Isolation, Identification, and Bioinformatic Analysis of Antibacterial Proteins and Peptides from Immunized Hemolymph of Red Palm Weevil Rhynchophorus ferrugineus

Stanisław Knutelski,Natalia Łukasz,Justyna Śmiałek,Mona Awad,Piotr Suder,Michał Bukowski,Paweł Mak,Grzegorz Dubin

doi:10.3390/biom11010083

Stanisław Knutelski, Natalia Łukasz + Show 6 more

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https://doi.org/10.3390/biom11010083

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Abstract

Red palm weevil (Rhynchophorus ferrugineus Olivier, 1791, Coleoptera: Curculionidae) is a destructive pest of palms, rapidly extending its native geographical range and causing large economic losses worldwide. The present work describes isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from the immunized hemolymph of this beetle. In total, 17 different bactericidal or bacteriostatic compounds were isolated via a series of high-pressure liquid chromatography steps, and their partial amino acid sequences were determined by N-terminal sequencing or by mass spectrometry. The bioinformatic analysis of the results facilitated identification and description of corresponding nucleotide coding sequences for each peptide and protein, based on the recently published R. ferrugineus transcriptome database. The identified compounds are represented by several well-known bactericidal factors: two peptides similar to defensins, one cecropin-A1-like peptide, and one attacin-B-like protein. Interestingly, we have also identified some unexpected compounds comprising five isoforms of pheromone-binding proteins as well as seven isoforms of odorant-binding proteins. The particular role of these factors in insect response to bacterial infection needs further investigation.

Highlights

There is a worldwide rapid increase in the number of pathogenic bacteria with resistance to the array of available antibiotics, which poses a growing threat to human and animal health [1,2]
We compared the chromatographic profiles of hemolymph samples collected from both control insects and infected beetles 24 h after injection of a mixture of Gram-negative and Gram-positive bacteria (Escherichia coli and Micrococcus luteus) into the hemocoel
The antibacterial radial diffusion assay performed on fractions collected during separation of immunized hemolymph distinguished a group of peaks that indicated bactericidal or bacteriostatic activity towards the standard bacterial strains used: E. coli and Staphylococcus intermedius

Summary

Introduction

There is a worldwide rapid increase in the number of pathogenic bacteria with resistance to the array of available antibiotics, which poses a growing threat to human and animal health [1,2] To overcome this problem, antimicrobial peptides/proteins (AMPs) have come to the forefront as potential antibiotic surrogates with robust killing activity against a wide spectrum of bacterial species, including drug-resistant strains [3]. Antimicrobial peptides/proteins (AMPs) have come to the forefront as potential antibiotic surrogates with robust killing activity against a wide spectrum of bacterial species, including drug-resistant strains [3] They exert an antimicrobial effect mainly by disrupting the microbial membrane, which makes microbes unable to develop resistance against these compounds [4,5,6].

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