Anti-lipopolysaccharide Factor from Crucifix Crab Charybdis feriatus , Cf- ALF2: Molecular Cloning and Functional Charac
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Anti‑lipopolysaccharide Factor from Crucifix Crab Charybdis feriatus, Cf‑ALF2: Molecular Cloning and Functional Characterization of the Recombinant Peptide K. S. Sruthy1,2 · Rosamma Philip1 Accepted: 1 October 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Antilipopolysaccharide factors (ALFs) are important effectors of innate immunity in crustaceans with broad spectrum antimicrobial activity. Present study deals with the molecular and functional characterization of a 98-amino acid ALF isoform from, crucifix crab, Charybdis feriatus termed as Cf-ALF2. The ALF isoform Cf-ALF2 exhibits characteristic features of an AMP including a cationic net charge of + 9 and a total hydrophobic ratio of 34%. Recombinant peptide rCf-ALF2 showed remarkable antimicrobial activity against Gram-negative and Gram-positive bacteria especially against Staphylococcus aureus (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 5 µM) and Escherichia coli (MIC 10 µM and MBC 20 µM). Using scanning electron microscopy, bacterial membrane blebbing, disruption, and cell content leakage were observed in peptide treated E. coli. The recombinant peptide was found to be non-hemolytic and non-cytotoxic in NCI-H460 cell line at the highest tested concentration (20 µM). Thus, this study identified a novel isoform of ALF from C. feriatus and revealed the potent antimicrobial property of the recombinant peptide Cf-ALF2 and the future prospects of using the peptide for therapeutic applications in the future. Keywords Antimicrobial peptides · Innate immunity · Charybdis feriatus · Anti-lipopolysaccharide factor · Recombinant protein
Introduction Crustaceans mainly depend on innate immunity for protection against extraneous invasions [1], and the antimicrobial peptides (AMPs) act as critical immune effectors keenly engaged in exclusion of pathogens and are usually non-cytotoxic to the host at concentrations enough for killing microorganisms [2]. Antilipopolysaccharide factors (ALFs) are single domain amphipathic peptides encompassing 114–124 amino acid residues with a 16–26 amino acid residue containing short signal peptide * Rosamma Philip [email protected]; [email protected] 1
Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, Kochi 682016, Kerala, India
Department of Molecular Reproduction, Development and Genetics, New Biological Sciences Building, Indian Institute of Science, Bangalore, Karnataka, India
2
sequence at the N-terminal region followed by a mature peptide with conserved bacterial lipopolysaccharide (LPS)-binding domain [3]. The first ALF isoform was identified from the amoebocytes of the limulids, Tachypleus tridentatus and Limulus polyphemus [4], and as it could bind to the LPS to inhibit the coagulation cascade, it was called ALF. Later, more ALF homologues were identified from decapod crustaceans including penaeid shrimps [5, 6], non-penaeid shrimps [7], lobsters [8, 9], crab
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