Molecular Cloning, Functional and Biophysical Characterization of an Antimicrobial Peptide from Rhizosphere Soil.

Abstract

This study was designed to screen and identify an antimicrobial peptide from rhizosphere soil. The study was further focused towards overexpression, purification and characterization of this antimicrobial peptide, and to functionally validate its efficiency and efficacy as an antimicrobial agent. Yet, the study was further aimed at corroborating structural and functional studies using biophysical tools. Antimicrobial resistance is emerging as one of the top 10 global health crisis, it is multifaceted and the second largest cause of mortality. According to the World Health Organization (WHO), around the world, an estimated 700,000 people die each year from infection caused by antibiotic-resistant microbes. Antimicrobial peptides offer the best alternative to combat and overcome this crisis. In this manuscript, we report cloning, expression, purification and characterization of an antimicrobial peptide discovered from rhizosphere soil. Objectives of this study include construction, screening and identification of antimicrobial peptide from metagenome followed by its expression, purification and functional and biophysical investigation. Yet another objective of the study was to determine antimicrobial efficacy and efficiency as an antimicrobial peptide against MRSA strains. In this study, we used an array of molecular biology tools that include genetic engineering, PCR amplification, construction of an expression construct and NI-NTA based purification of the recombinant peptide. We have also carried out antimicrobial activity assay to determine MIC (minimum inhibitory concentration) and IC50 values of antimicrobial peptide. To establish the structural and functional relationship, circular dichroism, and both extrinsic and intrinsic fluorescence spectroscopy studies were carried out. Screening of metagenomic library resulted in the identification of gene (~500bp) harbouring an open reading frame (ORF) consisting of 282 bp. Open reading frame identified in gene encodes an antimicrobial peptide which had shared ~95% sequence similarity with the antimicrobial peptide of Bacillus origin. Purification of recombinant protein using Ni-NTA column chromatography demonstrated a purified protein band of ~11 kDa on 14% SDS-PAGE, which is well corroborated to theoretical deduced molecular weight of peptide from its amino acids sequence. Interestingly, the peptide exhibited antimicrobial activity in a broad range of pH and temperature. MIC determined against gram positive Bacillus sp. was found to be 0.015mg/ml, whereas, in the case of gram negative E. coli, it was calculated to be 0.062mg/ml. The peptide exhibited IC50 values corresponding to ~0.25mg/ml against Bacillus and ~0.5 mg/ml against E. coli. Antimicrobial susceptibility assay performed against methicillin resistant Staphylococcus aureus strain ATCC 3412 and standard strain of Staphylococcus aureus ATCC 9144 revealed its strong inhibitory activity against MRSA, whereby we observed a ~16mm clearance zone at higher peptide concentrations ~2mg/ml (~181.8μM). Biophysical investigation carried out using Trp fluorescence, ANS fluorescence and circular dichroism spectroscopy further revealed conformational stability in its secondary and tertiary structure at a wide range of temperature and pH. Altogether, the peptide discovered from rhizosphere metagenome holds potential in inhibiting the growth of both gram positive and gram negative bacteria, and was equally effective in inhibiting the multidrug resistant pathogenic strains (MRSA).