Strongylocin 1

Sea urchins possess an innate immune system and are regarded as a potential source for the discovery of new antimicrobial peptides (AMPs). Here we report the purification and characterization of two novel antibacterial peptides (5.6 and 5.8kDa) from coelomocyte extracts of the green sea urchin, Strongylocentrotus droebachiensis. These are the first reported AMPs isolated from sea urchins. The cDNA encoding the peptides and genomic sequences was isolated and sequenced. The two peptides (named strongylocins 1 and 2) have putative isoforms (1b and 2b), similar to two putative proteins from the purple sea urchin S. purpuratus. The native strongylocins are cationic, defensin-like peptides (cysteine-rich), but show no similarity to other known AMPs concerning the cysteine distribution pattern. Strongylocin 1 consists of 83 amino acids that include a preprosequence of 35 amino acids, whereas strongylocins 2a and 2b are composed of 89 and 90 amino acids, respectively, where 38 amino acids represent a preprosequence. No introns were found in the cloned gene of strongylocin 1b, whereas three introns and four exons were found in strongylocins 1a and 2a/b. The latter gene organization was also found in genes coding for putative strongylocins in S. purpuratus. The molecular mass difference between the native peptide and the deduced strongylocin 2 suggests that the first amino acid is bromotryptophan. The native peptides display potent activities against Gram-negative and Gram-positive bacteria.

Antimicrobial peptides (AMPs) play a crucial role in innate immunity. We have previously reported the isolation and characterization of the AMPs, strongylocins 1 and 2, and centrocin 1, from coelomocyte extracts of Strongylocentrotus droebachiensis. Here we show that these AMPs were expressed in phagocytes. In addition, transcripts of strongylocin 1 were detected in vibratile cells and/or colorless spherule cells, while transcripts of strongylocin 2 were found in red spherule cells. Results from immunoblotting and immunocytochemistry studies showed that centrocin 1 was produced by phagocytes and stored in granular vesicles. Co-localization of centrocin 1 and phagocytosed bacteria suggests that the granular vesicles containing centrocin 1 may be involved in the formation of phagolysosomes. We also analyzed the temporal and spatial expression of AMPs throughout larval development. Strongylocins were expressed in the early pluteus stage, while centrocin 1 was expressed in the mid pluteus stage. The spatial expression pattern showed that centrocin 1 was mainly located in blastocoelar cells (BCs) around the stomach and the esophagus. In addition, a few patrolling BCs were detected in some larval arms. Together, these results suggest that AMPs are expressed in different types of coelomocytes and that centrocin 1 is involved in response against bacteria. Furthermore, the expression of AMPs in larval pluteus stage, especially in BCs, indicates that AMPs and BCs are engaged in the larval immune system.

In echinoderms, the immune system plays a relevant role in defense against infection by pathogens. Particularly, in sea urchins, the immune system has been shown to be complex, especially in terms of the variety of immune genes and molecules described. A key component of the response to external pathogens are the Toll-like receptors (TLRs), which are a well-characterized class of pattern recognition receptors (PRRs) that participate in the recognition of pathogen-associated molecular patterns (PAMPs). Despite the fact that TLRs have been described in several sea urchin species, for the red sea urchin (Loxechinus albus), which is one of the most important sea urchins across the world in terms of fisheries, limited information on the TLR-mediated immune response exists. In the present study, for the first time, we evaluated the effect of thermal stress, LPS and poly I:C treatment on the coelomocyte immune response of Loxechinus albus to determine how these factors modulate TLR and strongylocin (antimicrobial peptides of echinoderms) responses. We show that the tlr3-like, tlr4-like, tlr6-like and tlr8-like transcripts are modulated by poly I:C, while LPS only modulates the tlr4-like response; there was no effect of temperature on TLR expression, as evaluated by RT-qPCR. Additionally, we showed that strongylocin-1 and strongylocin-2 are modulated in response to simulated viral infection with poly I:C, providing the first evidence of strongylocin expression in L. albus. Finally, we determined that temperature and LPS modify the viability of coelomocytes, while poly I:C treatment did not affect the viability of these cells. This study contributes to the knowledge of immune responses in sea urchins to improve the understanding of the role of TLRs and strongylocins in echinoderms.