Glucan Binding Research Articles

Biofilm targeting with chitosan-based nanohydrogel containing Quercus infectoria G. Olivier extract against Streptococcus mutans: new formulations of a traditional natural product

BackgroundBiofilm formation has a crucial role in the cariogenic virulence of Streptococcus mutans, which leads to resistance to common antibacterials. The antimicrobial resistance crisis has led to increased research about traditional natural products.PurposeQuercus infectoria extract (QI extract) and nano hydrogels containing QI extract (QI-NH) and tannic acid (TA-NH) were evaluated against this pathogen.MethodsQI extract was analyzed by HPLC and the physiological characteristics of nanohydrogels were assessed by SEM, FTIR, zeta potential, DLS and determination of release kinetics and encapsulation efficiency. Determination of MIC and MBC of the material and their anti-biofilm effect was done by the microtiter method and on the extracted tooth surface. The properties of extracts and nano hydrogels in the expression of genes codifying glucosyltransferases (gtfB, gtfC and gtfD) and glucan binding protein B (gbpB) were quantified. Their toxicity was tested by the MTT method against the KB cell line.ResultsAccording to HPLC, 55.18% of QI extract contained TA. The encapsulation efficiency of QI-NH and TA-NH was equal to 60% and 80%, respectively. SEM and FTIR exhibited that QI extract and TA were successfully entrapped in the networks resulting from the chemical bonding of chitosan and TPP. The average size of QI-NH and TA-NH was 70.45 and 58.43 nm, and their zeta potential was 6.17 ± 2.58 and 0.25 ± 0.03 mv, respectively. PDI < 0.3 of nano hydrogels indicated the favorable polydispersity of nanohydrogels. MIC of QI extract, QI-NH and TA-NH were 937.5, 30 and 10 µg/ml, respectively. Also their MBIC50 was 35.1, 2.1 and 0.95 µg/ml, respectively, and the extracts and nano hydrogels restrained the biofilm maturation on enamel. The pivotal genes of S. mutans in biofilm formation were significantly less expressed by treatment with QI-NH and TA-NH than others. Based on the MTT test, QI-NH had less acute toxicity than QI extract and TA-NH. IC50 of QI-NH was calculated as 775.4 µg/ml, while it was equal to 3.12 µg/ml for chlorhexidine as a common antibacterial agent.ConclusionQI-NH, a new formulation derived from traditional anti-caries, can be a safe and efficient option to combat dental biofilm.

Aromatic residues in N-terminal domain of archaeal trehalase affect the folding and activity of catalytic domain

Considering the structure of the bacterial GH15 family glucoamylase (GA), Thermoplasma trehalase Tvn1315 may be composed of a β-sandwich domain (BD) and a catalytic domain (CD). Tvn1315 BD weakly binds to insoluble β-glucans, such as cellulose, and helps fold CD. To determine how aromatic residues contribute to proper folding and enzyme activity, we performed alanine scanning for 32 aromatic residues in the BD. The study did not identify a single residue involved in glucan binding. However, several aromatic residues were found to be involved in BD or CD folding and in modulating the activity of the full-length enzyme. Among those aromatic residue mutations, the W43A mutation led to reduced solubility of the BD and full-length protein and resulted in a full-length enzyme with significantly lower activity. The activity of W43F and W43Y was significantly higher than that of W43A. In addition, Ala substitutions of Tyr83, Tyr113, and Tyr17 led to a reduction in trehalase activity, but Phe substitutions of these residues could be tolerated, as these mutants maintained activities similar to WT activity. Thus, these aromatic residues in BD may interact with CD and modulate enzyme activity.Key points• Aromatic residues in the BD are involved in BD and CD folding.• Aromatic residues in the BD near the CD active site modulate enzyme activity.• BD interacts with CD and closely modulates enzyme activity.

In Situ Biofilm Affinity-Based Protein Profiling Identifies the Streptococcal Hydrolase GbpB as the Target of a Carolacton-Inspired Chemical Probe.

