Structural and Functional Characterization of a Novel Recombinant Antimicrobial Peptide from Hermetia illucens

Abstract

Antibiotics are commonly used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms raising the challenge to find new alternative drugs. Antimicrobial peptides (AMPs) are small/medium molecules ranging 10-60 residues synthesized by all living organisms and playing important roles in the defense systems. These features, together with the inability of microorganisms to develop resistance against the majority of AMPs, suggest that these molecules might represent effective alternatives to classical antibiotics. Because of their high biodiversity, with over one million described species, and their ability to live in hostile environments, insects represent the largest source of these molecules. However, production of insect AMPs in native forms is challenging. In this work we investigate a defensin-like antimicrobial peptide identified in the Hermetia illucens insect through a combination of transcriptomics and bioinformatics approaches. The C-15867 AMP was produced by recombinant DNA technology as a glutathione S-transferase (GST) fusion peptide and purified by affinity chromatography. The free peptide was then obtained by thrombin proteolysis and structurally characterized by mass spectrometry and circular dichroism analyses. The antibacterial activity of the C-15867 peptide was evaluated in vivo by determination of the minimum inhibitory concentration (MIC). Finally, crystal violet assays and SEM analyses suggested disruption of the cell membrane architecture and pore formation with leaking of cytosolic material.

Keywords: Hermetia illucens; antibacterial activity; antimicrobial peptides; defensins; insects.

Figure 1

(a) C-15867 ribbon model: the N-ter loop, the α-helix region and the C-ter β-sheet are shown in different colors; (b) hydrophobicity surface of the C-15867 peptide. Hydrophobic residues are in orange-red while hydrophilic residues are highlighted in blue. The figure was generated with UCSF CHIMERA software [45]; (c) Cys3, Cys15, Cys19, Cys30, Cys36, Cys38 residues present in C-15867 peptide; (d) disulfide bonds pattern of C15867 predicted through the DISULFIND server. The figure was generated with UCSF CHIMERA software [45].

Figure 2

Growth curve of E. coli BL₂₁ in the presence and in the absence of IPTG 0.4 mM for the over expression of GST-peptide. The error bars on the graphs stand for the standard deviation from the mean of three experiments.

Figure 3

(a) SDS-PAGE of the purified recombinant GST-peptide fusion product (2 µg). The band at about 29 kDa corresponds to the expected product while the band at about 25 kDa represents free GST. The amount of GST-peptide fusion product is equal to 3.2 mg per liter of culture. (b) SDS-PAGE (12.5%) of recombinant C-15867 after cleavage with thrombin (2 µg). The peptide yield is 1.8 mg per liter of culture.

Figure 4

CD spectrum of the native C-15867 peptide in PBS buffer.

Figure 5

Determination of C-15867 minimum inhibitory concentration (MIC). All bacteria cells were grown in the presence of serial dilution of peptide from 248 µM to 0.56 µM and incubated at 37 °C for 24 h. The error bars on the graphs stand for the standard deviation from the mean of three experiments.

Figure 6

Effect of C-15867 on E. coli BL₂₁ membrane permeability. The uptake of crystal violet was calculated in the presence of sub-mic concentration (9 µM) of the peptide.

Figure 7

Scanning electron microscopy of E. coli BL₂₁ cells following 3 h treatment with a sub-mic concentration (9 µM) of the antimicrobial peptide C-15867 (d–f) in comparison with untreated cells (a–c). The scale bar was 2 µm in panel (a), 500nm in panel (b,c) and 1µm in panel (d–f).