Identification And Bioactivity Of Antimicrobial Peptides In Amphioxus Branchiostoma Japonicum

Amphioxus is an important model organism, which is usually used to study the origin and evolution of vertebrates. Antimicrobial peptides (AMPs) are important innate immune components, acting as the first defense line of organisms against the invasion of microorganisms. AMPs have a special mode of action, which is different from the traditional antibiotcs. They have attracted much attention, and were supposed to be substitutes for traditional antibiotcs to deal with the drug resistance of bacteria. AMPs in amphioxus have been little investigated.In this study, an antimicrobial peptide cDNA (termed Bjampl) was identified from the amphioxus Branchiostoma japonicum. A predicted mature antimicrobial peptide (termed mBjAMP1) encoded by Bjampl was synthesized chemically with an amidated C-terminus. Subsequently, the antibacterial activity and mode of action of mBjAMP1 were investigated.qRT-PCR showed that Bjampl was predominantly expressed in the gill,1hepatic caecum, hind-gut, muscle and notochord, while little expression was observed in the ovary and testis. Challenge with LPS and LTA resulted in a significantly enhanced expression of Bjampl in all the tissues tested, including the hepatic caecum, notochord and muscle. These suggest that BjAMPl plays a role in the immune responses in B. japonicum.The cDNA of Bjampl obtained contained an open reading frame (ORF) of 294 bp encoding a deduced polypeptide of 97 amino acids. In the deduced polypeptide, N-terminus was a 24 amino acids long signal peptide followed by an anionic region and a cationnic C-terminus extension. By bioinformatics analysis, we predicted the final mature functional polypeptide was composed of the last 21 amino acids of the C-terminus. We synthesized this 21 amino acids long polypeptide with its C-terminus amidated at a purity grade Of>95% and named it mBjAMP1.To investigate alterations of the secondary structure of the mBjAMP1, CD spectroscopy was conducted in PBS with or without TFE, SDS. The results indicated that mBjAMP1 showed a conformational transition from a random coil pattern in PBS to an a-helical conformation in membrane-like conditions.We performed an antibacterial activity test of mBjAMP1 and found that mBjAMP1 had broad-spectrum antibacterial effect. It could inhibit the growth of both Gram-negative bacterium Escherichia coli (E. coli), Vibrio anguillarum (V. anguillarum) and Gram-positive bacterium Staphylococcus aureus (S. aureus), Micrococcus luteus (M. Luteus) with MIC of 6.3μg/ml,12.5μg/ml,6.3μg/ml, 6.3μg/ml respectively.To reveal the mode of action of mBjAMP1,the following tests were conducted. The cytoplasmic membrane depolarization assay of mBjAMP1 were performed with the membrane potential-sensitive dye diSC3-5 on E. coli and S. aureus. When treated with mBjAMP1, fluorescence intensity of the cell suspensions increased significantly in a dose-dependent manner and reach a maximum after about 15 min of treatment, indicating that mBjAMP1 caused depolarization of the cytoplasmic membrane. The results was implied that mBjAMP1 inhibit the growth of bacteria by a membrane active mechanism.Permeabilization assay was carried out by flow cytometry with fluorescent dye PI on E. coli and S. Aureus. We found that few E. coli and S. aureus cells treated with BSA showed PI fluorescent signal, suggesting that they had intact and viable cell membranes, by contrast, a significant proportion of E. coli and S. aureus cells treated with mBjAMP1 displayed PI fluorescent signal in a dose-depended manner. This indicated that mBjAMP1 was able to permeabilize the membrane integrity of bacteria.An enzyme-linked immunosorbent assay (ELISA) was carried out to investigate the mechanisms of binding activity. We found that mBjAMP1 had a significantly stronger affinity to LPS from Gram-negative bacteria, LTA from Gram-positive bacteria compared with BSA.In the transmission electron microscopy(TEM) test, after treated with mBjAMP1, the cell wall of E. coli and S. Aureus were damaged seriously. Cytoplasmic membrane thinning/transparency were also observed.It was found in the hemolytic activity test that mBjAMP1 had no hemolytic effect on human RBCs. In the MTT assay, mBjAMP1 showed no cytotoxicity to murine RAW264.7 cells.In summary, this study reports the identification of an uncharacterized AMP from amphioxus via in silico and functional analyses. This new AMP kills a broad spectrum of microbes via membrane active mechanism,while it is non-cytotoxic to mammalian cells, rendering it a promising template for the design of novel peptide antibiotics against microbes.