A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression

Abstract

Abstract Background Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. Results This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four β sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. Conclusion Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

Keywords

• Anti-lipopolysaccharide factor
• Portunus pelagicus
• Crustacean immunity
• Cytotoxicity
• Antimicrobial peptides