Arasin-likeSp

Antimicrobial peptides (AMPs) are some of the important host molecules required to resist pathogen infection. Two novel AMPs (arasin-likeSp and GRPSp) were identified from the hemocytes of the mud crab, Scylla paramamosain, by analysis of a hemocyte expressed sequence tag library. The deduced open reading frames of the arasin-likeSp and GRPSp cDNAs are 198 and 168 bp, and encode for predicted peptides of 65 and 55 amino acid residues, respectively. The calculated molecular mass of the mature peptides was 4373 and 2995 Da with an estimated isoelectric point (pI) of 11.03 and 9.66, respectively. The mature peptide of arasin-likeSp is predicted to contain an N-terminal region rich in glycine and arginine and a C-terminal region containing four cysteine residues. Its amino acid sequence has an overall sequence identity of 53% to arasin-2 from the spider crab, Hyas araneus. The mature protein of GRPSp contains two cysteine residues at the C-terminus and two glycine-rich repeats (GGYG and GYGG). In healthy crabs, both arasin-likeSp and GRPSp transcript levels were found to be high in the hemocytes and were further increased at 3 h after challenge with the bacterium, Aerococcus viridans. A synthetic arasin-likeSp peptide revealed the antimicrobial activity against both Gram-positive and Gram-negative bacteria including some crustacean pathogens (A. viridans, Vibrio harveyi and V. anguillarum), whilst the synthetic GRPSp peptide exhibited antibacterial activity against some Gram-positive (A. viridans and Micrococcus luteus), but not Gram-negative, bacteria. These results indicate that arasin-likeSp and GRPSp are potentially novel AMPs involved in the immune responses of mud crab, S. paramamosain.

Antibiotic resistance is a major public health threat to both humans and animals. There is an urgent need for antimicrobial agents with novel modes of action. Antimicrobial peptides (AMPs) with broad-spectrum antimicrobial activity become the ideal alternative to traditional antibiotics. Here, the ELP-intein self-cleavage system was used to produce antimicrobial peptide arasin-likeSp in Escherichia coli. The tagged target protein (ELP-intein-arasin-likeSp) was mainly expressed in soluble, separated from cell lysates by the inverse transition cycling (ITC), and the arasin-likeSp was further purified by the self-cleavage of intein and the second round of ITC. The final yield of arasin-likeSp was about 3.56 mg/L. Purified arasin-likeSp exhibited significant antibacterial activities against the Gram-positive Bacillus subtilis and Gram-negative Vibrio harveyi bacteria. FE-SEM and PI staining analysis revealed that the arasin-likeSp treatment altered the morphology and membrane permeability of Bacillus subtilis and Vibrio harveyi. Collectively, these data suggest that arasin-likeSp is a candidate AMP for effective inhibition of Vibrio harveyi, a significant bacterial pathogen infecting marine fish and invertebrates. The ELP-intein self-cleavage system described here is a low-cost, simple and potential method for producing antimicrobial peptides, which lays foundations for the large-scale production of antimicrobial peptides in the future.