Bacterial Polysaccharides Research Articles

Manganese salts function as potent adjuvants

Aluminum-containing adjuvants have been used for nearly 100 years to enhance immune responses in billions of doses of vaccines. To date, only a few adjuvants have been approved for use in humans, among which aluminum-containing adjuvants are the only ones widely used. However, the medical need for potent and safe adjuvants is currently continuously increasing, especially those triggering cellular immune responses for cytotoxic T lymphocyte activation, which are urgently needed for the development of efficient virus and cancer vaccines. Manganese is an essential micronutrient required for diverse biological activities, but its functions in immunity remain undefined. We previously reported that Mn2+ is important in the host defense against cytosolic dsDNA by facilitating cGAS-STING activation and that Mn2+ alone directly activates cGAS independent of dsDNA, leading to an unconventional catalytic synthesis of 2′3′-cGAMP. Herein, we found that Mn2+ strongly promoted immune responses by facilitating antigen uptake, presentation, and germinal center formation via both cGAS-STING and NLRP3 activation. Accordingly, a colloidal manganese salt (Mn jelly, MnJ) was formulated to act not only as an immune potentiator but also as a delivery system to stimulate humoral and cellular immune responses, inducing antibody production and CD4+/CD8+ T-cell proliferation and activation by either intramuscular or intranasal immunization. When administered intranasally, MnJ also worked as a mucosal adjuvant, inducing high levels of secretory IgA. MnJ showed good adjuvant effects for all tested antigens, including T cell-dependent and T cell-independent antigens, such as bacterial capsular polysaccharides, thus indicating that it is a promising adjuvant candidate.

Contribution of bacterial extracellular polymeric substances (EPS) in surface water purification.

Naturally present aquatic microorganisms play an important role in water purification systems, such as the self-purification of surface waters. In this study, two water sources representing polluted surface water (Olympic Green; OG) and unpolluted surface water (Jingmi river; JM), were used to explore the self-purification of surface water by bacteria under different environmental conditions. The dominant bacterial community of OG and JM waters (both are Firmicutes and Proteobacteria) were isolated, cultured, and then used to carry out flocculation tests. Results showed that the flocculation ability of the dominant bacteria and extracellular polymeric substances (EPS) obtained from OG isolation was significantly greater than that from JM. Further examination illustrated that the main components of EPS were polysaccharides, which played an important role in improving the flocculation ability of bacteria. EPS from dominant cultural bacteria strains (OG1 and JM3) isolated from the two different sources lacked hydrophilic groups (e.g. COOH) and had a networked structure which are the main reasons to enhance the flocculation ability. The bacterial diversity and redundancy analysis (RDA) results also showed that microbial community composition is determined by water quality (SS, TOC, and NH4+), and different Bacteroidetes, Actinobacteria and Proteobacteria community structures can improve the water body's ability to remove environmental pollutants (such as SS, humic acid and fulvic acid). These findings provide new information showing how bacterial communities change with environmental factors while maintaining the purity of surface water.

Making the Enterobacterial Common Antigen Glycan and Measuring Its Substrate Sequestration.

The enterobacterial common antigen (ECA), a three-sugar repeat unit polysaccharide produced by Enterobacteriaceae family members, impacts bacterial outer membrane permeability, and its biosynthesis affects the glycan landscape of the organism. ECA synthesis impacts the production of other polysaccharides by reducing the availability of shared substrates, the most notable of which is the 55-carbon polyisoprenoid bactoprenyl phosphate (BP), which serves as a carrier for the production of numerous bacterial glycans including ECA, peptidoglycan, O-antigen, and more. Here, using a combination of in vitro enzymatic synthesis and liquid chromatography-mass spectrometry (LC-MS) analysis of bacterial lysates, we provide biochemical evidence for the effect on endogenous polyisoprenoid pools from cell culture that arises from glycan pathway disruption. In this work, we have cloned and expressed each gene involved in ECA repeat unit biosynthesis and reconstituted the pathway in vitro, providing LC-MS characterized standards for the investigation of cellular glycan-linked intermediates and BP. We then generated ECA deficient mutants in genes associated with production of the polysaccharide, which we suspected would accumulate materials identical to our standards. We found that indeed accumulated products from these cells were indistinguishable from our enzymatically prepared standards, and moreover we observed a concomitant decrease in cellular BP levels with each mutant. This work provides the first direct biochemical evidence for the sequestration of BP upon the genetic disruption of glycan biosynthesis pathways in bacteria. This work also provides methods for the direct assessment of both the ECA glycan, and a new understanding of the dynamic interdependence of the bacterial polysaccharide repertoire.

