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Anti-lipopolysaccharide Factor from Crucifix Crab Charybdis feriatus, Cf-ALF2: Molecular Cloning and Functional Characterization of the Recombinant Peptide

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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.

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Acknowledgments

The authors are grateful to the Director, Centre for Marine Living Resources and Ecology (CMLRE) and Ministry of Earth Sciences (MoES), Govt. of India, for the research grant (MoES/10-MLR/01/2012) and scientific support for the work. Authors also thank Cochin University of Science and Technology and the Coordinator, National Centre for Aquatic and Animal Health (NCAAH), for scientific support. The first author gratefully acknowledges KSCSTE (Kerala State Council for Science, Technology and Environment) for the award of research fellowship.

Funding

The study was funded by Ministry of Earth Sciences (MoES), Govt. of India (MoES/10-MLR/01/2012).

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  1. Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, Kochi, 682016, Kerala, India

    K. S. Sruthy & Rosamma Philip

  2. Department of Molecular Reproduction, Development and Genetics, New Biological Sciences Building, Indian Institute of Science, Bangalore, Karnataka, India

    K. S. Sruthy

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All authors contributed to the conceptualization and design of the study. Under supervision of Rosamma Philip, all experiments were performed and analyzed by K. S. Sruthy. The manuscript was written by K. S. Sruthy and reviewed and edited by Rosamma Philip. All authors read and approved the final manuscript.

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Sruthy, K.S., Philip, R. Anti-lipopolysaccharide Factor from Crucifix Crab Charybdis feriatus, Cf-ALF2: Molecular Cloning and Functional Characterization of the Recombinant Peptide. Probiotics & Antimicro. Prot. 13, 885–898 (2021). https://doi.org/10.1007/s12602-020-09716-w

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