NMR Analyses of the Enzymatic Degradation End-Products of Diabolican: The Secreted EPS of Vibrio diabolicus CNCM I-1629.

Diabolican, or HE800, is an exopolysaccharide secreted by the non-pathogenic Gram-negative marine bacterium Vibrio diabolicus (CNCM I-1629). This polysaccharide was enzymatically degraded by the Bacteroides cellulosilyticus WH2 hyaluronan lyase. The end products were purified by size-exclusion chromatography and their structures were analyzed in depth by nuclear magnetic resonance (NMR). The oligosaccharide structures confirmed the possible site of cleavage of the enzyme showing plasticity in the substrate recognitions. The production of glycosaminoglycan-mimetic oligosaccharides of defined molecular weight and structure opens new perspectives in the valorization of the marine polysaccharide diabolican.

Structure of the Polysaccharide Secreted by Vibrio alginolyticus CNCM I-5035 (Epidermist 4.0TM).

Vibrio alginolyticus (CNCM I-5035) secretes an exopolysaccharide used as ingredient in cosmetic industry under the trademark Epidermist 4.0TM. It is appreciated for its ability to improve the physical and chemical barrier functions of the skin by notably increasing the keratinocyte differentiation and epidermal renewal. Composition analyses and in depth characterization of the polysaccharides as well as oligosaccharides obtained by mild acid hydrolyses revealed that it was composed of a repetition unit of three residues: d-galactose (d-Gal), d-N-acetylglucosamine (GlcNAc) and l-N-acetylguluronic acid, of which 30% (M/M) was acetylated in position 3. The complete structure of the polysaccharide was resolved giving the repetition unit: [→3)-α-d-Gal-(1→4)-α-l-GulNAcA/α-l-3OAc-GulNAcA-(1→4)-ß-d-GlcNAc-(1→].

Structure of the Exopolysaccharide Secreted by a Marine Strain Vibrio alginolyticus.

Vibrio alginolyticus (CNCM I-4151) secretes an exopolysaccharide whose carbohydrate backbone is decorated with amino acids, likely conferring its properties that are appreciated in cosmetics. Here, the secreted polysaccharide of another strain of V. alginolyticus (CNCM I-5034) was characterized by chromatography and one- and two-dimensional NMR spectroscopy experiments. The structure was resolved and shows that the carbohydrate backbone is made of four residues: D-galactose (Gal), D-galacturonic acid (GalA) D-N-acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA), forming a tetrasaccharide repetition unit [→4)-ß-d-GlcA-(1→3)-α-d-Gal-(1→3)-α-d-GalA-(1→3)-ß-GlcNAc(1→]. GlcA is derivatized with a lactate group giving 'nosturonic acid', and GalA is decorated with the amino acid alanine.

Depolymerization of Pseudomonas stutzeri exopolysaccharide upon fermentation as a promising production process of antibacterial compounds.

Many researchers have focused on high molecular weight (Mw) exopolysaccharides (EPS) as a source of potentially bioactive lower Mw derivatives. Therefore, it is of interest to find means for efficient and safe production of depolymerized-polymer derivatives. Exopolysaccharide-depolymerization products (EDP) varying in molecular weight were recovered from fermentative depolymerization of a native EPS produced by Pseudomonas stutzeri AS22. Following their purification and physicochemical characterization, the antibacterial activity of EDP on food spoilage and food poisoning microorganisms was evaluated through the measurement of the inhibition zone diameter, the half maximal (IC50) and the minimal (MIC) inhibitory concentrations. Our results indicate that the lower the Mw, the higher will be the effectiveness of EDP on reducing Gram-negative bacteria growth and the opposite trend was observed in the case of Gram-positive bacteria. EDP bioactivities may provide novel insights into the potentiality of P. stutzeri EPS and its derivatives to be used as functional-food components.

Structure of an Amino Acid-Decorated Exopolysaccharide Secreted by a Vibrio alginolyticus Strain.

Vibrio alginolyticus (CNCM I-4994) secretes an exopolysaccharide that can be used as an ingredient in cosmetic applications. The structure was resolved using chromatography and one- and two-dimensional NMR spectroscopy experiments. The results show that the carbohydrate backbone is made of two residues: d-galacturonic acid and N-acetyl-d-glucosamine (GlcNac), which together constitute a tetrasaccharide repetition unit: [→3)-α-d-GalA-(1→4)-α-d-GalA-(1→3)-α-d-GalA-(1→3)-ß-GlcNAc(1→]. Two amino acids, alanine and serine, are linked to GalA residues via amido linkages. The position and the distribution of the amino acids were characterized by two-dimensional NMR spectroscopy. To our knowledge, this is the first description of a structure for a marine exopolysaccharide decorated with an amino acid.

