Haemocoel injection of PirA1B1 to Galleria mellonella larvae leads to disruption of the haemocyte immune functions.

Gongqing Wu,Yunhong Yi

doi:10.1038/srep34996

Abstract

The bacterium Photorhabdus luminescens produces a number of insecticidal proteins to kill its larval prey. In this study, we cloned the gene coding for a binary toxin PirA1B1 and purified the recombinant protein using affinity chromatography combined with desalination technology. Furthermore, the cytotoxicity of the recombinant protein against the haemocytes of Galleria mellonella larvae was investigated. We found that the protein had haemocoel insecticidal activity against G. mellonella with an LD50 of 131.5 ng/larva. Intrahaemocoelic injection of PirA1B1 into G. mellonella resulted in significant decreases in haemocyte number and phagocytic ability. In in vitro experiments, PirA1B1 inhibited the spreading behaviour of the haemocytes of G. mellonella larvae and even caused haemocyte degeneration. Fluorescence microscope analysis and visualization of haemocyte F-actin stained with phalloidin-FITC showed that the PirA1B1 toxin disrupted the organization of the haemocyte cytoskeleton. Our results demonstrated that the PirA1B1 toxin disarmed the insect cellular immune system.

Highlights

The bacterium Photorhabdus luminescens produces a number of insecticidal proteins to kill its larval prey
In a recent study, toxin complexes (Tcs) toxins inhibited the phagocytic activity of haemocytes from Galleria mellonella, and the biologically active components of the Tc toxins were characterized as adenosine diphosphate (ADP)-ribosyltransferases, which modify unusual amino acids and cause actin polymerization[15]
The gene coding for the binary toxin PirA1B1 was cloned from P. luminescens and expressed in E.coli M15

Summary

Introduction

The bacterium Photorhabdus luminescens produces a number of insecticidal proteins to kill its larval prey. The fourth class, Photorhabdus insect-related toxins (PirAB), are binary toxins that exhibit injectable and oral toxicity against mosquitos and lepidopterans[6]. The symbiotic bacteria of entomopathogenic nematodes that enter the insect haemocoel must fight with the haemocytes and AMPs. Many toxic proteins produced by symbiotic bacteria are reported to target haemocytes, which affects the host immune system by reducing vitality[12] and by inducing apoptosis and cytolysis[13,14]. In a recent study, Tc toxins inhibited the phagocytic activity of haemocytes from Galleria mellonella, and the biologically active components of the Tc toxins were characterized as adenosine diphosphate (ADP)-ribosyltransferases, which modify unusual amino acids and cause actin polymerization[15]. In a recent study, we demonstrated that the PirA2B2 toxin attacks haemocytes and decreases the cellular immunity of G. mellonella larvae following a haemocoel injection of the toxin[18]. Whether the PirA1B1 toxins suppress the immune system of insects is a question to be investigated

Methods

Results

Conclusion