title: 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.
authors.affiliation: Centro Investigação Recursos Naturais do Centro de Biotecnologia dos Açores, Associate Laboratory of Institute for Biotechnology and Bioengineering, Department of Biology, University of Azores, Ponta Delgada, Portugal.
authors.forename: Duarte
authors.initials: D
authors.lastname: Toubarro
authors.forename: Mónica M
authors.initials: MM
authors.lastname: Avila
authors.forename: Youjin
authors.initials: Y
authors.lastname: Hao
authors.forename: Natesan
authors.initials: N
authors.lastname: Balasubramanian
authors.forename: Yingjun
authors.initials: Y
authors.lastname: Jing
authors.forename: Rafael
authors.initials: R
authors.lastname: Montiel
authors.forename: Tiago Q
authors.initials: TQ
authors.lastname: Faria
authors.forename: Rui M
authors.initials: RM
authors.lastname: Brito
authors.forename: Nelson
authors.initials: N
authors.lastname: Simões
mesh_terms: D000818:Animals; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism*; D006458:Hemolymph / Q000187:drug effects / Q000378:metabolism; D019476:Insect Proteins / Q000378:metabolism; D007313:Insecta / Q000378:metabolism / Q000469:parasitology*; D017168:Rhabditida / Q000378:metabolism* / Q000472:pathogenicity; D015843:Serpins / Q000235:genetics / Q000378:metabolism*
chemical_list: D015801:Helminth Proteins; D019476:Insect Proteins; D015843:Serpins