Abstract
Steinernema carpocapsae is a nematode pathogenic in a wide variety of insect species. The great pathogenicity of this nematode has been ascribed to its ability to overcome the host immune response; however, little is known about the mechanisms involved in this process. The analysis of an expressed sequence tags (EST) library in the nematode during the infective phase was performed and a highly abundant contig homologous to serine protease inhibitors was identified. In this work, we show that this contig is part of a 641-bp cDNA that encodes a BPTI-Kunitz family inhibitor (Sc-KU-4), which is up-regulated in the parasite during invasion and installation. Recombinant Sc-KU-4 protein was produced in Escherichia coli and shown to inhibit chymotrypsin and elastase activities in a dose-dependent manner by a competitive mechanism with Ki values of 1.8 nM and 2.6 nM, respectively. Sc-KU-4 also inhibited trypsin and thrombin activities to a lesser extent. Studies of the mode of action of Sc-KU-4 and its effects on insect defenses suggest that although Sc-KU-4 did not inhibit the activation of hemocytes or the formation of clotting fibers, it did inhibit hemocyte aggregation and the entrapment of foreign particles by fibers. Moreover, Sc-KU-4 avoided encapsulation and the deposition of clotting materials, which usually occurs in response to foreign particles. We show by protein-protein interaction that Sc-KU-4 targets recognition proteins of insect immune system such as masquerade-like and serine protease-like homologs. The interaction of Sc-KU-4 with these proteins explains the ability of the nematode to overcome host reactions and its large pathogenic spectrum, once these immune proteins are well conserved in insects. The discovery of this inhibitor targeting insect recognition proteins opens new avenues for the development of S . carpocapsae as a biological control agent and provides a new tool to study host-pathogen interactions.
Highlights
Steinernema carpocapsae is an entomopathogenic nematode (EPN) that is currently used to control insect pests, owing to its high virulence against a wide variety of insects [1]
Molecular Characterization of Sc-KU-4 A full-length 641 bp cDNA was produced using primers designed based in the expressed sequence tags (EST) sequence predicted for a serine protease inhibitor of S. carpocapsae
The alignment of gDNA and cDNA suggests that the sc-ku-4 gene has five exons containing 43, 72, 123, 140, and 175 bp, which are coded for by residues 1-43, 92-163, 211-333, 380-520 and 566-741, respectively, all flanked by the typical splice signals AG/GT
Summary
Steinernema carpocapsae is an entomopathogenic nematode (EPN) that is currently used to control insect pests, owing to its high virulence against a wide variety of insects [1]. In the encapsulation are participating soluble proteins from the haemocoel, proteins released from activated hemocytes and the hemocytes themselves [4]. This process involves three main events: cell activation, clot formation and activation of phenoloxidase [4,8]. The clot formation involves the activation of soluble proteins in the hemocoel, such as transglutaminase, lipophorin, hexamerins, and fondue and proteins derived from hemocytes, for instance hemolectin and tiggrin, that lead to the clotting of hemolymph forming a network of fibers that bind together to isolate the foreign body [11,12]. The three systems work together leading to the formation of hard clots that efficiently protect from invasive pathogens [14]
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