing a huge but underappreciated burden on human health. One of these, the opportunistic fungus Cryptococcus neoformans, kills hundreds of thousands of patients annually, disproportionately affecting people in resource-limited areas. This yeast is distinguished from other pathogenic fungi by a polysaccharide capsule that is displayed on the cell surface. The capsule consists of two complex polysaccharide polymers: a mannan substituted with xylose and glucuronic acid, and a galactan with galactomannan side chains that bear variable amounts of glucuronic acid and xylose. The cell wall, with which the capsule is associated, is a matrix of alpha and beta glucans, chitin, chitosan, and mannoproteins. In this review, we focus on synthesis of the wall and capsule, both of which are critical for the ability of this microbe to cause disease and are distinct from structures found in either model yeasts or the mammals afflicted by this infection. Significant research effort over the last few decades has been applied to defining the synthetic machinery of these two structures, including nucleotide sugar metabolism and transport, glycosyltransferase activities, polysaccharide export, and assembly and association of structural elements. Discoveries in this area have elucidated fundamental biology and may lead to novel targets for antifungal therapy. In this review, we summarize the progress made in this challenging and fascinating area, and outline future research questions.

Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation.

Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying transcription factors (TFs) involved in a specific process and used it to map regulation of the key virulence factor of a deadly fungus-its capsule. The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, midlevel, and "foreman" TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. These resources and analyses provide the first integrated, systems-level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to laboratories focused on specific physiological processes.

Conjugation of Inulin Improves Anti-Biofilm Activity of Chitosan.

Bacteria biofilm helps bacteria prevent phagocytosis during infection and increase resistance to antibiotics. Staphylococcus aureus is a Gram-positive pathogenic bacterium and is tightly associated with biofilm-related infections, which have led to great threat to human health. Chitosan, the only cationic polysaccharide in nature, has been demonstrated to have antimicrobial and anti-biofilm activities, which, however, require a relative high dosage of chitosan. Moreover, poor water solubility further restricts its applications on anti-infection therapy. Inulins are a group of polysaccharides produced by many types of plants, and are widely used in processed foods. Compared to chitosan, inulin is very soluble in water and possesses a mild antibacterial activity against certain pathogenic bacteria. In order to develop an effective strategy to treat biofilm-related infections, we introduce a method by covalent conjugation of inulin to chitosan. The physicochemical characterization of the inulin⁻chitosan conjugate was assayed, and the anti-biofilm activity was evaluated against S. aureus biofilm. The results indicated that, as compared to chitosan, this novel polysaccharide⁻polysaccharide conjugate significantly enhanced activities against S. aureus either in a biofilm or planktonic state. Of note, the conjugate also showed a broad spectrum anti-biofilm activity on different bacteria strains and low cellular toxicity to mammalian cells. These results suggested that chitosan conjugation of inulin was a viable strategy for treatment against biofilm-related infections. This finding may further spread the application of natural polysaccharides on treatments of infectious disease.

Cryptococcus neoformans dual GDP-mannose transporters and their role in biology and virulence.

Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro. We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis.

Unveiling the inverse antimicrobial impact of a hetero-chitooligosaccharide on Candida tropicalis growth and biofilm formation.

Chitosan and chitooligosaccharide (COS) are renowned for their potent antimicrobial prowess, yet the precise antimicrobial efficacy of COS remains elusive due to scant structural information about the utilized saccharides. This study delves into the antimicrobial potential of COS, spotlighting a distinct hetero-chitooligosaccharide dubbed DACOS. In contrast to other COS, DACOS remarkably fosters the growth of Candida tropicalis planktonic cells and fungal biofilms. Employing gradient alcohol precipitation, DACOS was fractionated, unveiling diverse structural characteristics and differential impacts on C. tropicalis. Notably, in a murine model of systemic candidiasis, DACOS, particularly its 70 % alcohol precipitates, manifests a promotive effect on Candida infection. This research unveils a new pathway for exploring the intricate nexus between the structural attributes of chitosan oligosaccharides and their physiological repercussions, underscoring the imperative of crafting chitosan and COS with meticulously defined structural configurations.

