Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis.

Luís Felipe Costa Ramos,Rodrigo Almeida,Fábio Nogueira,Livia Castilho,Fabio Gomes,Guilherme Andrade,João Rangel,Anderson Pinheiro,Danielle Oliveira,Cristiane Anobom,Carolina Lixa

doi:10.1590/1678-9199-jvatitd-2020-0127

Abstract

BackgroundInsects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. ResultsAgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. ConclusionsThe first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.

Highlights

Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria
The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform
We identified a cecropin B-like peptide from the soybean caterpillar A. gemmatalis

Summary

Introduction

Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. AMPs are cationic peptides usually ranging from 20 to 50 amino acid residues [6] that exhibit broad activity against bacteria, fungi, as well as certain parasites and viruses [7,8,9] In insects, these peptides are mainly synthesized in the fat body [10,11], hemocytes, and digestive tract [12]. Cecropins are a well-studied class of antimicrobial peptides, with an α helical structure, first described in Hyalophora cecropia hemolymph [13,14] and later described in other lepidopterans and dipterans [15,16,17,18] They belong to a large family of cationic AMPs, which is structured the α-helicoidal secondary structure when they encounter microorganism surfaces [19]. These peptides have shown antimicrobial activity against a wide spectrum of bacteria [20,21,22,23] and fungi [24]

Methods

Results

Discussion

Conclusion