Accumulation of nanoparticles in "jellyfish" mucus: a bio-inspired route to decontamination of nano-waste.

The economic and societal impacts of nano-materials are enormous. However, releasing such materials in the environment could be detrimental to human health and the ecological biosphere. Here we demonstrate that gold and quantum dots nanoparticles bio-accumulate into mucus materials coming from natural species such as jellyfish. One strategy that emerges from this finding would be to take advantage of these trapping properties to remove nanoparticles from contaminated water.

The marine bacteria Cobetia marina DSMZ 4741 synthesizes an unexpected K-antigen-like exopolysaccharide.

We have studied the exopolysaccharide produced by Cobetia marina DSMZ 4741, a marine bacterium isolated from coastal seawater. This strain is able to produce a polysaccharide in presence of carbon sources as glucose, mannitol and alginate. The maximum production occurs in aerobic condition, during the end of the exponential phase. The polymer is a non-viscous, acidic heteropolysaccharide of 270kDa constituted of a repeating unit of: This kind of chemical structure is generally related to K-antigen polysaccharide of pathogenic Escherichia coli strains. This is the first time this type of EPS is described from a marine bacterium. Moreover the polysaccharide exhibits a pyruvate substitution on its 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue never encountered before. The discovery of such an unexpected EPS with high biotechnological potential is a new incentive for a better exploration of bioactive marine resources.

Characterization and structure of the polysaccharide produced by Pseudomonas fluorescens strain TF7 isolated from an arid region of Algeria.

Many bacteria possess a natural ability to synthesize and excrete exopolysaccharides which are widely varied in structure and function. These bacteria have the ability to solubilize inorganic phosphorus, which is important to promote growth and increase crop yields. The objective of this study is to select an adaptive strain to the constraints of erratic rainfall and large temperature variations and to determine the possible synergistic effects of its EPS and organic acid on tricalcium phosphate (TCP) solubilization. The strain TF7 isolated from an arid region of Algeria was characterized on the basis of its morphological and physiological traits. Polysaccharide production and the phosphate-solubilizing activity of the strain were evaluated using sucrose and tricalcium phosphate. This EPS was studied by sugar analysis as well as proton NMR spectra. The 16S rRNA gene sequence of this strain shared a similarity of more than 96% with Pseudomonas fluorescens. The maximum polysaccharide productivity was estimated at 4.5g·L(-1) after 5 days. The analyzed sugar was comprised of fructose, glucose, and mannose in a ratio of 4:1:0.6. NMR spectra indicated that the polysaccharide produced by the strain was levan with ß-(2→6)-linked fructose units in accordance with the generally accepted structure. The strain TF7 solubilizes phosphate and forms a clear halo around the colony. The phosphate-solubilizing index is 2.33.

Exopolysaccharide biosynthesis and biodegradation by a marine hydrothermal Alteromonas sp. strain.

Alteromonas macleodii subsp. fijiensis biovar deepsane is a deep-sea ecotype exopolysaccharide-producing bacteria isolated from the polychaete annelid Alvinella pompejana. The high molecular weight biopolymer HYD657 produced by this strain, is the first marine exopolysaccharide (EPS) to be commercialized for cosmetic use. Depolymerization methods are necessary to elucidate the complete structure of this EPS and to generate potentially bioactive oligosaccharides. Enzymatic methods are useful for elucidating polysaccharide structure because they specifically cleave glycosidic bonds and do not require harsh chemical conditions. The HYD657 EPS is structurally complex and no commercially available enzymes are able to effectively degrade it. Here, we present the first results on the endogenous enzymatic depolymerization of a marine EPS of biotechnological interest by the producing strain. Enzymatic activity was detected in the bacterial lysate and was able to decrease the apparent molecular size of the EPS, releasing mainly oligosaccharides. The reduced form of the native polysaccharide showed a slightly modified osidic composition, particularly in terms of molar ratio. Several exoglycosidase activities were measured in the bacterial lysate using paranitrophenyl-osides.

Rhelogical, dermal wound healing and in vitro antioxidant properties of exopolysaccharide hydrogel from Pseudomonas stutzeri AS22.

The in vitro antioxidant activity and the in vivo wound healing performance of the exopolysaccharide EPS22, produced by Pseudomonas stutzeri AS22, were investigated. Antioxidant activity was evaluated by three different tests. The scavenging effect on DPPH radicals at a concentration of 1mg/ml was 80±1.41%. The reducing power reached a maximum of 1.26±0.02 at 2 mg/ml. Moreover, EPS22 showed good chelating ability and chelated almost 88.5±0.7% of ferrous ions at 0.75 mg/ml. The rheological characterization of EPS22 gel (0.5%) showed a pseudoplastic behavior, high elasticity, good mechanical strength and stability with high water-absorption ability. The application of the EPS22 gel on dermal full-thickness excision wounds in a rat model every two days, enhanced significantly wound healing activity and a total closure was achieved after 12 days of wound induction. Further, histological examination of biopsies showed advanced tissue regeneration, characterized by the presence of well-organized stratum of both derma and epidermis.