Contamination and Biofilm Formation of Foodborne and Opportunistic Pathogens in Yellow-Feathered Chicken Carcass.

This study aims to analyze the contamination and biofilm formation of foodborne and opportunistic pathogens in yellow-feathered chicken carcasses sampled in different seasons and to prove the relationship between biofilm-forming ability and bacterial extracellular polysaccharide (EPS) production. A total of 78 strains were isolated from chicken samples. The strains consisted of 30.8% Escherichia coli, 14.1% Pseudomonas aeruginosa, 12.8% Salmonella enteritidis, 12.8% Klebsiella pnenmoniae, 10.2% Enterobacter cloacae, 8.9% Proteus mirabilis, 5.1% Klebsiella oxytoca, 1.3% Staphylococcus aureus, and 1.3% Citrobacter braakii. Crystal violet staining assay revealed six strains with strong biofilm-forming ability, namely, E. coli S7, K. oxytoca B12, K. pnenmoniae B6, S. enteritidis H4, P. aeruginosa M5, and S. aureus G1, which showed had high abilities of cell motility and EPS production. Confocal laser scanning microscopy and scanning electron microscopy showed that all six strains can form mature biofilm architectures after 5 d of cultivation. This study may serve as a reference to control the contamination of foodborne pathogens in yellow-feathered chicken and enhance the quality and shelf life of these chicken products.

Changing expression patterns of TonB-dependent transporters suggest shifts in polysaccharide consumption over the course of a spring phytoplankton bloom

Algal blooms produce large quantities of organic matter that is subsequently remineralised by bacterial heterotrophs. Polysaccharide is a primary component of algal biomass. It has been hypothesised that individual bacterial heterotrophic niches during algal blooms are in part determined by the available polysaccharide substrates present. Measurement of the expression of TonB-dependent transporters, often specific for polysaccharide uptake, might serve as a proxy for assessing bacterial polysaccharide consumption over time. To investigate this, we present here high-resolution metaproteomic and metagenomic datasets from bacterioplankton of the 2016 spring phytoplankton bloom at Helgoland island in the southern North Sea, and expression profiles of TonB-dependent transporters during the bloom, which demonstrate the importance of both the Gammaproteobacteria and the Bacteroidetes as degraders of algal polysaccharide. TonB-dependent transporters were the most highly expressed protein class, split approximately evenly between the Gammaproteobacteria and Bacteroidetes, and totalling on average 16.7% of all detected proteins during the bloom. About 93% of these were predicted to take up organic matter, and for about 12% of the TonB-dependent transporters, we predicted a specific target polysaccharide class. Most significantly, we observed a change in substrate specificities of the expressed transporters over time, which was not reflected in the corresponding metagenomic data. From this, we conclude that algal cell wall-related compounds containing fucose, mannose, and xylose were mostly utilised in later bloom stages, whereas glucose-based algal and bacterial storage molecules including laminarin, glycogen, and starch were used throughout. Quantification of transporters could therefore be key for understanding marine carbon cycling.

OBTAINING OF BACTERIAL POLYSACCHARIDES

The aim of this work was to develop a technology for obtaining bacterial exopolysaccharides. The problem was solved by using the bacterial strain Halovibrio variabilis UzAS3 as a polysaccharide producer. The optimal parameters of bacterial cultivation have been determined. A technological scheme for obtaining bacterial polysaccharides has been developed, which includes such stages as the cultivation of bacteria, the separation of bacterial biomass from the culture liquid, desalting, precipitation, washing and drying. The obtained results show that bacteria have a high potential for the yield of bacterial exopolysaccharides (7.1-9.5 mg/ml).

