Molecular cloning, genomic structure and antimicrobial activity of PtALF7, a unique isoform of anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus

Abstract

Anti-lipopolysaccharide factors (ALFs), as the potent antimicrobial peptides, are becoming predominant candidates for potential therapeutic agents of bacterial and viral diseases. In this study, a unique isoform of ALF (PtALF7) was identified from hemocytes cDNA library of the swimming crab Portunus trituberculatus. The PtALF7 cDNA contained an open reading frame (ORF) of 372 bp encoding 123 amino acids. The deduced peptide of PtALF7 shared high similarity with our previously reported PtALF1–3 but low with PtALF4–6. The PtALF7 gene consisted of three exons interrupted by two introns, and was clearly transcribed from different genomic loci compared with other PtALF isoforms. Totally 128 SNPs including 12 in coding region and 116 in noncoding region were detected in PtALF7 gene by direct sequencing of 20 samples. The mRNA expression of PtALF7 transcript was primarily observed in hemocytes followed by gill and eyestalk, but barely detectable in hepatopancreas. After challenge with Vibrio alginolyticus, a main pathogen causing high mortality in P. trituberculatus, the PtALF7 transcript in hemocytes showed a clear time-dependent response expression pattern with obvious decrease at 6 h and significant increase at 24 h. The recombinant PtALF7 protein exhibited antimicrobial activity against the test Gram-negative and Gram-positive bacteria, but did not inhibit the growth of fungus Pichia pastoris. These results together indicate a potential involvement for PtALF7 in the innate immune response of P. trituberculatus.