Date Published: February 22, 2019
Publisher: Public Library of Science
Author(s): Maria Eugenia Nuñez-Valdez, Anne Lanois, Sylvie Pagès, Bernard Duvic, Sophie Gaudriault, Erjun Ling.
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.
Insects rely on innate immune responses to defend themselves against foreign microorganisms. Their cellular defense mechanisms are mediated by hemocytes, the immunity cells of insects. Hemocytes play a key role in the phagocytosis, nodulation and encapsulation of intruding pathogens. The main humoral mechanisms involve antimicrobial peptides and the prophenoloxidase (PO) system . The PO system is responsible for melanization, a process in which an insoluble brown-black pigment, melanin, is synthesized and deposited. Melanization takes place in three steps. The first of these steps is the recognition of pathogen-associated molecular patterns (PAMPs), such as the peptidoglycans or lipopolysaccharides of bacteria and the β-1,3-glucans of fungi. In the second step, a precursor, prophenoloxidase, is cleaved by a serine protease cascade to generate the active enzyme, phenoloxidase. In the third step, phenoloxidase catalyzes the oxidation of phenolic compounds, which then polymerize to form melanin. Melanin seals the wound (hemolymph clotting) and traps the intruding microorganisms (nodulation and encapsulation) [2–5]. Moreover, the polymerization of melanin generates redox-active melanogenic intermediates. These intermediates, alone or together with reactive intermediates of oxygen and nitrogen, are highly cytotoxic .
We show here that the X. nematophila rhabduscin gene cluster is not necessary for bacterial pathogenicity in insects of the genus Spodoptera. However, this cluster is sufficient for inhibition of the S. frugiperda (Sf) phenoloxidase, a major component of the humoral immune system of the insect. This phenotype was confirmed in X. nematophila ATCC19061T (Xn), but also by heterologous expression of the Xn rhabduscin gene cluster in species of Enterobacteriaceae devoid of any natural ability to inhibit phenoloxidase.