Natural products are important precursors for antibiotic drug design. These chemical scaffolds serve as synthetic inspiration for chemists who leverage their structures to develop novel antibacterials and chemical probes. We have previously studied carolacton, a natural product macrolactone fromSorangium cellulosum, and discovered a simplified derivative, A2, that maintained apparent biofilm inhibitory activity, although the biological target was unknown. Herein, we utilize affinity-based protein profiling (AfBPP) in situ during biofilm formation to identify the protein target using a photoexcitable cross-linking derivative of A2. From these studies, we identified glucan binding protein B (GbpB), a peptidoglycan hydrolase, as the primary target of A2. Further characterization of the interaction between A2 and GbpB, as well as PcsB, a closely related homologue from the more pathogenic S. pneumoniae, revealed binding to the catalytic CHAP (cysteine, histidine, aminopeptidase) domain. To the best of our knowledge, this is the first report of a small-molecule binder of a conserved and essential bacterial CHAP hydrolase, revealing its potential as an antibiotic target. This work also highlights A2 as a useful tool compound for streptococci and as an initial scaffold for the design of more potent CHAP binders.

Suppression of Drosophila antifungal immunity by a parasite effector via blocking GNBP3 and GNBP-like 3, the dual receptors for β-glucans

The tactics used by animal pathogens to combat host immunity are largely unclear. Here, we report the depiction of the virulence-required effector Tge1 deployed by the entomopathogen Metarhizium robertsii to suppress Drosophila antifungal immunity. Tge1 can target both GNBP3 and GNBP-like 3 (GL3), and the latter can bind to β-glucans like GNBP3, whereas the glucan binding by both receptors can be attenuated by Tge1. As opposed to the surveillance GNBP3, GL3 is inducible in Drosophila depending on the Toll pathway via a positive feedback loop mechanism. Losses of GNBP3 and GL3 genes result in the deregulations of protease cascade, Spätzle maturation, and antimicrobial gene expressions in Drosophila upon fungal challenges. Fly survival assays confirm that GL3 plays a more essential role than GNBP3 in combating fungal infections. In addition to evidencing the gene-for-gene interactions between fungi and insects, our data advance insights into Drosophila antifungal immunity.

Gossypium hirsutum gene of unknown function Gohir.A02G131900 encodes a potential plant-specific, dual-domain exo-1,3-β-glucosidase.

A gene of unknown function, Gohir.A02G131900.1, identified in Gossypium hirsutum was studied using computational sequence and structure bioinformatic tools. The encoded protein GhGH5BG-A0A1U8NW40 (UniProt A0A1U8NW40) is predicted to be secreted and localized to the cell wall. Homology and conserved residues indicate it belongs to a plant-specific subgroup of the glycoside hydrolase family 5 and likely has exo-1,3-β-glucosidase activity. This subgroup is unique in containing a fascin-like domain which may have evolved a unique glucan binding site of interest for further research.

Structural Determination of Glucosyltransferase C by Cryo-Electron Microscopy.

Biofilm formation is a critical factor in the development of cariogenic virulence of Streptococcus mutans. S. mutans has evolved a concerted mechanism to synthesize a biofilm matrix from dietary sugars by a family of glucosyltransferases (Gtfs). Three Gtfs, GtfB, C, and D utilize sucrose to form a sticky polymer consisting of insoluble and soluble glucans, a biofilm foundation. Each Gtf possesses two distinct domains, an N-terminal enzyme catalytic domain and a C-terminal glucan binding domain. X-ray crystallographic studies have determined a three-dimensional structure of the catalytic domain of GtfC, however, the structure of the C-terminal domain and the overall structure of Gtfs are unknown. Here, we provided a protocol that will guide us to solve Gtf structures using cryo-electron microscopy (cryo-EM). Cryo-EM and X-ray crystallography are two widely used techniques for determining protein structures, and both methods have their own advantages and limitations. Additionally, we also predicted the full-length GtfC structural using AlphaFold2. Overall, the combination of AlphaFold 2 with experimental approaches offers a powerful and synergistic strategy for protein structure determination, accelerating the pace of scientific discovery and enabling new insights into the molecular basis that govern the biosynthetic activities of Gtfs, which may inform the design of more effective treatments for cariogenic biofilms and associated diseases.

Plasma β-1,3 Glucan Binding Protein Mediated Opsono-Phagocytosis by Hemocytes In Vitro of Marine Mussel Perna viridis.