Photoprotective effect of the fucose-containing polysaccharide FucoPol

This study reports the performance of FucoPol, a fucose-containing bacterial polysaccharide, as a photostable agent with high absorption yield at concentrations as low as 0.02 % (w/v). FucoPol is non-cytotoxic, efficiently protects from UVA and UVB at concentrations of 0.02–2 % (w/v) and 0.2–2 % (w/v), respectively, has over 94 % overall photostability up to 1.5 h of irradiation time. The lowest concentration studied (0.02 %, w/v) shows a sun protection factor (SPF) of 2.61 ± 0.08. The SPF-to-concentration unit ratio showed that FucoPol is about 60-fold more photoprotective than combinations of common organic and inorganic UV filters. In vitro radiation exposure experiments of adhered Vero epithelial and PM1 keratinocytic cells in the presence of 0.25 % (w/v) FucoPol further showed that cell viability was preserved, and delayed radiation-induced cell death was prevented. Overall, FucoPol outperforms common cosmetic biopolymers like xanthan and fucogel. These results are very promising for the development of bio-based sunscreen formulations.

The PTS Components in Klebsiella pneumoniae Affect Bacterial Capsular Polysaccharide Production and Macrophage Phagocytosis Resistance.

Capsular polysaccharide (CPS) is a crucial virulence factor for Klebsiella pneumoniae infection. We demonstrated an association of CPS production with two phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs). Deficiency of crr, encoding enzyme IIA of PTS, in K. pneumoniae enhanced the transcriptional activities of galF, wzi and gnd, which are in the cps gene cluster, leading to high CPS production. A crr mutant exhibited a higher survival rate in 1% hydrogen peroxide than the wild-type. The crr mutant showed less sensitivity to engulfment by macrophage (RAW 264.7) than the wild-type by observing the intracellular bacteria using confocal laser scanning microscopy (CLSM) and by calculating the colony-forming units (CFU) of intracellular bacteria. After long-term incubation, the survival rate of the intracellular crr mutant was higher than that of the wild-type. Deficiency of crr enhanced the transcriptional activities of etcABC which encodes another putative enzyme II complex of a PTS. Deletion of etcABC in the crr mutant reduced CPS production and the transcriptional activities of galF compared to those of the crr mutant. These results indicated that one PTS component, Crr, represses CPS production by repressing another PTS component, EtcABC, in K. pneumoniae. In addition, PTS plays a role in bacterial resistance to macrophage phagocytosis.

Development of a technology for polysaccharide-based film coating

This work describes the design of a film coating based on polysaccharides of bacterial and plant origin with several methods of application (by spraying, brushing and wrapping) on food. We used the bacterial polysaccharide xanthan as a thickener, the plant polysaccharide carboxymethyl cellulose (CMC) as a structurant, and the biological additive lecithin as a plasticizer. Depending on the film coating application method, concentrations of the studied polymers were selected with the same component composition. A technology for preparing the film coating was developed. The influence of heating (40, 50, 60, and 70 ° C) on the solubility of polysaccharide solutions with several component ratios was studied; the temperature of 50 ° C was chosen as resulted in a strong, stable, but not too viscous gel. The effect of concentration on the appearance and thickness of the film coatings was studied.

Injectable cuttlefish HAP and macromolecular fibroin protein hydrogel for natural bone mimicking matrix for enhancement of osteoinduction progression

The biological macromolecules like silk fibroin (FIB) based injectable hydrogel on mimicking the extracellular matrix for effective tissue regeneration. Fabricated hydrogel composed of bacterial polysaccharides, hyaluronic acid (HA), which networks filled with hexapeptides, osteogenic stimulants, trace elements in the form of hydroxyapatite (HAP) and FIB protein. Natural HAP is isolated from the abundant sources of cuttlefish bone, and it conjugate with HA. The formation of HA-HAP and HA-HAP-FIB confirms by the FTIR and RAMAN studies by a change in the carboxyl group. Hydrogel exhibited the porous structure due to the bending of FIB betta structure, and even distribution of HAP particles confirms by SEM investigation. The biocompatible effect of HA-HAP-FIB hydrogel, improves the cell growth for 96%, and it was high cell survival compared to HA and HA-HAP. The highest cell proliferation occurs at the range of 8 μg/mL hydrogel due to faster biomineralization of cuttlefish HAP. Hydrogel antibacterial properties against S. aureus and E.coli with the zone of inhibition above 20 mm were obtained. The internalized nano-HAP, fibroin matrix dynamic structure, and hydrophilic polymer anchoring support make composite hydrogel to mimic native ECM. The newly formed injectable nanocomposite hydrogels, suggesting to fill irregular bone defects and improves the tissue architecture.

