Klara Stensvåg

Ascidians are a group of marine invertebrates, most of which are sessile and soft-bodied. Their lack of an adaptive immune system makes them rely on innate immune responses to detect and eliminate invading microbes. Antimicrobial peptides (AMPs) play an essential part in this process. In this paper, we present the isolation, structure elucidation, and bioactivities of two new cysteine-rich peptides (CRPs) from the Arctic marine ascidian Synoicum turgens. The sequences and structures of the peptides were determined by Edman degradation sequencing, mass spectrometry, and NMR analysis. This revealed two novel 2 kDa peptides, St-CRP-1 and St-CRP-2, with neutral net charge and C-terminal amidation. St-CRP-1 consisted of 18 amino acids and displayed selective and moderate growth inhibition of two Gram-positive bacterial strains (Bacillus subtilis and Corynebacterium glutamicum) at 24.6 µM, whereas St-CRP-2 consisted of 19 amino acids and inhibited the growth of B. subtilis at 49.2 µM. St-CRP-1 had no effect on two mammalian cell lines or the brine shrimp Artemia salina at the highest concentration tested. Structural analysis of the St-CRPs indicated a Cys1–Cys6, Cys2–Cys4, and Cys3–Cys5 disulfide connectivity, which is also found in alpha-defensins. The results from this study show that Arctic marine ascidians are a rich source of novel bioactive peptides.

Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological states. The crude venom of the sea anemone Metridium senile showed the ability to interact with nAChRs. Four novel peptides (Ms11a-1–Ms11a-4) with nAChR binding activity were isolated. These peptides stabilized by three disulfide bridges have no noticeable homology with any known peptides. Ms11a-1–Ms11a-4 showed different binding activity towards the muscle-type nAChR from the Torpedo californica ray. The study of functional activity and selectivity for the most potent peptide (Ms11a-3) revealed the highest antagonism towards the heterologous rat α9α10 nAChR compared to the muscle and α7 receptors. Structural NMR analysis of two toxins (Ms11a-2 and Ms11a-3) showed that they belong to a new variant of the inhibitor cystine knot (ICK) fold but have a prolonged loop between the fifth and sixth cysteine residues. Peptides Ms11a-1–Ms11a-4 could represent new pharmacological tools since they have structures different from other known nAChRs inhibitors.

“Sea urchin lesion syndrome” is known as sea urchin disease with the progressive development of necrotic epidermal tissue and loss of external organs, including appendages on the outer body surface. Recently, a novel strain, Vibrio echinoideorum has been isolated from the lesion of green sea urchin (Strongylocentrotus droebachiensis), an economically important mariculture species in Norway. V. echinoideorum has not been reported elsewhere in association with green sea urchin lesion syndrome. Therefore, in this study, an immersion based bacterial challenge experiment was performed to expose sea urchins (wounded and non-wounded) to V. echinoideorum, thereby mimicking a nearly natural host–pathogen interaction under controlled conditions. This infection experiment demonstrated that only the injured sea urchins developed the lesion to a significant degree when exposed to V. echinoideorum. Pure cultures of the employed bacterial strain were recovered from the infected animals and its identity was confirmed by the MALDI-TOF MS spectra profiling. Additionally, the hemolytic phenotype of V. echinoideorum substantiated its virulence potential towards the host, and this was also supported by the cytolytic effect on red spherule cells of sea urchin. Furthermore, the genome sequence of V. echinoideorum was assumed to encode potential virulence genes and were subjected to in silico comparison with the established virulence factors of Vibrio vulnificus and Vibrio tasmaniensis. This comparative virulence profile provided novel insights about virulence genes and their putative functions related to chemotaxis, adherence, invasion, evasion of the host immune system, and damage of host tissue and cells. Thus, it supports the pathogenicity of V. echinoideorum. In conclusion, the interaction of V. echinoideorum with injured sea urchin facilitates the development of lesion syndrome and therefore, revealing its potentiality as an opportunistic pathogen.