We have shown in the past decade, for the first time in a bivalve mollusc, detection, isolation, and purification of β-1,3 glucan binding protein (β-GBP) in the plasma of the marine mussel Perna viridis and demonstrated its role in a nonself-induced activation of plasma prophenoloxidase system. In this study, we present evidence for its ability to function as an opsonin during phagocytosis of trypsinized yeast cells by the hemocytes of P. viridis. The in vitro pretreatment of target cells (trypsinized yeast cells) with β-GBP enhanced the phagocytic response of hemocytes. Such β-GBP-mediated enhanced phagocytic response appeared to be dose dependent. This opsono-phagocytic response could be inhibited by the presence of laminarin (a polymer of β-1,3 glucans), glucose, as well as polyclonal antibodies raised against β-GBP. These observations clearly indicate that the plasma β-GBP can possibly recognize and bind to β-1,3 glucans on the surface of targets and facilitate hemocyte recognition processes possibly by forming a bridge between the hemocytes and the target, consequently leading to opsono-phagocytosis. These observations together with our earlier annotations indicate the multifunctional potential of plasma β-GBP in the marine mussel P. viridis.

The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors

Toxoplasma gondii is an intracellular parasite that generates amylopectin granules (AGs), a polysaccharide associated with bradyzoites that define chronic T. gondii infection. AGs are postulated to act as an essential energy storage molecule that enable bradyzoite persistence, transmission, and reactivation. Importantly, reactivation can result in the life-threatening symptoms of toxoplasmosis. T. gondii encodes glucan dikinase and glucan phosphatase enzymes that are homologous to the plant and animal enzymes involved in reversible glucan phosphorylation and which are required for efficient polysaccharide degradation and utilization. However, the structural determinants that regulate reversible glucan phosphorylation in T. gondii are unclear. Herein, we define key functional aspects of the T. gondii glucan phosphatase TgLaforin (TGME49_205290). We demonstrate that TgLaforin possesses an atypical split carbohydrate-binding-module domain. AlphaFold2 modeling combined with hydrogen–deuterium exchange mass spectrometry and differential scanning fluorimetry also demonstrate the unique structural dynamics of TgLaforin with regard to glucan binding. Moreover, we show that TgLaforin forms a dual specificity phosphatase domain–mediated dimer. Finally, the distinct properties of the glucan phosphatase catalytic domain were exploited to identify a small molecule inhibitor of TgLaforin catalytic activity. Together, these studies define a distinct mechanism of TgLaforin activity, opening up a new avenue of T. gondii bradyzoite biology as a therapeutic target.

Molecular interaction analysis of β-1, 3 glucan binding protein with Bacillus licheniformis and evaluation of its immunostimulant property in Oreochromis mossambicus

Analyzing the health benefits of any two immunostimulants (synbiotics) in combined form and information on their interactions gain more visibility in the usage of synbiotics in aquafarms. With this intention, the current work explores the immunostimulant effect and structural interaction of synbiotic (β-1, 3 glucan binding protein from marine crab, Portunus pelagicus (Ppβ-GBP) and Bacillus licheniformis) on Oreochromis mossambicus. The experimental diet was prepared with Ppβ-GBP and B. licheniformis, and nourished to the fingerlings of O. mossambicus for 30 days. After the experimental trial, a higher growth rate and immune reactions (lysozyme, protease, myeloperoxidase and alkaline phosphatase activity) were noticed in the fish nourished with synbiotic (B. licheniformis and Ppβ-GBP) enriched diet. Moreover, the synbiotic enriched diet elevated the antioxidant responses like glutathione peroxidase (GSH-Px) and catalase (CAT) activity in the experimental diet-nurtured fish. At the end of the feed trial, synbiotic diet nourished fish shows an increased survival rate during Aeromonas hydrophila infection, reflecting the disease resistance potential of experimental fish. Also, the interaction between Ppβ-GBP and Bacillus licheniformis was analyzed through computational approaches. The results evidenced that, Ppβ-GBP interacts with the B. licheniformis through sugar-based ligand, β-glucan through a hydrogen bond with a good docking score. Thus, the synbiotic diet would be an effective immunostimulant to strengthen the fish immune system for better productivity.