Structure of a full-length bacterial polysaccharide co-polymerase

Lipopolysaccharides are important components of the bacterial cell envelope that among other things act as a protective barrier against the environment and toxic molecules such as antibiotics. One of the most widely disseminated pathways of polysaccharide biosynthesis is the inner membrane bound Wzy-dependent pathway. Here we present the 3.0 Å structure of the co-polymerase component of this pathway, WzzB from E. coli solved by single-particle cryo-electron microscopy. The overall architecture is octameric and resembles a box jellyfish containing a large bell-shaped periplasmic domain with the 2-helix transmembrane domain from each protomer, positioned 32 Å apart, encircling a large empty transmembrane chamber. This structure also reveals the architecture of the transmembrane domain, including the location of key residues for the Wzz-family of proteins and the Wzy-dependent pathway present in many Gram-negative bacteria, explaining several of the previous biochemical and mutational studies and lays the foundation for future investigations.

Specificity profile of αGal antibodies in αGalT KO mice as probed with comprehensive printed glycan array: Comparison with human anti-Galili antibodies.

The α1,3-galactosyltransferase gene-knockout (GalT KO) mice are able to produce natural anti-αGal antibodies apparently without any specific immunization. GalT KO mice are commonly used as a model immunological system for studying anti-αGal responses to Gal-positive xenografts in human. In this study, we compared the specificity of mouse and human αGal antibodies to realize the adequacy of the murine model. Using hapten-specific affinity chromatography antibodies against Galα1-3Galβ1-4GlcNAcβ epitope were isolated from both human and GalT KO mice blood sera. Specificity of isolated antibodies was determined using a printed glycan array (PGA) containing 400 mammalian glycans and 200 bacterial polysaccharides. The quantity of isolated specific anti-Galα antibodies corresponds to a content of <0.2% of total Ig, which is an order of magnitude lower than that generally assumed for both human and murine peripheral blood immunoglobulin, with a high predominance of IgM over IgG (95% vs 5%). Analysis using a printed glycan array has demonstrated that (a) antibodies from both species bind not only the Galα1-3Galβ1-4GlcNAcβ epitope, but also unrelated glycans; (b) particularly, for human (but not mouse) antibodies the best binders appear to be bacterial polysaccharides; (c) the profile of mouse antibodies is broader, it is noteworthy that they recognize a variety of human blood group B epitopes and even glycans without the α-galactosyl residue. We believe that the mouse model should be used cautiously in xenotransplantation experiments when the fine epitope specificity of antibodies is critical.

Incorporation of Apigenin and tt-Farnesol into dental composites to modulate the Streptococcus mutans virulence

ObjectivesThe aim of this in vitro study was to incorporate two anti-caries agents, Apigenin and tt-Farnesol, to resin composite and resin cement to reduce the virulence of Streptococcus mutans around dental restorations. MethodsApigenin (Api, 5 mM) and tt-Farnesol (Far, 5 mM) were added alone, together, and combined with fluoride (F). Biofilm of S. mutans was grown on composite discs, and the dry-weight, bacterial viability, and the polysaccharides (alkali-soluble, intracellular and water-soluble) were quantified. CLSM images of the S. mutans biofilm were obtained after three years of water-storage. The effect of the additions on the physicochemical properties and the composite colorimetric parameters were also analyzed. ResultsThe additions did not affect bacterial viability. Api alone and combined with Far or combined with Far and F decreased the bacterial dry-weight, alkali-soluble and intracellular polysaccharides. After three years, the composites containing the additions presented a greater EPS matrix on the top of biofilm. Statistical difference was obtained for the degree of conversion; however, the maximum polymerization rate and curing kinetics were unaffected by the additions. No difference was observed for the water-soluble polysaccharides, flexural strength, and elastic modulus. Api increased the yellowness of the composites. SignificanceApi, alone and combined, reduced the expression of virulence of S. mutans without jeopardizing the physicochemical properties of the composites.

The structure of human dermatan sulfate epimerase 1 emphasizes the importance of C5-epimerization of glucuronic acid in higher organisms.

