Paralithocins, Antimicrobial Peptides with Unusual Disulfide Connectivity from the Red King Crab, Paralithodes camtschaticus.

As part of an ongoing exploration of marine invertebrates as a source of new antimicrobial peptides, hemocyte extracts from the red king crab, Paralithodes camtschaticus, were studied. Three cationic cysteine (Cys)-rich peptides, named paralithocins 1-3, were isolated by bioassay-guided purification, and their amino acid sequences determined by Edman degradation and expressed sequences tag analysis. Disulfide bond mapping was performed by high-resolution tandem mass spectrometry. The peptides (38-51 amino acids in length) share a unique Cys motif composed of eight Cys, forming four disulfide bridges with a bond connectivity of (Cys relative position) Cys1-Cys8, Cys2-Cys6, Cys3-Cys5, and Cys4-Cys7, a disulfide arrangement that has not been previously reported among antimicrobial peptides. Thus, paralithocins 1-3 may be assigned to a previously unknown family of antimicrobial peptides within the group of Cys-rich antimicrobial peptides. Although none of the isolated peptides displayed antimicrobial activity against the target strains Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus, they inhibited the growth of several marine bacterial strains with minimal inhibitory concentrations in the 12.5-100 µM range. These findings corroborate the hypothesis that marine organisms are a valuable source for discovering bioactive peptides with new structural motifs.

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.

Expression of antimicrobial peptides from Hyas araneus haemocytes following bacterial challenge in vitro.

Circulating haemocytes play major roles in the host defense reactions of decapods, including the synthesis and release of antimicrobial peptides (AMPs). Unlike the AMPs from insects, those in decapods are constitutively expressed. This study aims to establish primary cell cultures of the three main haemocyte types in Hyas araneus haemocytes, and to measure the in vitro expression of AMP genes in the cells following microbial challenge. The haemocyte populations were separated on Percoll gradients and cultured in modified L-15 medium. Expression analysis by real-time RT-PCR showed that the granular cells are the main producers of crustin, hyastatin and arasin 1 AMPs, but the hyaline cells and semigranular cells also show some expression of these genes. Incubating the cell populations with Aerococcus viridans var. homari (a Gram-positive bacterium) or Listonella anguillarum (a Gram-negative pathogen) provoked no dramatic changes in the gene expression of any of the AMP, and although there was a small (single doubling) significant increase in expression of the crustin gene in granular cells 24h after exposure to L. anguillarum, it is unclear if this is biologically relevant under in vitro conditions. The results presented in this study are in accordance with several in vivo studies.

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.

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.

Arasin 1, a proline-arginine-rich antimicrobial peptide isolated from the spider crab, Hyas araneus.

Antimicrobial peptides (AMPs) are considered to play an important role as host-defense molecules in both vertebrates and invertebrates. This work was undertaken to characterize AMPs from hemocyte extracts of the small spider crab, Hyas araneus. A novel proline-arginine-rich AMP of 37 amino acids was isolated and characterized. The peptide, named arasin 1, has a chimeric structure with an N-terminal domain rich in proline and arginine and a C-terminal domain containing two disulfide linkages. The peptide precursor of 64 amino acids, deduced from a cDNA library, contained a hydrophobic pre-region of 25 amino acids, directly followed by the mature peptide. C-terminally, this precursor had two additional amino acids, which seem to be cleaved off post-translationally. Synthetic arasin 1 showed antibacterial activity. A putative isoform of arasin 1, named arasin 2, was found at the genetic level, and both transcripts were shown by real-time RT-PCR to be expressed mainly in hemocytes.