A Screening Approach to Assess the Impact of Various Commercial Sources of Crude Marine λ-Carrageenan on the Production of Oligosaccharides with Anti-heparanase and Anti-migratory Activities.

Oligosaccharides derived from λ-carrageenan (λ-COs) are gaining interest in the cancer field. They have been recently reported to regulate heparanase (HPSE) activity, a protumor enzyme involved in cancer cell migration and invasion, making them very promising molecules for new therapeutic applications. However, one of the specific features of commercial λ-carrageenan (λ-CAR) is that they are heterogeneous mixtures of different CAR families, and are named according to the thickening-purpose final-product viscosity which does not reflect the real composition. Consequently, this can limit their use in a clinical applications. To address this issue, six commercial λ-CARs were compared and differences in their physiochemical properties were analyzed and shown. Then, a H2O2-assisted depolymerization was applied to each commercial source, and number- and weight-averaged molar masses (Mn and Mw) and sulfation degree (DS) of the λ-COs produced over time were determined. By adjusting the depolymerization time for each product, almost comparable λ-CO formulations could be obtained in terms of molar masses and DS, which ranged within previously reported values suitable for antitumor properties. However, when the anti-HPSE activity of these new λ-COs was screened, small changes that could not be attributed only to their small length or DS changes between them were found, suggesting a role of other features, such as differences in the initial mixture composition. Further structural MS and NMR analysis revealed qualitative and semi-quantitative differences between the molecular species, especially in the proportion of the anti-HPSE λ-type, other CARs types and adjuvants, and it also showed that H2O2-based hydrolysis induced sugar degradation. Finally, when the effects of λ-COs were assessed in an in vitro migration cell-based model, they seemed more related to the proportion of other CAR types in the formulation than to their λ-type-dependent anti-HPSE activity.

Factors other than metalloprotease are required for full virulence of French Vibrio tubiashii isolates in oyster larvae.

Vibrio tubiashii is a marine pathogen isolated from larval and juvenile bivalve molluscs that causes bacillary necrosis. Recent studies demonstrated the isolation of this species in a French experimental hatchery/nursery affecting Crassostrea gigas spat in 2007. Here, using larvae of C. gigas as an interaction model, we showed that the French V. tubiashii is virulent to larvae and can cause bacillary necrosis symptoms with an LD50 of about 2.3 × 10(3) c.f.u. ml(-1) after 24 h. Moreover, complete or gel permeation HPLC fractionated extracellular products (ECPs) of this strain appeared toxic to larvae. MS-MS analysis of the different ECP fractions revealed the existence of an extracellular metalloprotease and other suspected virulence factors. This observation is also supported by the expression level of some potential virulence factors. The overall results suggest that the pathology caused by the French V. tubiashii in C. gigas oysters is caused by a group of toxic factors and not only the metalloprotease.

First description of French V. tubiashii strains pathogenic to mollusk: II. Characterization of properties of the proteolytic fraction of extracellular products.

Extracellular products (ECPs) of the French Vibrio tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1,10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Taken together, our results suggest that this (these) zinc metalloprotease(s) might contribute to the impairment of hemocyte immunological functions; however, their direct involvement in ECPs toxicity remains to be demonstrated.

First description of French V. tubiashii strains pathogenic to mollusk: I. Characterization of isolates and detection during mortality events.

Nine dominant bacterial isolates were obtained from different batches of Crassostrea gigas spat experiencing high mortality rates in a French experimental hatchery/nursery in 2007. Using phenotypic analysis combined with multilocus sequence analysis, the isolates were shown to be genetically close to the Vibrio tubiashii type strain. Based on (1) analyses of the recA gene sequences; (2) the results of DNA-DNA hybridization assays between 07/118 T2 (LMG 27884=CECT 8426), which is a representative strain, and the V. tubiashii type strain (69%); and (3) phenotypic traits, the bacteria were classified in a group close to American V. tubiashii strain. Its virulence (70% of mortalities) and the toxicity of the extracellular products of 07/118 T2 was demonstrated (41% of mortalities). Moreover, a QPCR diagnostic tool targeting the gyrB gene was developed to investigate the epidemiological significance of V. tubiashii in French oyster mortality outbreaks recorded by the national surveillance network. Of the 21 batches originating from hatcheries, only two were positive, whereas V. tubiashii DNA could not be detected in any of the batches of moribund animals collected in field/outdoor facilities. These results demonstrate the existence of a group of virulent V. tubiashii in France that episodically infect C. gigas.