Characterization of a new homologous anti-lipopolysaccharide factor SpALF7 in mud crab Scylla paramamosain

Invertebrates, such as mud crab Scylla paramamosain, rely only on innate immunity. Anti-lipopolysaccharide factors (ALFs), are one of the typical antimicrobial peptides (AMPs) commonly found in crustaceans, playing a vital role in the immune defense against pathogens. In this study, a new homologous ALF, named SpALF7, was identified from S. paramamosain. Its full-length cDNA sequence consists of 640 bp, including 121 deduced amino acid residues. The homology between SpALF7 and the other six reported ALFs in S. paramamosain is about 24.69%-43.59%. Absolute quantitative PCR (qPCR) analysis showed that SpALF7 was widely distributed throughout the embryonic and larval developmental stages and the tissues of adult crabs, with the highest transcription level in the seminal vesicles of adult male crabs. Not surprisingly, SpALF7 gene could be significantly upregulated in hepatopancreas under LPS and Vibrio alginolyticus challenge analyzed by relative qPCR. Through the Pichia pastoris eukaryotic expression system, the SpALF7 recombinant protein (rSpALF7) was successfully obtained and a truncated peptide containing the putative LPS binding domain (sSpALF7) was chemically synthesized. Both of them exhibited potent antibacterial activity against several Gram-positive and Gram-negative bacteria. The bactericidal kinetic curves produced by rSpALF7 showed that it could kill more than 90% Pseudomonas stutzeri and Shigella flexneri within 5 min and 120 min at a concentration of 1 mu M and 6 mu M, respectively. After rSpALF7 treatment, the disruption of cell membrane integrity in S. flexneri and P. stutzeri was observed by scanning electron microscope, which was further confirmed by the flow cytometry assay with SYTO9 and propidium iodide (PI) stainning. In addition, it was worth noting that although rSpALF7 had no bactericidal effect on the crab pathogen V. alginolyticus, it could significantly reduce the bacterial endotoxin levels. Furthermore, the in vivo studies showed that rSpALF7 could obviously improve the survival of crabs infected by V. alginolyticus. Taken together, these results suggested that SpALF7 might play an important role in the innate immunity of S. paramamosain, and has potential applications in aquaculture.