Synthesis of a Peptide Derivative of MicrocinJ25 and Evaluation of Antibacterial and Biological Activities

Abstract

MicrocinJ25 (MccJ25) is a small ribosomally synthesized antimicrobial peptide that is produced by Enterobacteriacea family especially E. coli. The present study focuses on preparation and evaluation of in-vitro antimicrobial and biological properties of a new peptide derived from MccJ25. We prepared a MccJ25-derived peptide containing 14 amino acids and a single intra-molecular disulfide bond according to solid phase synthesis strategy. The purified peptide was characterized by Liquid chromatography-mass spectrometry (LC-MS) and Fourier Transform Infrared (FTIR) spectroscopy. 96-well microdilution plate assay was exerted for determination of minimum inhibitory concentration (MIC) of peptide against different bacterial strains. Cytotoxicity of the peptide derivative on HT-29 cell line assayed using MTT test. The final peptide successfully was prepared with purity more than 99.8% as determined by analytical HPLC. The evaluation of antibacterial activity of the peptide against Gram-positive and Gram- negative bacteria revealed that the peptide was very effective against E. coli 35218 with minimum inhibitory concentration (MIC) at dose 3.9 µM. The hemolytic activity toward human erythrocytes was very minimal below 0.3%. The cell viability percentage of HT-29 cell line after 24 h of contact with the peptide was more than 83%. The high sensitivity of E. coli strain to this new peptide derived from MccJ25 and through minimal toxicity to cancerous cell, suggesting that above synthesized peptide could be considered as a bioactive compound for further investigations.

Keywords: Biological activity; Cytotoxicity; E. coli; MicrocinJ25 derivative; Synthesis.

Figure 1

Amino acid sequences of MccJ25 (a) and derived peptide (b). Structure of the synthetic derived peptide (c)

Figure 2

Reverse phase HPLC chromatogram of purified MccJ25-peptide derivative

Figure 3

Mass spectrum analysis of the prepared peptide derived from MccJ25 in positive electrospray ionization (ESI) mode. Peaks at m/z 1519.9 and 1560.8 correspond to molecular ions [M+H] + and [M+CH3CN+H] + respectively

Figure 4

Infrared spectrum of the prepared peptide derived from MccJ25. (the details for IR spectrum characterization have been described in the text)

Figure 5

Inhibition of bacterial growth by the synthesized peptide at concentration of 250 µM after 24 h incubation at 35-37 ˚C

Figure 6

Cell viability (%) of HT-29 cell line after incubation with different concentrations of the synthesized peptide for 24 h incubation at 37 °C in 5% CO₂ atmosphere. The values were calculated as mean ± SD of triplicate independent experiments

Figure 7

Hemolysis of human erythrocytes by different concentrations of the synthesized peptide. The values were calculated as mean ± SD of triplicate independent experiments