Klara Stensvåg

Antibacterial activity in Strongylocentrotus droebachiensis (Echinoidea), Cucumaria frondosa (Holothuroidea), and Asterias rubens (Asteroidea).

A search for antibacterial activity in different body parts of the green sea urchin Strongylocentrotus droebachiensis, the common starfish Asterias rubens, and the sea cucumber Cucumaria frondosa was conducted. Antibacterial activity was detected in extracts from several tissues in all species tested, but mainly in the coelomocyte and body wall extracts. Relatively high antibacterial activity could also be detected in gastrointestinal organs and eggs from A. rubens and in eggs from C. frondosa. Differences between active extracts regarding hydrophobicity and sensitivity to heat and proteinase K treatment indicated that several different compounds were responsible for the antibacterial activities detected. Lysozyme-like activity could be detected in several tissues from A. rubens. Haemolytic activity could be detected in all species tested, especially in the body wall extracts. Results from the current study suggest that marine echinoderms are a potential source for the discovery of novel antibiotics.

Strongylocins, novel antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis

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 from marine invertebrates: Challenges and perspectives in marine antimicrobial peptide discovery

The emergence of pathogenic bacteria resistance to conventional antibiotics calls for an increased focus on the purification and characterization of antimicrobials with new mechanisms of actions. Antimicrobial peptides are promising candidates, because their initial interaction with microbes is through binding to lipids. The interference with such a fundamental cell structure is assumed to hamper resistance development. In the present review we discuss antimicrobial peptides isolated from marine invertebrates, emphasizing the isolation and activity of these natural antibiotics. The marine environment is relatively poorly explored in terms of potential pharmaceuticals, and it contains a tremendous species diversity which evolved in close proximity to microorganisms. As invertebrates rely purely on innate immunity, including antimicrobial peptides, to combat infectious agents, it is believed that immune effectors from these animals are efficient and rapid inhibitors of microbial growth.

Synoxazolidinones A and B: novel bioactive alkaloids from the ascidian Synoicum pulmonaria.

Bioassay-guided fractionation of the sub-Arctic ascidian Synoicum pulmonaria collected off the Norwegian coast led to the isolation of a novel family of brominated guanidinium oxazolidinones named synoxazolidinones A and B (1 and 2). The backbone of the compounds contains a 4-oxazolidinone ring rarely seen in natural products. The structure of the compounds was determined by spectroscopic methods. The synoxazolidinones exhibited antibacterial and antifungal activities.

The antibacterial effect of a polyhydroxylated fucophlorethol from the marine brown alga, Fucus vesiculosus

An antibacterial compound was isolated from the brown alga Fucus vesiculosus. Purification consisted of extraction of plant material with 0.05% trifluoroacetic acid, concentration on a C18 cartridge, and reverse-phase high performance liquid chromatography on a C18 semi-preparative column. The isolated compound exhibited antibacterial activity against both the Gram-positive and the Gram-negative bacteria tested. Killing studies conducted indicated that the activity was bactericidal. The compound showed no haemolytic effect against human red blood cells. Results obtained by electrospray ionization mass spectrometry indicated that the antibacterial activity was caused by a polyhydroxylated fucophlorethol.

Hyastatin, a glycine-rich multi-domain antimicrobial peptide isolated from the spider crab (Hyas araneus) hemocytes

Marine invertebrates are a rich source for the discovery of novel antimicrobial peptides, compounds regarded as important defense components in the host defense system. Here we report the purification and characterization of an 11.7kDa Gly-rich peptide, named hyastatin, from the hemocytes of Hyas araneus. It consists of three distinctly different domains: an N-terminal region enriched in Gly residues, a short Pro/Arg-rich region, and a C-terminal region containing six Cys residues with a Cys pattern resembling the one found in penaeidins. The C-terminus of the mature peptide is presumably amidated. The hyastatin transcript is constitutively expressed and mainly found in hemocytes. Hyastatin shows antimicrobial activity against yeasts, and Gram-positive and Gram-negative bacteria. The N-terminal region, devoid of the Cys-containing region, was recombinantly expressed in Escherichia coli cells, and shows only weak activity against the Gram-positive bacteria Corynebacterium glutamicum. Both hyastatin and the N-terminal region had the ability to bind chitin. Conclusively, this indicates the importance of the Cys-containing region for the antimicrobial activity, and a possible multifunctional character of hyastatin.