In Vitro and In Silico Evaluation of Biological Properties of Some 1, 3, 4-Oxadiazole Derivatives Against Streptococcus mutans and Their Interaction With Gbp-C by Molecular Docking

Background: The need to replace new drug structures for the treatment of resistant strains has become essential. Streptococcus mutans is one of the most important factors in causing tooth decay. Glucan binding protein-C (Gbp-C) is a crucial mobileular floor protein that is worried in biofilm formation, and 1, 3, 4-oxadiazoles are new antibacterial structures. Accordingly, this study focused on assessing in vitro and in silico activity of our previously synthesized compounds of 1, 3, 4-oxadiazole against S. mutans. Methods: To this end, our previously synthesized derivatives were re-synthesized and prepared, and then antibacterial susceptibility tests were used for inhibition zone, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) test values. The molecular docking method was also applied to confirm the effect of compounds in interaction with the Gbp-C of S. mutans. Results: All compounds showed different effects against the bacterial sample. Among these, the most effective ones were related to naphthalene (4d), fluorophenyl (4e), and dimethoxyphenyl (4h) derivatives against S. mutans, respectively. Other compounds also had antibacterial properties but to a lesser extent. In the molecular part, compounds 4d and 4h had the highest affinity to inhibit the GbpC-protein. compound 4d with amino acids ASP and GLN established 402 and 391 hydrogen bonds, respectively, and compound 4h with amino acids SER, GLU, THR, and TRP established 347, 360, 449, and 451 hydrogen bonds, respectively. Conclusions: In general, 1, 3, 4-oxadiazoles containing naphthalene and dimethoxy phenyl functional groups in high concentrations can be good alternatives to the existing drugs for eliminating caries-causing tooth mutants that have drug resistance. It seems that more inhibitory effects can be observed on clinical specimens by adding different purposeful groups and increasing the destructive power of oxadiazole-based compounds.

Characterization of a fungal competition factor: Production of a conidial cell-wall associated antifungal peptide.

Competition is one of the fundamental driving forces of natural selection. Beauveria bassiana is a soil and plant phylloplane/root fungus capable of parasitizing insect hosts. Soil and plant environments are often enriched with other fungi against which B. bassiana competes for survival. Here, we report an antifungal peptide (BbAFP1), specifically expressed and localized to the conidial cell wall and is released into the surrounding microenvironment inhibiting growth of competing fungi. B. bassiana strains expressing BbAFP1, including overexpression strains, inhibited growth of Alternaria brassicae in co-cultured experiments, whereas targeted gene deletion of BbAFP1 significantly decreased (25%) this inhibitory effect. Recombinant BbAFP1 showed chitin and glucan binding abilities, and growth inhibition of a wide range of phytopathogenic fungi by disrupting membrane integrity and eliciting reactive oxygen species (ROS) production. A phenylalanine residue (F50) contributes to chitin binding and antifungal activity, but was not required for the latter. Expression of BbAFP1 in tomato resulted in transgenic plants with enhanced resistance to plant fungal pathogens. These results highlight the importance of fungal competition in shaping primitive competition strategies, with antimicrobial compounds that can be embedded in the spore cell wall to be released into the environment during the critical initial phases of germination for successful growth in its environmental niche. Furthermore, these peptides can be exploited to increase plant resistance to fungal pathogens.

Defining the Regulatory Roles of Glucan Phosphatases in Transitory Starch Degradation

Plants generate starch during the day and metabolize it at night to store and use for cellular energy. The glucan phosphatase Starch EXcess4 (SEX4) plays a crucial role in transitory starch degradation via its involvement in the reversible starch phosphorylation mechanism. Previous structural studies have shown that SEX4 preferentially binds to and dephosphorylates starch at the C6 position of glucosyl residues, but there remains little data on how SEX4 navigates and interacts with the complex starch granule. Further, enzyme kinetics and regulatory mechanisms of SEX4 are currently not well understood. We employed both generic and substrate specific phosphatase assays to determine kinetics and substrate binding mechanisms of SEX4. Upon use of a generic phosphatase substrate, p‐nitro phenyl phosphate (p‐NPP), SEX4 followed Michaelis‐Menten kinetics. A sigmoidal curve observed for the physiological substrate amylopectin suggests a non‐ Michaelis‐Menten behavior for SEX4 and provides experimental evidence for a possible allosteric regulatory mechanism of SEX4. The enhanced dephosphorylation rates obtained for generic phosphatase assays with SEX4 preincubated with different domains of starch structure strongly suggest a conformational change within the active site of SEX4 upon glucan binding. Collectively, our finding suggests a novel posttranslational regulatory mechanism of SEX4 that is necessary for the regulation of transitory starch degradation at night.

Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10-13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

Sucrose and Xylitol-Induced Streptococcus mutans Biofilm Adherence

Objective: To study the adherence of Streptococcus mutans biofilm after induction with sucrose and xylitol. Material and Methods: Laboratory experimental study incorporating posttest-only control group design. S. mutans biofilm was generated for 24 hours at a temperature of 37°C using BHIB with 5% sucrose and BHIB with 1% xylitol. An adherence assay was conducted in accordance with the method applied previously. The quantity of adhered bacteria was measured by means of a spectrophotometer at 570 nm. The data were presented as mean and standard deviation. Results: A biofilm induced with sucrose has a higher adherence level (0.9294 ± 0.0431) compared with one induced with xylitol (0.5095 ± 0.0392). Sucrose induces adherence levels by increasing glucan binding protein and glucosyltransferase of the bacteria, whereas xylitol will inhibit the glycolysis process of the bacteria. Conclusion: The adherence of sucrose-induced S. mutans biofilm is higher than that of xylitol-induced S. mutans biofilm.

Suppressive Effects of Octyl Gallate on Streptococcus mutans Biofilm Formation, Acidogenicity, and Gene Expression.

The accumulation of biofilm by Streptococcus mutans bacteria on hard tooth tissues leads to dental caries, which remains one of the most prevalent oral diseases. Hence, the development of new antibiofilm agents is of critical importance. The current study reports the results from testing the effectiveness of octyl gallate (C8-OG) against: (1) S. mutans biofilm formation on solid surfaces (polystyrene, glass), (2) acidogenicity, (3) and the expression of biofilm-related genes. The amount of biofilm formed by S. mutans bacteria was evaluated using the colorimetric method and optical profilometry. The pH of the biofilm growth medium was measured with microelectrode. A quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to assess the expression of genes encoding glucan binding protein B (gbpB), glucosyltransferases B, -C, -D (gtfB, -C, -D), and the F-ATPase β subunit of the F1 protein (atpD). The results show that C8-OG significantly diminished biofilm formation by exposed S. mutans on solid surfaces and suppressed acidogenicity in a dose-dependent manner, compared to unexposed bacteria (p < 0.05). The C8-OG concentration of 100.24 µM inhibited S. mutans biofilm development on solid surfaces by 100% and prevented a decrease in pH levels by 99%. In addition, the RT-qPCR data demonstrate that the biofilm-producing bacteria treated with C8-OG underwent a significant reduction in gene expression in the case of the four genes under study (gbpB, gtfC, gtfD, and atpD), and there was a slight decrease in expression of the gtfB gene. However, C8-OG treatments did not produce significant expression change compared to the control for the planktonic cells, although there was a significant increase for the atpD gene. Therefore, C8-OG might be a potent antibiofilm and/or anticaries agent for oral formulations that aim to reduce the prevalence of dental caries.

LIKE SEX4 1 Acts as a β-Amylase-Binding Scaffold on Starch Granules during Starch Degradation.

In Arabidopsis (Arabidopsis thaliana) leaves, starch is synthesized during the day and degraded at night to fuel growth and metabolism. Starch is degraded primarily by β-amylases, liberating maltose, but this activity is preceded by glucan phosphorylation and is accompanied by dephosphorylation. A glucan phosphatase family member, LIKE SEX4 1 (LSF1), binds starch and is required for normal starch degradation, but its exact role is unclear. Here, we show that LSF1 does not dephosphorylate glucans. The recombinant dual specificity phosphatase (DSP) domain of LSF1 had no detectable phosphatase activity. Furthermore, a variant of LSF1 mutated in the catalytic cysteine of the DSP domain complemented the starch-excess phenotype of the lsf1 mutant. By contrast, a variant of LSF1 with mutations in the carbohydrate binding module did not complement lsf1 Thus, glucan binding, but not phosphatase activity, is required for the function of LSF1 in starch degradation. LSF1 interacts with the β-amylases BAM1 and BAM3, and the BAM1-LSF1 complex shows amylolytic but not glucan phosphatase activity. Nighttime maltose levels are reduced in lsf1, and genetic analysis indicated that the starch-excess phenotype of lsf1 is dependent on bam1 and bam3 We propose that LSF1 binds β-amylases at the starch granule surface, thereby promoting starch degradation.

