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Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, Antheraea assamensis

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Abstract

Antimicrobial peptides (AMPs) are produced in all living organisms including insects in a non-specific manner, and act as innate immune defense arsenal against the invading pathogens. Muga silkworm (Antheraea assamensis) larvae were injected with Candida albicans and AMPs were isolated from the hemolymph after extracting with methanol, acetic acid and water mixture (90:1:9) and evaluated for antimicrobial activity against fungal and bacterial pathogens. Further purification was done through successive semipreparative and analytical reversed phase HPLC using C-18 column. The obtained fractions were collected, lyophilized and tested for antimicrobial activity. Among the HPLC fractions, one showed highest activity with MIC value of 64 µg/ml against Gram-negative bacteria, Escherichia coli and Enterobacter cloacae. Purity of this isolated peptide was confirmed by SDS-PAGE and TLC, and its molecular mass was determined as 9.052 kDa by MALDI-TOF mass spectrometry. From the mass fingerprinting analysis of this peptide after trypsin digestion a peptide fragment with molecular mass of 2622.7 Da was obtained. De novo sequencing of this peptide fragment following MS/MS analysis identified few amino acid residues as “KSGGGGWGS” with a total score of 46.9 with gloverin peptide of A. mylitta. The peptide inhibited biofilm formation of the Gram-negative bacterial pathogens. SEM study revealed that peptide disrupted bacterial cell wall to leach out intracellular materials and may be the major target for its antimicrobial activity.

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Abbreviations

RP-HPLC:

Reverse phase-high performance liquid chromatograpy

MALDI-TOF:

Matrix-assisted laser desorption/ionization-time of flight

MIC:

Minimum inhibitory concentration

SEM:

Scanning electron microscope

LPS:

Lipopolysaccharide

MS:

Mass spectroscopy

AMPs:

Antimicrobial peptides

CFU:

Colony-forming unit

TLC:

Thin layer chromatography

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

PMSF:

Phenyl methane sulfonyl fluoride

TFA:

Trifluoroacetic acid

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Acknowledgements

The work was supported from a grant from Department of Biotechnology, Government of India (No: BT/464/NE/TBP/2013).

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Authors and Affiliations

  1. Department of Biotechnology, Central Research Facility, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Tilak Nayak, Santi M. Mandal & Ananta K. Ghosh

  2. Central Muga Eri Research and Training Institute, Central Silk Board, Ministry of Textiles, Lahdoigarh, Jorhat, Assam, 785700, India

    Kartik Neog

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  1. Tilak Nayak
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  2. Santi M. Mandal
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  4. Ananta K. Ghosh
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Correspondence to Ananta K. Ghosh.

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Nayak, T., Mandal, S.M., Neog, K. et al. Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, Antheraea assamensis . Int J Pept Res Ther 24, 337–346 (2018). https://doi.org/10.1007/s10989-017-9618-0

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