Identification of a group D anti-lipopolysaccharide factor (ALF) from kuruma prawn (Marsupenaeus japonicus) with antibacterial activity against Vibrio parahaemolyticus

Anti-lipopolysaccharide factor (ALF), which belongs to the antimicrobial peptide (AMP) family, has become a relatively new weapon to combat severe infections and has been demonstrated to be active against bacteria, fungi and some viruses. In the present study, a new ALF of group D (MjALF-D; GenBank accession No. MN416688) from Marsupenaeus japonicus was detected. MjALF-D encodes a polypeptide with 124 aa, and the peptide contains a 26-residue signal peptide and a lipopolysaccharide-binding domain (LBD). The structure of MjALF-D was found to consist of three α-helices, four β-sheets and random coils. qRT-PCR analysis revealed that MjALF-D expression was primarily observed in the stomach and was universally upregulated in both the gill and stomach after challenge by lipopolysaccharide (LPS) and Vibrio parahaemolyticus. Moreover, rMjALF-D can inhibit the growth of V. parahaemolyticus. rMjALF-D could destroy the bacterial membrane and lead to cytoplasmic leakage investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which may be the mechanism by which rMjALF-D inhibits V. parahaemolyticus. Additionally, rMjALF-D showed distinct binding or antibacterial ability after direct incubation with V. parahaemolyticus or bacterial genomic DNA and a certain effect on the protein expression of it. Together, these results indicated that rMjALF-D possessed the antibacterial activity against V. parahaemolyticus and the potential involvement in the innate immune response of M. japonicus.