Structural Studies of a Novel Glucan Phosphatase from the Red Alga Cyanidioschyzon Merolae

BackgroundGlucan phosphatases are a unique subset of the Dual Specificity Phosphatase (DSP) family that binds and dephosphorylates carbohydrate substrates. They are found in widely divergent organisms ranging from extremophilic single‐cell red algae to humans and act on different carbohydrate (starch or glycogen) substrates. Red algae, Cyanidioschyzon merolae (Cm) possesses a single glucan phosphatase with three structural domains which are different in family and organization compared to other known glucan phosphatase family members. Further, bioinformatics analysis suggested that it is most similar to a vertebrate glucan phosphatase rather than plant glucan phosphatases. This is intriguing, since its endogenous substrate is starch rather than glycogen.HypothesisCm‐laforin is uniquely stable and active glucan phosphatase and can be an important partner with other catabolic enzymes in the process of starch metabolism.ObjectiveExplore the structural basis for Cm‐laforin's function as a novel glucan phosphatase.MethodsWe expressed and purified the full, tandem, and isolated carbohydrate binding (CBM) and phosphatase (DSP) domains of Cm‐laforin to study their thermal stability, phosphatase function, and domain coupling. We utilized para‐nitrophenylphosphate (p‐NPP) substrate for generic and amylopectin in malachite green assay for specific phosphatase activities determination. Differential scanning fluorimetry (DSF) and starch binding assays were utilized for glucan binding studies. The oligomeric state of laforin was determined by Size Exclusion Chromatography with Multi‐Angle static Light Scattering (SEC‐MALS). X‐ray crystallography was utilized for structural studies, which allowed structure‐guided mutagenesis.ResultsCm‐laforin is found to be a stable and highly active glucan phosphatase. The full three‐domain protein was found to be highly active and have maximum phosphatase activity at 55°C. Further, it possesses 2‐fold higher specific glucan phosphatase activity compared to vertebrate laforin orthologs. Utilizing the tools of structural biology, we explored the importance of different amino acids of the DSP domain required for its stability. We determined the structure of the DSP domain at 1.5Å and found that Cm‐laforin utilizes a significant number of unique electrostatic interactions centered on D89 to maintain its significantly higher stability. We also found that CBM1 and DSP domain can be expressed and function in isolation, but CBM1 and linker region between CBM2 and DSP are required for the proper folding and stability of CBM2. We further demonstrate that Cm‐laforin exists as a monomer, and that the tandem CBM domains are required for high affinity glucan binding and enzymatic coupling during starch degradation.ConclusionCm‐laforin is enzymatically highly active and stable phosphatase with unique structural and functional features that underlie its role as a specific glucan phosphatase.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibitory Effects of Ethyl Gallate on Streptococcus mutans Biofilm Formation by Optical Profilometry and Gene Expression Analysis.