“Sea urchin lesion syndrome” is known as sea urchins disease with the progressive development of necrotic epidermal tissue and loss of external organs, including appendages on the outer body surface. Recently, a novel strain, Vibrio echinoideorum has been isolated from the lesions of green sea urchin (Strongylocentrotus droebachiensis), an economically important mariculture species in Norway. V. echinoideorum has not been reported elsewhere in association of with green sea urchin lesion syndrome. Therefore, in this study, an immersion based bacterial challenge experiment was performed to expose sea urchins (wounded and non-wounded) to V. echinoideorum, thereby mimicking a nearly natural host-pathogen interaction under controlled conditions. This infection experiment demonstrated that only the injured sea urchins developed the lesion to a significant degree when exposed to V. echinoideorum. Pure cultures of the employed bacterial strain was recovered from the infected animals and its identity was confirmed by the MALDI-TOF MS spectra profiling. Additionally, the hemolytic phenotype of V. echinoideorum substantiated its virulence potential towards the host, and this was also supported by the cytolytic effect on red spherule cells of sea urchins. Furthermore, the genome sequence of V. echinoideorum was assumed to encode potential virulence genes and were subjected for in silico comparison with the established virulence factors of Vibrio vulnificus and Vibrio tasmaniensis. This comparative virulence profile provided novel insights about virulence genes and their putative functions related to chemotaxis, adherence, invasion, evasion of the host immune system, and damage of host tissue and cells. Thus, it supports the pathogenicity of V. echinoideorum. In conclusion, the interaction of V. echinoideorum with injured sea urchins appears to be essential for the development of lesion syndrome and therefore, revealing its potentiality as an opportunistic pathogen.

Red spherule cells (RSCs) are considered one of the prime immune cells of sea urchins, but their detailed biological role during immune responses is not well elucidated. Lack of pure populations accounts for one of the major challenges of studying these cells. In this study, we have demonstrated that live RSCs exhibit strong, multi-colour autofluorescence distinct from other coelomocytes, and with the help of fluorescence-activated cell sorting (FACS), a pure population of live RSCs was successfully separated from other coelomocytes in the green sea urchin, Strongylocentrotus droebachiensis. This newly developed RSCs isolation method has allowed profiling of the naphthoquinone content in these cells. With the use of ultra high-performance liquid chromatography, UV absorption spectra, and high-resolution tandem mass spectrometry, it was possible to identify sulphated derivatives of spinochrome C, D, E and spinochrome dimers, which suggests that the RSCs may play an important biological role in the biogenesis of naphthoquinone compounds and regulating their bioactivity.

This study reports the isolation of two novel cysteine-rich antibacterial peptides, turgencin A and turgencin B, along with their oxidized derivatives, from the Arctic marine colonial ascidian Synoicum turgens. The peptides are post-translationally modified, containing six cysteines with an unusual disulfide connectivity of Cys¹-Cys⁶, Cys²-Cys⁵, and Cys³-Cys⁴ and an amidated C-terminus. Furthermore, the peptides contain methionine residues resulting in the isolation of peptides with different degrees of oxidation. The most potent peptide, turgencin A_Mox1 with one oxidized methionine, displayed antimicrobial activity against both Gram-negative and Gram-positive bacteria with a minimum inhibitory concentration (MIC) as low as 0.4 µM against selected bacterial strains. In addition, the peptide inhibited the growth of the melanoma cancer cell line A2058 (IC₅₀ = 1.4 µM) and the human fibroblast cell line MRC-5 (IC₅₀ = 4.8 µM). The results from this study show that natural peptides isolated from marine tunicates have the potential to be promising drug leads.

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide (P6) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide (P6) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide (P6) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide (P6) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.

A Gram-stain-negative, facultatively anaerobic Vibrio strain, designated NFH.MB010T, was isolated from an epidermal lesion on the test (hard shell skeleton) of a green sea urchin (Strongylocentrotus droebachiensis) collected from northern Norway. Cells of strain NFH.MB010T were rod shaped and motile by means of a single, long polar flagellum. Growth was observed at 1–5% NaCl (w/v) and at 4 °C, but not above 28 °C. Phylogenetic analyses based on eight-gene multilocus sequence analysis (16S rRNA, atpA, gyrB, mreB, pyrH, recA, rpoA and rpoD) suggested novelty at the species level. In silico DNA–DNA hybridization and orthologous average nucleotide identity estimates showed percentage genomic resemblances to its closest relative, Vibrio splendidus , that were well below the established same species threshold values. Phenotypically, utilization of glycogen and gentiobiose, inability of acetoin production, and undetectable valine arylamidase and trypsin activity discriminated strain NFH.MB010T from the closely related reference strains. Protein spectra generated by maldi-tof mass spectrometry further consolidated the species level uniqueness of strain NFH.MB010T. Based on the described polyphasic approach, strain NFH.MB010T therefore appears as a novel species within the Splendidus clade of the genus Vibrio , and the name Vibrio echinoideorum sp. nov. is proposed, with NFH.MB010T (=DSM 107264T=LMG 30656T) as the type strain.