Dermatan sulfate epimerase 1 (DS-epi1, EC 5.1.3.19) catalyzes the conversion of d-glucuronic acid to l-iduronic acid on the polymer level, a key step in the biosynthesis of the glycosaminoglycan dermatan sulfate. Here, we present the first crystal structure of the catalytic domains of DS-epi1, solved at 2.4 Å resolution, as well as a model of the full-length luminal protein obtained by a combination of macromolecular crystallography and targeted cross-linking mass spectrometry. Based on docking studies and molecular dynamics simulations of the protein structure and a chondroitin substrate, we suggest a novel mechanism of DS-epi1, involving a His/double-Tyr motif. Our work uncovers detailed information about the domain architecture, active site, metal-coordinating center and pattern of N-glycosylation of the protein. Additionally, the structure of DS-epi1 reveals a high structural similarity to proteins from several families of bacterial polysaccharide lyases. DS-epi1 is of great importance in a range of diseases, and the structure provides a necessary starting point for design of active site inhibitors.

Reduction of free polysaccharide contamination in the production of a 15-valent pneumococcal conjugate vaccine

Glycoconjugate vaccines are vaccines in which a bacterial polysaccharide antigen is conjugated to a carrier protein to enhance immunogenicity by promoting T cell-dependent immune response. However, the free (unreacted) polysaccharides remaining after the conjugation process can inhibit the immunogenicity of a conjugate vaccine. Thus, we aimed to reduce the unbound free polysaccharides in the polysaccharide-protein conjugation process for the development of a new 15-valent pneumococcal conjugate vaccine (PCV15) by varying some factors that may affect the conjugation results such as polysaccharide/protein ratio, polysaccharide size, and concentration of a coupling agent in a conjugation reaction mixture. Concentrations of a coupling agent, carbodiimide (EDAC), and a carrier protein (CRM197) used in PCV15 production, during the conjugation process, had little effect on the content of free polysaccharides. However, the size of the polysaccharide was identified as the critical factor to control the free polysaccharide content, with an inverse relationship observed between the molecular weight of the polysaccharide and the residual free polysaccharide content after conjugation. Based on these results, a new PCV15 with low free polysaccharide contamination was produced and tested for immunogenicity using a rabbit model to show that it induces similar level of immune responses in rabbits compared to a comparator vaccine Prevnar13®.

Gel point determination of gellan biopolymer gel from DC electrical conductivity

Abstract Gellan is an anionic bacterial polysaccharide, which in aqueous solution dissociates into a charged gellan polymer molecule containing carboxyl ions and counter ions and forms thermoreversible gel under appropriate conditions. In this study, we investigated the effect of polymer concentration, the concentration of added monovalent metallic ion, and temperature on the DC electrical conductivity of the gellan. Results suggest that the DC conductivity decreases with the increasing polymer concentrations and the added monovalent metallic ions. Such a decrease in DC conductivity can be attributed to the reduction of the mobility of counter ions due to the increase in the crosslinking density of the gellan network. DC conductivity of gellan gels was increased with temperature, which is interpreted as the dissolution of physically cross-linked networks, thus increasing the mobility of counter ions. The behavior of temperature variation of DC electrical conductivity reveals an abrupt change at a specific temperature, which can be considered a way to determine the gel point or sol–gel transition temperature T c of this thermoreversible biopolymer gel. This result agrees with that of rheological measurements where the viscosity showed a similar trend with temperature and diverges to infinity at the temperature close to T c.

A thermostable bacterial lytic polysaccharide monooxygenase with high operational stability in a wide temperature range

BackgroundLytic polysaccharide monooxygenases (LPMOs) are oxidative, copper-dependent enzymes that function as powerful tools in the turnover of various biomasses, including lignocellulosic plant biomass. While LPMOs are considered to be of great importance for biorefineries, little is known about industrial relevant properties such as the ability to operate at high temperatures. Here, we describe a thermostable, cellulose-active LPMO from a high-temperature compost metagenome (called mgLPMO10).ResultsMgLPMO10 was found to have the highest apparent melting temperature (83 °C) reported for an LPMO to date, and is catalytically active up to temperatures of at least 80 °C. Generally, mgLPMO10 showed good activity and operational stability over a wide temperature range. The LPMO boosted cellulose saccharification by recombinantly produced GH48 and GH6 cellobiohydrolases derived from the same metagenome, albeit to a minor extent. Cellulose saccharification studies with a commercial cellulase cocktail (Celluclast®) showed that the performance of this thermostable bacterial LPMO is comparable with that of a frequently utilized fungal LPMO from Thermoascus aurantiacus (TaLPMO9A).ConclusionsThe high activity and operational stability of mgLPMO10 are of both fundamental and applied interest. The ability of mgLPMO10 to perform oxidative cleavage of cellulose at 80 °C and the clear synergy with Celluclast® make this enzyme an interesting candidate in the development of thermostable enzyme cocktails for use in lignocellulosic biorefineries.

