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

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 (Δ isnAB and Δ GT 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 Δ isnAB mutant was unable to inhibit phenoloxidase, whereas Δ GT 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 P tet 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.

Xenorhabdus spp.: An Overview of the Useful Facets of Mutualistic Bacteria of Entomopathogenic Nematodes

“…All tested X. nematophila strains showed insecticidal activity against representative pests of three insect orders; the cabbage white caterpillar Pieris brassicae (Lepidoptera: Pieridae), the mosquito larva Aedes aegypti (Diptera: Culicidae), and the mustard beetle Phaedon cochleariae (Coleoptera: Chrysomelidae) [52]. In this study and others [40,44,55,56], an important note is the variation in the abilities of different Xenorhabdus species/strains to kill/inhibit the growth of the intended pest or pathogen. These variations are based either on the ability of each Xenorhabdus species/strain to generate effective metabolites or the relative susceptibility/tolerance of the targeted pest/pathogen.…”

“…Therefore, it can be mostly presumed that the distribution and diversity of the EPN species is only an artefact of the linked sampling efforts [18]. However, growing interest is mainly dedicated to these bacteria when applied to suppress pests and pathogens independently, i.e., without the EPN partners [5,[40][41][42][43][44].…”

“…Generally, physical parameters, especially pH, temperature, and sodium chloride, could variably affect the mortality percentage induced by these metabolites to the G. mellonella larvae [101]. Nematophin Antibacterial and antifungal [114] Xenocin Antibacterial [87] Xenorhabdicin (R-type bacteriocins) Antibacterial [44] Xenocoumacins Antibacterial, antifungal, and antiulcer [115] Xenortides Antiprotozoal and cytotoxic [116] Rhabdopeptides Antiprotozoal, insecticidal, and cytotoxic [92] Xenematides Antibacterial and insecticidal [117] Rhabducin Insecticidal [117] Oxindole and Benzylidene-acetone Antibacterial, immuno-suppressant, and insecticidal [41,85] Antimicrobial multidrug-resistant pathogens [120] * Compiled from recent references [22,41,42].…”

Taxonomy of Non-sporulating Bacteria Tested in Biological Control Against Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae)

“…There are new molecules that can act against the pest. Rhabduscin synthesized by Xenorhabdus nematophila (non-spore forming) is an isocyanide that acts at nanomolar-level as an inhibitor of phenoloxidase, a key component of the insect innate immune system (Nuñez-Valdez et al, 2019). Thus, this drug is able to kill the insect-pest once it cannot fight against this and others applied in addition a (Crawford et al, 2012).…”

Nematophilic bacteria associated with entomopathogenic nematodes and drug development of their biomolecules

“…Despite the following grouping of biomolecules from both Xenorhabdus and Photorhabdus according to the similarity of ecological function, their biosynthesis is species-specific. The first grouping is insecticidal toxins, and these can be divided into insect immune suppressors via inhibition of phenoloxidase pathway:-1,2-benzene dicarboxylic acid (PA) ( Ullah et al, 2014 ), benzylideneacetone (BZA) ( Song et al, 2011 ), rhabduscin ( Crawford et al, 2012 ; Eugenia Nuñez-Valdez et al, 2019 ), and 1,3-dihydroxy-2-(isopropyl)-5-(2-phenylethenyl)benzene ( Eleftherianos et al, 2007 ); hemocyte pore-forming complexes: Xenorhabdus particulate toxins (Xpt) ( Sheets et al, 2011 ), toxin complex toxins (Tc) ( Blackburn et al, 1998 ), and Xenorhabdus α-xenorhabdolysin toxins (Xax) ( Vigneux et al, 2007 ); apoptosis inducers: make caterpillar floppy toxins (Mcf) ( Daborn et al, 2002 ; Dowling et al, 2004 ) and PaTox toxins ( Jank et al, 2016 ); and those with yet unknown modes of action: PirAB ( Yang et al, 2017 ) and xenocyloin ( Proschak et al, 2014 ). Another ecological function of secreted metabolites is bioconversion by enzymes such as lipases, proteases, amylases, and proteases—their respective genes are enriched in Xenorhabdus and Photorhabdus genomes ( Chaston et al, 2011 )—creating a rich nutrient pool.…”