Characterization of the secretomes of two vibrios pathogenic to mollusks.

Vibrio tapetis causes the brown ring disease in the Japanese clam Ruditapes philippinarum while Vibrio aestuarianus is associated with massive oyster mortalities. As extracellular proteins are often associated with the virulence of pathogenic bacteria, we undertook a proteomic approach to characterize the secretomes of both vibrios. The extracellular proteins (ECPs) of both species were fractionated by SEC-FPLC and in vitro assays were performed to measure the effects of each fraction on hemocyte cellular parameters (phagocytosis and adhesion). Fractions showing a significant effect were subjected to SDS-PAGE, and proteins were identified by nano LC-MS/MS. 45 proteins were identified for V. aestuarianus and 87 for V. tapetis. Most of them belonged to outer membrane or were periplasmic, including porins or adhesins that were already described as virulence factors in other bacterial species. Others were transporter components, flagella proteins, or proteins of unknown function (14 and 15 respectively). Interestingly, for V. aestuarianus, we noted the secretion of 3 extracellular enzymes including the Vam metalloprotease and two other enzymes (one putative lipase and one protease). For V. tapetis, we identified five extracellular enymes, i.e. two different endochitinases, one protease, one lipase and an adhesin. A comparison of both secretomes also showed that only the putative extracellular lipase was common to both secretomes, underscoring the difference in pathogenicity mechanisms between these two species. Overall, these results characterize for the first time the secretomes of these two marine pathogenic vibrios and constitute a useful working basis to further analyze the contribution of specific proteins in the virulence mechanisms of these species.

Purification and structural data of a highly substituted exopolysaccharide from Pseudomonas stutzeri AS22.

Pseudomonas stutzeri AS22, when grown on media containing starch and yeast extract and incubated at 30 °C and 200 rpm for 24h, was found to produce an acidic and high-molecular mass exopolysaccharide (EPS22). The EPS22 was purified and a yield of 1.3g/l was achieved. The average molecular mass of the EPS22 was determined by high-performance size-exclusion chromatography (HPSEC) and showed an average molecular mass of 9.9 × 10(5)Da and a polydispersity index Mw/Mn (Mw, weight-average and Mn, number-average) of 1.197 ± 0.015. Structural data of this EPS22 were determined using a combination approach including monosaccharide composition (HPAEC-PAD and GLC), methylation analysis (GC-MS) and NMR spectroscopy analysis. EPS22 was found to be a complex heteropolysaccharide with a repeating unit mainly composed of glucose, mannose and lactyl rhamnose in a molar ratio of 1:1.1:0.7. The acidic nature of the polysaccharide is due to the presence of three non-osidic substituents consisting of a lactyl, acetyl, and pyruvyl groups.

Exopolysaccharides isolated from hydrothermal vent bacteria can modulate the complement system.

The complement system is involved in the defence against bacterial infection, or in the elimination of tumour cells. However, disturbances in this system contributes to the pathogenesis of various inflammatory diseases. The efficiency of therapeutic anti-tumour antibodies is enhanced when the complement system is stimulated. In contrast, cancer cells are able to inhibit the complement system and thus proliferate. Some marine molecules are currently being developed as new drugs for use in humans. Among them, known exopolyssacharides (EPSs) generally originate from fungi, but few studies have been performed on bacterial EPSs and even fewer on EPSs extracted from deep-sea hydrothermal vent microbes. For use in humans, these high molecular weight EPSs must be depolymerised. Furthermore, the over-sulphation of EPSs can modify their biological activity. The aim of this study was to investigate the immunodulation of the complement system by either native or over-sulphated low molecular weight EPSs isolated from vent bacteria in order to find pro or anti-activators of complement.

Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling

Oligosaccharides are implicated in the development of the immune response notably in complement activation. Anti-tumoural immunotherapy by monoclonal antibodies (mAbs) offers some advantages to chemotherapy including cell targeting but some of them are inefficient to generate cytotoxicity dependent complement (CDC) known to be important in the antibody’s efficacy. The aim of this study is to give a CDC activity of mAb by linkage of a complement activating oligosaccharide to this antibody via a hetero-bifunctional linker allowing control of the conjugation reaction. We worked on non Hodgkin Burkitt’s lymphoma as cancer source, Fab fragments of rituximab devoid of complement activity as mAb and the trisaccharide Gal alpha(1→3)Gal beta(1→4)GlcNAc as immunogenic glycan. The bioconjugate Fab-Gal was characterized by biochemical methods and we demonstrated that the α-Gal epitope was recognized by seric immunoglobulins. After checking the recognition capacity of the Fab-Gal conjugate for the CD20 epitope, in vitro assays were performed to evaluate the activation of the complement cascade by the Fab-Gal conjugate. The effect of this bioconjugate was confirmed by the evaluation of the proliferation response of Burkitt’s cell line. The relative facility realization of this strategy represents new approaches to increase activities of mAbs.