This study aimed to test the effectiveness of ethyl gallate (EG) against S. mutans biofilm formation on solid surfaces (polystyrene, glass) and acidogenicity, and to examine the effect on expression of related genes. The biofilm that is formed by S. mutans bacteria was evaluated using colorimetric assay and optical profilometry, while the pH of the biofilm growth medium was measured with microelectrode. The expression of genes encoding glucan binding protein B (gbpB), glucosyltranferases B, -C, -D (gtfB, -C, -D) and F-ATPase (atpD, atpF) was assessed using a quantitative reverse transcription-polymerase chain reaction (RT-qPCR). It was revealed that all of the EG concentrations significantly suppressed S. mutans biofilm build-up on polystyrene and glass surfaces, and inhibited acidogenicity, in a dose-dependent manner, compared to the activity of untreated bacteria (p < 0.05). The highest concentration of EG (3.53 mM) reduced biofilm formation on polystyrene and glass surfaces by 68% and more than 91%, respectively, and prevented a decrease in pH levels by 95%. The RT-qPCR data demonstrate that the biofilm-producing bacteria treated with EG underwent significant gene expression changes involving the gtfC (a 98.6 increase in fold change), gtfB gene (a 47.5 increase in fold change) and the gbpB gene (a 13.8 increase in fold change). However, for the other genes tested (gtfD, atpD and atpF), the EG treatments did not produce significant expression change compared to the control. EG produced significant gene expression change in three genes—gtfC, gtfB, and gbpB; it has the capacity to inhibit S. mutans biofilm formation on solid surfaces (polystyrene, glass), as well as acidogenicity. Therefore, EG might be used as an antibiofilm and/or anticaries agent for oral formulations in order to reduce the prevalence of dental caries.

β-1, 3 glucan binding protein based selenium nanowire enhances the immune status of Cyprinus carpio and protection against Aeromonas hydrophila infection

In the present study, immunoenhancing effect of β-1, 3 glucan binding protein based selenium nanowire (Phβ-GBP-SeNWs) in common carp, Cyprinus carpio was assessed. Biological based selenium nanoform was synthesized, using crustacean immune molecule β-GBP purified from the haemolymph of Paratelphusa hydrodromus. The morphological property of Phβ-GBP-SeNWs was analyzed through TEM which reveals, the synthesized nanowire exhibits approximately 30–50 nm width with smooth surface. For this current study, fish were fed with experimental diet includes Phβ-GBP, sodium selenite, selenomethionine and Phβ-GBP-SeNWs supplemented diet at different concentrations (0.5 mg, 1 mg and 2 mg) for 30 days. The growth performance, cellular and humoral immune responses (myeloperoxidase, reactive oxygen species, alkaline phosphatase and lysozyme activity) and antioxidant enzymes (glutathione peroxidase and catalase activity) in the fish fed with Phβ-GBP-SeNWs supplemented diet were significantly increased in dose-dependent manner, which was observed at two different interval period (15th and 30th day). Also, Phβ-GBP-SeNWs supplemented diet fed fish gain resistant after challenged with aquatic pathogen Aeromonas hydrophila and the relative survival percentage was increased. Agar disc diffusion and BacLight assay clearly demonstrated the antibacterial property of plasma of fish fed with Phβ-GBP-SeNWs supplemented diet against aquatic pathogen A. hydrophila, Vibrio parahaemolyticus and Vibrio alginolyticus. Moreover, confocal laser scanning microscopic analysis clearly showed that, Phβ-GBP-SeNWs supplemented diet fed fish plasma was more efficient in disrupting the architecture of bacterial colonies and thereby reduced the thickness of biofilm. Thus, the present study indicates that, incorporation of Phβ-GBP-SeNWs in the diet enhances the fish immune responses and disease resistance against aquatic pathogens.

Glucan Binding Protein C of Streptococcus mutans Mediates both Sucrose-Independent and Sucrose-Dependent Adherence.

The high-resolution structure of glucan binding protein C (GbpC) at 1.14 Å, a sucrose-dependent virulence factor of the dental caries pathogen Streptococcus mutans, has been determined. GbpC shares not only structural similarities with the V regions of AgI/II and SspB but also functional adherence to salivary agglutinin (SAG) and its scavenger receptor cysteine-rich domains (SRCRs). This is not only a newly identified function for GbpC but also an additional fail-safe binding mechanism for S. mutans Despite the structural similarities with S. mutans antigen I/II (AgI/II) and SspB of Streptococcus gordonii, GbpC remains unique among these surface proteins in its propensity to adhere to dextran/glucans. The complex crystal structure of GbpC with dextrose (β-d-glucose; Protein Data Bank ligand BGC) highlights exclusive structural features that facilitate this interaction with dextran. Targeted deletion mutant studies on GbpC's divergent loop region in the vicinity of a highly conserved calcium binding site confirm its role in biofilm formation. Finally, we present a model for adherence to dextran. The structure of GbpC highlights how artfully microbes have engineered the lectin-like folds to broaden their functional adherence repertoire.