The Diverse Transformer (Trf) Protein Family in the Sea Urchin Paracentrotus lividus Acts through a Collaboration between Cellular and Humoral Immune Effector Arms.

Sea urchins are long-living marine invertebrates with a complex innate immune system, which includes expanded families of immune receptors. A central immune gene family in sea urchins encodes the Transformer (Trf) proteins. The Trf family has been studied mainly in the purple sea urchin Strongylocentrotus purpuratus. Here, we explore this protein family in the Mediterranean Sea urchin Paracentrotus lividus. The PlTrf genes and predicted proteins are highly diverse and show a typical Trf size range and structure. Coelomocytes and cell-free coelomic fluid from P. lividus contain different PlTrf protein repertoires with a shared subset, that bind specifically to E. coli. Using FACS, we identified five different P. lividus coelomocyte sub-populations with cell surface PlTrf protein expression. The relative abundance of the PlTrf-positive cells increases sharply following immune challenge with E. coli, but not following challenge with LPS or the sea urchin pathogen, Vibrio penaeicida. Phagocytosis of E. coli by P. lividus phagocytes is mediated through the cell-free coelomic fluid and is inhibited by blocking PlTrf activity with anti-SpTrf antibodies. Together, our results suggest a collaboration between cellular and humoral PlTrf-mediated effector arms in the P. lividus specific immune response to pathogens.

Phylogeny and Antagonistic Activities of Culturable Bacteria Associated with the Gut Microbiota of the Sea Urchin (Paracentrotus lividus).

In this study, we have investigated the phylogeny and the antagonistic interactions of culturable bacteria isolated from the sea urchin Paracentrotus lividus collected from Aber and Morgat, both located in Crozon peninsula, France. Bacteria were isolated from the gastrointestinal tracts of ten specimens by using conventional culture-dependent method and then investigated by using phylogenetic analysis based on 16S rRNA gene sequence comparisons. Assays for antagonistic interactions among the bacterial strains were performed; bacteria (including at least one strain representative of each OTU identified) were screened for antimicrobial substance production. So, 367 bacterial strains were isolated on marine-agar. On the basis of morphological characteristics, 180 strains were sequenced and 94 OTUs were classified. The dominant phyla were Proteobacteria, Firmicutes and Actinobacteria, with a high abundance of the strains belonging to the genus Psychrobacter. From the antagonistic interactions assays, it could be determined that 22.7% strains were positive for at least one antagonism interaction, 18.3% of them isolated from the sea urchins collected in Morgat. We hypothesize that the bacteria isolated in this study may represent the transitory microbiota of the gastrointestinal tract of P. lividus, and that this microbiota may be related to the diet of this marine invertebrate. Furthermore, our results suggest that chemical antagonism could play a significant role in shaping the bacterial communities within gastrointestinal tract of the sea urchins. In addition, most isolated bacteria may have promising biotechnology applications.

Composition and geographic variation of the bacterial microbiota associated with the coelomic fluid of the sea urchin Paracentrotus lividus.

In the present work, culture-based and culture-independent investigations were performed to determine the microbiota structure of the coelomic fluid of Mediterranean sea urchin Paracentrotus lividus individuals collected from two distinct geographical sites neighboring a high-density population bay and a nature reserve, respectively. Next Generation Sequencing analysis of 16S rRNA gene (rDNA) showed that members of the Proteobacteria, Bacteroidetes and Fusobacteria phyla, which have been previously reported to be commonly retrieved from marine invertebrates, dominate the overall population of microorganisms colonizing this liquid tissue, with minority bacterial genera exhibiting remarkable differences among individuals. Our results showed that there is a correlation between microbiota structure and geographical location of the echinoderm collection site, highlighting over-representation of metagenomic functions related to amino acid and bioactive peptides metabolism in specimens inhabiting the nature reserve. Finally, we also described the developmental delay and aberrations exhibited by sea urchin embryos exposed to distinct bacterial isolates, and showed that these defects rely upon hydrophilic compound(s) synthesized by the bacterial strains assayed. Altogether, our findings lay the groundwork to decipher the relationships of bacteria with sea urchins in their aquatic environment, also providing an additional layer of information to understand the biological roles of the coelomic fluid.

Shewanella harboring antimicrobial and copper resistance genes in sea urchins (Paracentrotus lividus) from the Crozon peninsula (Brittany, France).

Shewanella is a genus of aquatic non-fermenting Gram-negative bacteria with increasing numbers of reports of infections in humans and appearance of antimicrobial resistant strains. Cases of infection show a relatively strong association with seafood consumption or exposure to seawater. This study aimed to analyze Shewanella spp. isolated from the sea urchin Paracentrotus lividus collected from the Crozon peninsula (France) with the intention of obtaining insights into the role of this genus as a reservoir of antimicrobial and heavy metal resistance genes. Five among seven Shewanella isolates were resistant to antimicrobials, mainly to broad spectrum beta-lactams. Four isolates displayed multiple resistance to at least three of these antimicrobial classes: broad spectrum beta-lactams, aminoglycosides, macrolide, quinolones and/or tetracycline. Three antimicrobial resistance genes were detected in just one isolate encoding resistance to beta-lactam (blaSHV and blaTEM-1) and macrolide (ermB). In addition, the copper resistance gene cusB, was observed in this isolate which is also a plasmid carrier. Another copper resistance encoding gene, copA, was found among the isolates. These results indicate that the multidrug-resistant (MDR) Shewanella isolates and resistance genes could be potential risks to public health, due to the carrying of these MDR bacteria by sea urchins through human consumption.

Characterization of the bacterial communities associated with the bald sea urchin disease of the echinoid Paracentrotus lividus.

The microbial communities involved in the bald sea urchin disease of the echinoid Paracentrotus lividus are investigated using culture-independent techniques. Lesions of diseased specimens from two locations in France, La Ciotat (Mediterranean Sea) and Morgat (Atlantic Ocean), are examined by Scanning Electron Microscopy (SEM) and the diversity of their microbiota is analysed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene clones libraries construction. Microscopic observations demonstrated that only the central area of the lesions is invaded by bacteria but not the peripheral zone and the surrounding healthy tissues. Molecular analysis identified at least 24 bacterial genomospecies in bald sea urchin lesions: 5 are Alphaproteobacteria, 10 are Gammaproteobacteria, 8 are CFB bacteria and 1 is a Fusobacteria. Out of them, 4 are observed in both locations while 10 occur only in the Atlantic Ocean and 10 only in the Mediterranean Sea. Gammaproteobacteria are the most represented in clones libraries from both locations, with respectively 65% and 43% of the total clones. CFB and Alphaproteobacteria accounted for the majority of the remaining clones and were detected by DGGE in virtually all samples from both stations. Our results demonstrate that bacterial communities observed on diseased individuals of the same echinoid species but originating from distinct locations are not similar and thus support the hypothesis that bacteria involved in the worldwide echinoid disease commonly called the bald sea urchin disease are opportunistic and not specific.