A Serpin Released by an Entomopathogen Impairs Clot Formation in Insect Defense System

Abstract

Steinernema carpocapsae is an entomopathogenic nematode widely used for the control of insect pests due to its virulence, which is mainly attributed to the ability the parasitic stage has to overcome insect defences. To identify the mechanisms underlying such a characteristic, we studied a novel serpin-like inhibitor (sc-srp-6) that was detected in a transcriptome analysis. Recombinant Sc-SRP-6 produced in Escherichia coli had a native fold of serpins belonging to the α-1-peptidase family and exhibited inhibitory activity against trypsin and α-chymotrypsin with Ki of 0.42×10−7 M and 1.22×10−7 M, respectively. Functional analysis revealed that Sc-SRP-6 inhibits insect digestive enzymes, thus preventing the hydrolysis of ingested particles. Moreover, Sc-SRP-6 impaired the formation of hard clots at the injury site, a major insect defence mechanism against invasive pathogens. Sc-SRP-6 does not prevent the formation of clot fibres and the activation of prophenoloxidases but impairs the incorporation of the melanin into the clot. Binding assays showed a complex formation between Sc-SRP-6 and three proteins in the hemolymph of lepidopteran required for clotting, apolipophorin, hexamerin and trypsin-like, although the catalytic inhibition occurred exclusively in trypsin-like. This data allowed the conclusion that Sc-SRP-6 promotes nematode virulence by inhibiting insect gut juices and by impairing immune clot reaction.

The Global Transcription Factor Lrp Controls Virulence Modulation in Xenorhabdus nematophila

“…X. nematophila infects insects by virtue of its association with the nematode Steinernema carpocapsae. The nematode infects and releases X. nematophila into the insect's blood-containing body cavity, wherein both partners contribute to suppressing insect immunity and causing insect death (10)(11)(12)(13). Both partners multiply by consuming nutrients within the cadaver, putatively liberated by bacterially produced exoenzymes, such as proteases and lipases (14).…”

Partner-specific induction ofSpodoptera frugiperdaimmune genes in response to the entomopathogenic nematobacterial complexSteinernema carpocapsae-Xenorhabdus nematophila

“…In addition, the induction of coagulation responses is particularly interesting, since many clotting factors participate in D. melanogaster resistance to infestation by another type of NBC, the Heterorhabiditis bacteriophora-Photorhabdus luminescens association (91–94). Moreover, despite S. carpocapsae does not pierce the insects’ cuticles as H. bacteriophora (1), it has been shown to express at least two secreted proteases with inhibitory activities towards the formation of clot fibers and coagulation-associated pathogen trapping (26, 27). Once again, the induction of such immune responses is consistent with the previous identification of several virulence factors of the NBC targeting cellular immunity (26, 28, 29, 31–38).…”

“…These interactions have firstly been studied from the NBC point of view, which allowed the identification of a multitude of immunoevasive and immunosuppressive strategies. For instance, studies in Rhynchophorus ferrugineus and Galleria mellonella have respectively shown that the cuticle of S. carpocapsae is not recognized by the host’s immune system (24, 25) and that the nematode secretes protease inhibitors impairing the coagulation responses (26, 27). Studies in diverse insect models have also shown that both partners produce factors impairing melanization (28–31), hemocyte’s viability (32–36) and the production of cellular immune responses by several ways (27–29, 31, 37, 38).…”

The Imaginal Disc Growth Factors 2 and 3 participate in the Drosophila response to nematode infection

“…Understanding the molecular mechanisms of insect response to nematode infection is a crucial step towards interpreting the antiparasitic properties of the host innate immune system . Entomopathogenic (or insect pathogenic) nematodes have emerged as excellent models for studying the molecular basis of nematode parasitism and elucidating the function of molecules that promote nematode persistence in the host . A major advantage of entomopathogenic nematodes is that they are viable in the absence of their mutualistic bacteria (axenic nematodes); consequently, each partner in the mutualistic relationship can be separated and studied in isolation or in combination enabling host immune responses to be studied against each pathogen separately and against the nematode‐bacteria complex together …”

“…1 Entomopathogenic (or insect pathogenic) nematodes have emerged as excellent models for studying the molecular basis of nematode parasitism and elucidating the function of molecules that promote nematode persistence in the host. [2][3][4][5] A major advantage of entomopathogenic nematodes is that they are viable in the absence of their mutualistic bacteria (axenic nematodes) 6,7 ; consequently, each partner in the mutualistic relationship can be separated and studied in isolation or in combination enabling host immune responses to be studied against each pathogen separately and against the nematode-bacteria complex together. 6,8,9 The entomopathogenic nematode Steinernema carpocapsae forms a mutualistic relationship with the Gram-negative bacteria Xenorhabdus nematophila.…”