Arasin-1

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.

Arasin 1 from the spider crab Hyas araneus is a proline-rich antimicrobial peptide (PR-AMP), which kills target bacteria by a non-membranolytic mechanism. By using a fluorescent derivative of the peptide, we showed that arasin 1 rapidly penetrates into Escherichia coli cells without membrane damage. To unravel its mode of action, a knockout gene library of E. coli was screened and two types of mutants with a less susceptible phenotype to the arasin 1 fragment (1-23) were found. The first bore the mutation of sbmA, a gene coding for an inner membrane protein involved in the uptake of different antibiotic peptides. The second mutation was located in the ygdD gene, coding for a conserved inner membrane protein of unknown function. Functional studies showed that YgdD is required for the full susceptibility to arasin 1(1-25), possibly by supporting its uptake and/or intracellular action. These results indicated that different bacterial proteins are exploited by arasin 1(1-25) to exert its antibacterial activity and add new insights on the complex mode of action of PR-AMPs.

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 NH(2) 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.