Medium-throughput profiling method for screening polysaccharide-degrading enzymes in complex bacterial extracts.

Polysaccharides are the most abundant and the most diverse renewable materials found on earth. Due to the stereochemical variability of carbohydrates, polysaccharide-degrading enzymes - i.e. glycoside hydrolases and polysaccharide lyases - are essential tools for resolving the structure of these complex macromolecules. The exponential increase of genomic and metagenomic data contrasts sharply with the low number of proteins that have ascribed functions. To help fill this gap, we designed and implemented a medium-throughput profiling method to screen for polysaccharide-degrading enzymes in crude bacterial extracts. Our strategy was based on a series of filtrations, which are absolutely necessary to eliminate any reducing sugars not directly generated by enzyme degradation. In contrast with other protocols already available in the literature, our method can be applied to any panel of polysaccharides having known and unknown structures because no chemical modifications are required. We applied this approach to screen for enzymes that occur in Pseudoalteromonas carrageenovora grown in two culture conditions.

Floridoside extracted from the red alga Mastocarpus stellatus is a potent activator of the classical complement pathway.

Many biological properties of algae have been found to have useful applications in human health, particularly in the fields of oncology and immunology. Floridoside, extracted from the red alga Mastocarpus stellatus, has a structure similar to the xenoantigen Gal alpha 1-3 Gal. This xenoantigen has been described to induce a high immune response in human xenografts and is mediated by natural anti-gal antibodies that activate the classical complement pathway. Based on this property, we analyzed the potential activities of floridoside on the immune system. We demonstrated that floridoside activates a complement cascade via the classical complement pathway, through the recruitment and activation of natural IgM. This algal molecule could represent an important step in the development of a potent new anticomplementary agent for use in therapeutic complement depletion.

Characterization of mannuronan C-5-epimerase genes from the brown alga Laminaria digitata.

Alginate is an industrially important polysaccharide obtained commercially by harvesting brown algae. The final step in alginate biosynthesis, the epimerization of beta-1,4-d-mannuronic acid to alpha-1,4-l-guluronic acid, a structural change that controls the physicochemical properties of the alginate, is catalyzed by the enzyme mannuronan C-5-epimerase. Six different cDNAs with homology to bacterial mannuronan C-5-epimerases were isolated from the brown alga Laminaria digitata (Phaeophyceae). Hydrophobic cluster analysis indicated that the proteins encoded by the L. digitata sequences have important structural similarities to the bacterial mannuronan C-5-epimerases, including conservation of the catalytic site. The expression of the C-5-epimerase genes was examined by northern-blot analysis and reverse transcriptase-polymerase chain reaction in L. digitata throughout a year. Expression was also monitored in protoplast cultures by northern and western blot, reverse transcriptase-polymerase chain reaction, and activity measurements. From both the structural comparisons and the expression pattern, it appears that the cDNAs isolated from L. digitata encode functional mannuronan C-5-epimerases. The phylogenetic relationships of the bacterial and brown algal enzymes and the inferences on the origin of alginate biosynthetic machinery are discussed.

A novel, highly viscous polysaccharide excreted by an alteromonas isolated from a deep-sea hydrothermal vent shrimp.

A deep-sea, mesophilic, aerobic, and heterotrophic microorganism, able to produce an extracellular polysaccharide, was isolated from a shrimp collected near an active hydrothermal vent of the Mid-Atlantic Ridge. On the basis of phenotypic and phylogenetic analyses and DNA/DNA relatedness, this strain could be assigned to the species Alteromonas macleodii as a variant of the fijiensis subspecies. It was selected for its ability to exhibit a swarming mucoid phenotype on specific media. The bacterium secreted, under laboratory conditions, an extremely viscous exopolysaccharide consisting of glucose, galactose as neutral sugars, and glucuronic, galacturonic acids as uronic acids, along with pyruvate and acetate as main substituents.

Dynamic Light Scattering Evidence for a Ligand-Induced Motion between the Two Domains of Glucoamylase G1 of Aspergillus niger with Heterobivalent Substrate Analogues.

Heterobifunctional ligands that bind at the same time to the catalytic domain and to the starch-binding domain of glucoamylase induce a conformational change of the protein, as shown by dynamic light scattering. The ligands consist of acarbose and ß-cyclodextrin linked together by oligoethylene glycols of variable length (see the schematic diagram).