Covalent Immobilization of N-Acetylcysteine on a Polyvinyl Chloride Substrate Prevents Bacterial Adhesion and Biofilm Formation.

Biofilm formation and antimicrobial resistance at surgical implant sites result in high morbidity and mortality. Identifying novel molecules that inhibit biofilm formation to coat surgical biomaterials is essential. One such compound is N-acetylcysteine (NAC), a potent antioxidant precursor for glutathione, necessary in mammalian cells and known to disrupt/prevent biofilms. In this study, NAC was covalently immobilized onto functionalized polyvinyl chloride surfaces using plasma immersion ion implantation (PIII) treatment that achieves covalent binding without the need for linker groups. NAC immobilization was characterized using water contact angles, Fourier-transform infrared, and X-ray photoelectron spectroscopy techniques. Bacterial viability and biofilm formation on NAC surfaces were assessed using resazurin assays, phase contrast microscopy, and colony counting experiments. Effect of NAC on bacterial polysaccharide production and DNA cleaving was investigated using the phenol-sulfuric acid method and the Qubit fluorometer. Surface thermodynamics between the NAC coating and bacterial cells were measured using the Lewis acid-base method. Surface characterization techniques demonstrated superficial changes after PIII treatment and subsequent covalent NAC immobilization. NAC-coated surfaces significantly reduced biofilm viability and the presence of Gram-negative and Gram-positive bacteria. NAC also decreased polysaccharide production and degraded DNA. This led to unfavorable conditions for biofilm formation on NAC-coated surfaces, as demonstrated by surface thermodynamic analysis. NAC-coated surfaces showed no cytotoxicity to human fibroblast cells. This study has successfully utilized NAC as an antibiofilm coating, which may pave the way for improved prophylactic coatings on medical implant devices in the future.

Enhancing the geotechnical properties of soil using xanthan gum—an eco-friendly alternative to traditional stabilizers

Several soil stabilization techniques have been adopted to favorably modify the geotechnical properties like hydraulic conductivity, strength, and compressibility of soil. In this study, xanthan gum (XG), an anionic bacterial extracellular polysaccharide is used to modify the geotechnical properties of the soil, particularly its strength and hydraulic conductivity. The addition of xanthan gum to soil improves its strength and stiffness and also decreases its hydraulic conductivity. The addition of xanthan gum induces polymer cross-linking, forms interconnected network of hydrogels in the voids of the soil matrix and causes preferential adsorption of the biopolymer molecules and cations on the soil surface. These interactions between the soil and the biopolymer alter the geotechnical properties of the treated soil matrix favorably. The decrease in permeability is nearly 1000 times with a small addition of 0.25% xanthan gum to the soil. Xanthan gum tends to aggregate the particles at lower concentration and at higher concentrations forms more viscous hydrogels that fill the pore spaces and clogs the pores. Strength also shows a similar increase and hence xanthan gum can be recommended for soil stabilization.

DYNAMIC BEHAVIOR OF METHYLENE BLUE ONE. coliK30 CAPSULAR POLYSACCHARIDE USING LAYER BY LAYER SELF-ASSEMBLY TECHNIQUE

Layer-by-layer (LbL) self-assembly is one of the unique methods for fabrication of ultra-thin film. This technique has immense application aross many fields in today’s life. In this paper, we have presented the fabrication of the thin film comprising of antifungal and antibacterial cationic dye methylene blue with negatively charged E. coli K30 bacterial polysaccharide sharing with PAH. The multilayers were characterized by UV–Vis absorption spectra and AFM. Various photophysical behaviors were investigated. Such studies provide a new light in the research of layer by layer self-assembly technique.