The Skp1 protein from Toxoplasma is modified by a cytoplasmic prolyl 4-hydroxylase associated with oxygen sensing in the social amoeba Dictyostelium.

In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O(2) and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O(2) regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3(SCF) ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O(2) sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O(2) levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O(2) requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O(2). The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O(2) by detecting the levels of O(2)-regulated metabolites such as α-ketoglutarate.

Variations in metabolic enzymes cause differential changes of heparan sulfate and hyaluronan in high glucose treated cells on chip.

Glycocalyx dysfunction is believed as the first step in diabetic vascular disease. However, few studies have systematically investigated the influence of HG on the glycocalyx as a whole and its major constituent glycans towards one type of cell. Furthermore, most studies utilized traditional two-dimensional (2D) cultures in vitro, which can't provide the necessary fluid environment for glycocalyx. Here, we utilized vascular glycocalyx on chips to evaluate the changes of glycocalyx and its constituent glycans in HG induced HUVECs. Fluorescence microscopy showed up-regulation of hyaluronan (HA) but down-regulation of heparan sulfate (HS). By analyzing the metabolic enzymes of both glycans, a decrease in the ratio of synthetic/degradative enzymes for HA and an increase in that for HS were demonstrated. Two substrates (UDP-GlcNAc, UDP-GlcA) for the synthesis of both glycans were increased according to omics analysis. Since they were firstly pumped into Golgi apparatus to synthesize HS, less substrates may be left for HA synthesis. Furthermore, the differential changes of HA and HS were confirmed in vessel slides from db/db mice. This study would deepen our understanding of impact of HG on glycocalyx formation and diabetic vascular disease.

Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.

O(2) regulates multicellular development of the social amoeba Dictyostelium, suggesting it may serve as an important cue in its native soil environment. Dictyostelium expresses an HIFα-type prolyl 4-hydroxylase (P4H1) whose levels affect the O(2)-threshold for culmination implicating it as a direct O(2)-sensor, as in animals. But Dictyostelium lacks HIFα, a mediator of animal prolyl 4-hydroxylase signaling, and P4H1 can hydroxylate Pro143 of Skp1, a subunit of E3(SCF)ubiquitin-ligases. Skp1 hydroxyproline then becomes the target of five sequential glycosyltransferase reactions that modulate the O(2)-signal. Here we show that genetically induced changes in Skp1 levels also affect the O(2)-threshold, in opposite direction to that of the modification enzymes suggesting that the latter reduce Skp1 activity. Consistent with this, overexpressed Skp1 is poorly hydroxylated and Skp1 is the only P4H1 substrate detectable in extracts. Effects of Pro143 mutations, and of combinations of Skp1 and enzyme level perturbations, are consistent with pathway modulation of Skp1 activity. However, some effects were not mirrored by changes in modification of the bulk Skp1 pool, implicating a Skp1 subpopulation and possibly additional unknown factors. Altered Skp1 levels also affected other developmental transitions in a modification-dependent fashion. Whereas hydroxylation of animal HIFα results in its polyubiquitination and proteasomal degradation, Dictyostelium Skp1 levels were little affected by its modification status. These data indicate that Skp1 and possibly E3(SCF)ubiquitin-ligase activity modulate O(2)-dependent culmination and other developmental processes, and at least partially mediate the action of the hydroxylation/glycosylation pathway in O(2)-sensing.

A xylosylphosphotransferase of Cryptococcus neoformans acts in protein O-glycan synthesis.

Cryptococcal meningoencephalitis is an AIDS-defining illness caused by the opportunistic pathogen Cryptococcus neoformans. This organism possesses an elaborate polysaccharide capsule that is unique among pathogenic fungi, and the glycobiology of C. neoformans has been a focus of research in the field. The capsule and other cellular glycans and glycoconjugates have been described, but the machinery responsible for their synthesis remains largely unexplored. We recently discovered Xpt1p, an enzyme with the unexpected activity of generating a xylose-phosphate-mannose linkage. We now demonstrate that this novel activity is conserved throughout the C. neoformans species complex, localized to the Golgi apparatus, and functions in the O-glycosylation of proteins. We also present the first survey of O-glycans from C. neoformans.