Structure-Activity Relationships of the Antimicrobial Peptide Arasin 1 — And Mode of Action Studies of the N-Terminal, Proline-Rich Region

Arasin 1 is a 37 amino acid long proline-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. In this work the active region of arasin 1 was identified through structure-activity studies using different peptide fragments derived from the arasin 1 sequence. The pharmacophore was found to be located in the proline/arginine-rich NH2 terminus of the peptide and the fragment arasin 1(1–23) was almost equally active to the full length peptide. Arasin 1 and its active fragment arasin 1(1–23) were shown to be non-toxic to human red blood cells and arasin 1(1–23) was able to bind chitin, a component of fungal cell walls and the crustacean shell. The mode of action of the fully active N-terminal arasin 1(1–23) was explored through killing kinetic and membrane permeabilization studies. At the minimal inhibitory concentration (MIC), arasin 1(1–23) was not bactericidal and had no membrane disruptive effect. In contrast, at concentrations of 5×MIC and above it was bactericidal and interfered with membrane integrity. We conclude that arasin 1(1–23) has a different mode of action than lytic peptides, like cecropin P1. Thus, we suggest a dual mode of action for arasin 1(1–23) involving membrane disruption at peptide concentrations above MIC, and an alternative mechanism of action, possibly involving intracellular targets, at MIC.

Centrocins: isolation and characterization of novel dimeric antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis.

As immune effector molecules, antimicrobial peptides (AMPs) play an important role in the invertebrate immune system. Here, we present two novel AMPs, named centrocins 1 (4.5kDa) and 2 (4.4kDa), purified from coelomocyte extracts of the green sea urchin, Strongylocentrotus droebachiensis. The native peptides are cationic and show potent activities against Gram-positive and Gram-negative bacteria. The centrocins have an intramolecular heterodimeric structure, containing a heavy chain (30 amino acids) and a light chain (12 amino acids). The cDNA encoding the peptides and genomic sequences were cloned and sequenced. One putative isoform (centrocin 1b) was identified and one intron was found in the genes coding for the centrocins. The full length protein sequence of centrocin 1 consists of 119 amino acids, whereas centrocin 2 consists of 118 amino acids which both include a preprosequence of 51 or 50 amino acids for centrocins 1 and 2, respectively, and an interchain of 24 amino acids between the heavy and light chain. The difference of molecular mass between the native centrocins and the deduced sequences from cDNA indicates that the native centrocins contain a post-translational brominated tryptophan. In addition, two amino acids at the C-terminal, Gly-Arg, were removed from the light chains during the post-translational processing. The separate peptide chains of centrocin 1 were synthesized and the heavy chain alone was shown to be sufficient for antimicrobial activity. The genome of the closely related species, the purple sea urchin (S. purpuratus), was shown to contain two putative proteins with high similarity to the centrocins.

Characterization of crustins from the hemocytes of the spider crab, Hyas araneus, and the red king crab, Paralithodes camtschaticus.

Crustins are distributed across the decapods and are believed to play a significant part in the humoral defense system of their host. In this study, two crustin isoforms from Hyas araneus hemocytes were purified and tested for antimicrobial activity against selected microorganisms. They show both antibacterial and antifungal activity, with highest activity against the Gram-positive bacteria Corynebacterium glutamicum. Sequencing of the transcripts showed them to have a mature peptide of 90 amino acids and differing in three positions in the mature peptide. They were named CruHa1 and CruHa2. Real-time RT-PCR revealed that they mainly are expressed in hemocytes. Screening a cDNA library detected a crustin sequence in Paralithodes camtschaticus hemocytes, coding for a mature peptide of 98 amino acids. It was named CruPc. Based on phylogenetic inference and primary structure, CruHa1 and CruHa2 were placed within the Type I group of crustins, while CruPc belongs to the Type II.

Antifouling compounds from the sub-arctic ascidian Synoicum pulmonaria: synoxazolidinones A and C, pulmonarins A and B, and synthetic analogues.

The current study describes the antifouling properties of four members belonging to the recently discovered synoxazolidinone and pulmonarin families, isolated from the sub-Arctic sessile ascidian Synoicum pulmonaria collected off the Norwegian coast. Four simplified synthetic analogues were also prepared and included in the study. Several of the studied compounds displayed MIC values in the micro-nanomolar range against 16 relevant marine species involved in both the micro- and macrofouling process. Settlement studies on Balanus improvisus cyprids indicated a deterrent effect and a low toxicity for selected compounds. The two synoxazolidinones displayed broad activity and are shown to be among the most active natural antifouling bromotyrosine derivatives described. Synoxazolidinone C displayed selected antifouling properties comparable to the commercial antifouling product Sea-Nine-211. The pulmonarins prevented the growth of several bacterial strains at nanomolar concentrations but displayed a lower activity toward microalgae and no effect on barnacles. The linear and cyclic synthetic peptidic mimics also displayed potent antifouling activities mainly directed against bacterial adhesion and growth.