Search

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.

The identification of the mechanisms by which marine dissolved organic matter (DOM) is produced and regenerated is critical to develop robust prediction of ocean carbon cycling. Polysaccharides represent one of the main constituents of marine DOM and their degradation is mainly attributed to polysaccharidases derived from bacteria. Here, we report that marine viruses can depolymerize the exopolysaccharides (EPS) excreted by their hosts using 5 bacteriophages that infect the notable EPS producer, Cobetia marina DSMZ 4741. Degradation monitorings as assessed by gel electrophoresis and size exclusion chromatography showed that 4 out of 5 phages carry structural enzymes that depolymerize purified solution of Cobetia marina EPS. The depolymerization patterns suggest that these putative polysaccharidases are constitutive, endo-acting, and functionally diverse. Viral adsorption kinetics indicate that the presence of these enzymes provides a significant advantage for phages to adsorb onto their hosts upon intense EPS production conditions. The experimental demonstration that marine phages can display polysaccharidases active on bacterial EPS lead us to question whether viruses could also contribute to the degradation of marine DOM and modify its bioavailability. Considering the prominence of phages in the ocean, such studies may unveil an important microbial process that affects the marine carbon cycle.

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.

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.

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 alpha-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.

Aims: To identify Vibrio vulnificus, Vibrio cholerae and Vibrio alginolyticus using standardized DNA extraction method and real-time PCR assays, among a large number of bacterial strains isolated from marine environment. Methods and Results: Methods for DNA extraction and real-time PCR were standardized to identify a large number of Vibrio spp. strains isolated through regular collection campaigns of environmental samples. Three real-time PCR assays were developed from a multiplex PCR, targeting V. vulnificus, V. cholerae and V. alginolyticus on the dnaJ gene. After testing their specificity, these systems were applied for the identification of 961 strains isolated at 22°C (446 strains) and 37°C (515 strains) in September 2009. The predominance of V. alginolyticus (82·6%) among the Vibrio spp. strains isolated at 37°C was shown. At 22°C, only 1·6% of the strains were identified by PCR and they were V. alginolyticus. Conclusions: Reproducible and specific real-time PCR assays combined to a DNA extraction method on microplates were used to constitute a large environmental Vibrio strains collection and to identify and detect potential human pathogenic Vibrio isolated at 37°C. For environmental strains isolated at 22°C, because of the higher species diversity, other approaches, like sequencing, should be chosen for identification. Significance and Impact of the Study: The protocol developed in this study provides an appropriate and rapid screening tool to identify a large number of bacterial strains routinely isolated from the environment in long-term studies.

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.