Role of the Skp1 prolyl-hydroxylation/glycosylation pathway in oxygen dependent submerged development of Dictyostelium.

BACKGROUND: Oxygen sensing is a near universal signaling modality that, in eukaryotes ranging from protists such as Dictyostelium and Toxoplasma to humans, involves a cytoplasmic prolyl 4-hydroxylase that utilizes oxygen and α-ketoglutarate as potentially rate-limiting substrates. A divergence between the animal and protist mechanisms is the enzymatic target: the animal transcriptional factor subunit hypoxia inducible factor-α whose hydroxylation results in its poly-ubiquitination and proteasomal degradation, and the protist E3SCF ubiquitin ligase subunit Skp1 whose hydroxylation might control the stability of other proteins. In Dictyostelium, genetic studies show that hydroxylation of Skp1 by PhyA, and subsequent glycosylation of the hydroxyproline, is required for normal oxygen sensing during multicellular development at an air/water interface. Because it has been difficult to detect an effect of hypoxia on Skp1 hydroxylation itself, the role of Skp1 modification was investigated in a submerged model of Dictyostelium development dependent on atmospheric hyperoxia. RESULTS: In static isotropic conditions beneath 70-100% atmospheric oxygen, amoebae formed radially symmetrical cyst-like aggregates consisting of a core of spores and undifferentiated cells surrounded by a cortex of stalk cells. Analysis of mutants showed that cyst formation was inhibited by high Skp1 levels via a hydroxylation-dependent mechanism, and spore differentiation required core glycosylation of Skp1 by a mechanism that could be bypassed by excess Skp1. Failure of spores to differentiate at lower oxygen correlated qualitatively with reduced Skp1 hydroxylation. CONCLUSION: We propose that, in the physiological range, oxygen or downstream metabolic effectors control the timing of developmental progression via activation of newly synthesized Skp1.

Chitosan Oligosaccharides Regulate the Occurrence and Development of Enteritis in a Human Gut-On-a-Chip.

Past studies on the protective effects of chitosan oligosaccharides (COS) on inflammatory bowel disease (IBD) commonly rely on animal models, because traditional cell culture systems couldn't faithfully mimic human intestinal physiology. Here a novel human gut-on-a-chip microsystem was established to further explore the regulatory effects of COS on the occurrence and development of human enteritis. By constructing an intestinal injury model caused by dextran sodium sulfate (DSS) on the chip, this study proved that COS can reduce intestinal epithelial injury by promoting the expression of the mucous layer for the first time. By establishing an inflammatory bowel disease model on the chip caused by E. coli 11775, this study demonstrated that COS can protect the intestinal epithelial barrier and vascular endothelial barrier by inhibiting the adhesion and invasion of E. coli 11775 for the first time. In addition, similar to the results in vivo, COS can decrease the inflammatory response by reducing the expression of toll-like receptor 4 protein and reducing the nuclear DNA binding rate of nuclear factor kappa-B protein on this chip. In summary, COS can be used as a potential drug to treat human IBD and the human gut-on-a-chip would be used as a platform for quick screening drugs to treat human IBD in future.

High molecular weight chitosan oligosaccharide exhibited antifungal activity by misleading cell wall organization via targeting PHR transglucosidases.

The fungal cell wall is an ideal target for the design of antifungal drugs. In this study we used an analog of cell wall polymer, a highly deacetylated high molecular-weight chitosan oligosaccharide (HCOS), to test its effect against pathogenic Candida strains. Results showed that HCOS was successfully incorporated into the dynamic cell wall organization process and exhibited an apparent antifungal activity against both plankton and mature fungal biofilm, by impairing the cell wall integrity. Unexpectedly, mechanistic studies suggested that HCOS exerts its activity by interfering with family members of PHR ß-(1,3)-glucanosyl transferases and affecting the connection and assembly of cell wall polysaccharides. Furthermore, HCOS showed great synergistic activity with different fungicides against Candida cells, especially those in biofilm. These findings indicated HCOS has a great potential as an antifungal drug or drug synergist and proposed a novel antifungal strategy with structure-specific oligosaccharides mimicking cell wall polysaccharide fragments.