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Publication
Role on serine protease inhibitors in the pathogenisis of Steinernema carpocapsae
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Abstract(s)
Steinernema carpocapsae é um nemátodo entomopatogénico altamente eficaz no controlo de diversas pestes de insectos, contudo os mecanismos envolvidos no seu processo parasitário não são inteiramente conhecidos. O presente estudo averigua o papel de inibidores de serino proteases expressados pelo estado parasita de S. Carpocapsae como potenciais factores de virulência. Obteve‐se e analisou‐se o cADN completo de seis inibidores e procedeu‐se ao estudo exaustivo das funções de dois deles, pertences às famílias serpina e Kunitz. Estudos in vitro mostraram que ambos são inibidores competitivos de proteases, sendo que a serpina inibe tripsina (IC50 = 17.3 μM and Ki = 1.22 x 10‐7 M) enquanto a Kunitz inibe quimotripsina e elastase (Ki of 2.6 x 10‐9 M and of 1.8 x 10‐9 M). Ensaios biológicos permitiram concluir que, apesar de não suprimirem a actividade da fenoloxidase, ambos inibidores afectam outros mecanismos da resposta imunitária. A serpina reduziu a formação de coágulos rígidos, uma defesa fundamental do insecto contra patógenos, bloqueando a interacção entre as fibras de coágulo e a melanina. Por seu turno, a Kunitz inibiu a agregação de hemócitos e diminuiu o encapsulamento de partículas estranhas, presumivelmente através da interacção com proteínas de padrões de reconhecimento do insecto, contribuindo desta forma para a ausência de respostas imunitárias. Este é o primeiro trabalho que conclui com sucesso que inibidores de serino proteases participam no estado parasitário do S. carpocapsae e explica como estes interagem com o sistema de defesa de insectos. Isto abre novas possibilidades na melhoria selectiva deste nemátode como agente de controlo biológico de pragas e sua utilização como modelo de estudo da interacção hospedeiro‐parasita.
ABSTRACT: Steinernema carpocapsae is an entomopathogenic nematode highly effective in the control of several insect pests, however the mechanisms involved in its parasitic process are not entirely known. The present study ascertains the role of serine protease inhibitors expressed by the parasitic stage of S. carpocapsae as potential virulence factors. The full‐length cDNA of six inhibitors was obtained and analysed and the functions of two of them, belonging to families serpin and Kunitz, were extensively explored. In vitro studies showed that they are both competitive inhibitors of proteases with the serpin inhibiting trypsin (IC50 = 17.3 μM and Ki = 1.22 x 10‐7 M) while the Kunitz inhibited chymotrypsin and elastase (Ki of 2.6 x 10‐9 M and of 1.8 x 10‐9 M). Biological assays allowed concluding that, although they did not suppress phenoloxidase activity, both inhibitors affected other mechanisms of the insect’s immune response. The serpin impaired the formation of hard clots, a fundamental insect defense against pathogens, by blocking the interaction between clot fibers and melanin. On its part, the Kunitz inhibited the aggregation of haemocytes and impaired encapsulation of foreign particles, presumably by targeting insect pattern recognition proteins, therefore contributing to the absence of immunitary responses. This is the first work that successfully concludes that serine protease inhibitors participate in the parasitic stage of S. carpocapsae and explains how they interact with the defense system of insects. This opens new possibilities in the genetical improvement of this nematode as a biological control agent and its use as a model to study host‐parasite interaction.
ABSTRACT: Steinernema carpocapsae is an entomopathogenic nematode highly effective in the control of several insect pests, however the mechanisms involved in its parasitic process are not entirely known. The present study ascertains the role of serine protease inhibitors expressed by the parasitic stage of S. carpocapsae as potential virulence factors. The full‐length cDNA of six inhibitors was obtained and analysed and the functions of two of them, belonging to families serpin and Kunitz, were extensively explored. In vitro studies showed that they are both competitive inhibitors of proteases with the serpin inhibiting trypsin (IC50 = 17.3 μM and Ki = 1.22 x 10‐7 M) while the Kunitz inhibited chymotrypsin and elastase (Ki of 2.6 x 10‐9 M and of 1.8 x 10‐9 M). Biological assays allowed concluding that, although they did not suppress phenoloxidase activity, both inhibitors affected other mechanisms of the insect’s immune response. The serpin impaired the formation of hard clots, a fundamental insect defense against pathogens, by blocking the interaction between clot fibers and melanin. On its part, the Kunitz inhibited the aggregation of haemocytes and impaired encapsulation of foreign particles, presumably by targeting insect pattern recognition proteins, therefore contributing to the absence of immunitary responses. This is the first work that successfully concludes that serine protease inhibitors participate in the parasitic stage of S. carpocapsae and explains how they interact with the defense system of insects. This opens new possibilities in the genetical improvement of this nematode as a biological control agent and its use as a model to study host‐parasite interaction.
Description
Tese de Doutoramento, Biologia (Biologia Celular e Molecular), 18 de Novembro de 2013, Universidade dos Açores.
Keywords
Inibidores de Serino Proteases Nemátodo Entomopatogénico Controlo Biológico Steinernema carpocapsae Entomopathogenic Nematodes Serine Protease Inhibitors
Pedagogical Context
Citation
Martinez Ávila, Mónica. "Role on serine protease inhibitors in the pathogenisis of Steinernema carpocapsae". 2013. xvi, 115 p.. (Tese de Doutoramento, Biologia: Biologia Celular e Molecular) - Ponta Delgada: Universidade dos Açores, 2013.