Inhibition of Spodoptera frugiperda phenoloxidase activity by the products of the Xenorhabdus rhabduscin gene cluster.

Bernard Duvic,Sylvie Pagès,Maria Eugenia Nuñez-Valdez,Sophie Gaudriault,Anne Lanois

doi:10.1371/journal.pone.0212809

Abstract

We evaluated the impact of bacterial rhabduscin synthesis on bacterial virulence and phenoloxidase inhibition in a Spodoptera model. We first showed that the rhabduscin cluster of the entomopathogenic bacterium Xenorhabdus nematophila was not necessary for virulence in the larvae of Spodoptera littoralis and Spodoptera frugiperda. Bacteria with mutations affecting the rhabduscin synthesis cluster (ΔisnAB and ΔGT mutants) were as virulent as the wild-type strain. We then developed an assay for measuring phenoloxidase activity in S. frugiperda and assessed the ability of bacterial culture supernatants to inhibit the insect phenoloxidase. Our findings confirm that the X. nematophila rhabduscin cluster is required for the inhibition of S. frugiperda phenoloxidase activity. The X. nematophila ΔisnAB mutant was unable to inhibit phenoloxidase, whereas ΔGT mutants displayed intermediate levels of phenoloxidase inhibition relative to the wild-type strain. The culture supernatants of Escherichia coli and of two entomopathogenic bacteria, Serratia entomophila and Xenorhabdus poinarii, were unable to inhibit S. frugiperda phenoloxidase activity. Heterologous expression of the X. nematophila rhabduscin cluster in these three strains was sufficient to restore inhibition. Interestingly, we observed pseudogenization of the X. poinarii rhabduscin gene cluster via the insertion of a 120 bp element into the isnA promoter. The inhibition of phenoloxidase activity by X. poinarii culture supernatants was restored by expression of the X. poinarii rhabduscin cluster under the control of an inducible Ptet promoter, consistent with recent pseudogenization. This study paves the way for advances in our understanding of the virulence of several entomopathogenic bacteria in non-model insects, such as the new invasive S. frugiperda species in Africa.

Highlights

Insects rely on innate immune responses to defend themselves against foreign microorganisms
It has been suggested that the X. nematophila rhabduscin gene cluster contributes to pathogenesis in the larvae of the highly susceptible species G. mellonella [27]
We show here that the X. nematophila rhabduscin gene cluster is not necessary for bacterial pathogenicity in insects of the genus Spodoptera

Summary

Introduction

Insects rely on innate immune responses to defend themselves against foreign microorganisms. Photorhabdus and Xenorhabdus can interact directly with the insect immune system following their transfer from the nematode gut to the insect hemolymph [16,17,18] They target the hemocytes with hemolysins [19], block the activity of antimicrobial peptides [20], and inhibit prophenoloxidase activation and eicosanoid-mediated nodulation [21]. Spodoptera phenoloxidase and Xenorhabdus rhabduscin asymbiotica, which causes opportunistic infections in humans, acts as a potent inhibitor of the mammalian alternative complement pathway [29] These findings suggest an important role for the isnAB genes in host-pathogen interactions. We investigated the importance of rhabduscin for the process of insect infection further, by evaluating the impact of Xenorhabdus nematophila rhabduscin synthesis on virulence and phenoloxidase activity in insects of agronomic importance from the genus Spodoptera. We focused on X. poinarii, in which we observed pseudogenization of the endogenous X. poinarii rhabduscin genes through an insertion into the promoter region

Materials and methods

Results

Discussion