Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae

Autor(es)

Soares, Tatiane Sanches ^[1] ; Soares Torquato, Ricardo Jose ^[1] ; Gonzalez, Yamile Gonzalez ^[1] ; Alves Lemos, Francisco Jose ^[2] ; Tanaka, Aparecida Sadae ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Paulo, Dept Bioquim, Escola Paulista Med, BR-04044020 Sao Paulo, SP - Brazil

^[2] Univ Estadual Norte Fluminense, Biotecnol Lab, Rio De Janeiro, RJ - Brazil

• Tipo de documento: Artigo Científico
• Fonte: PARASITES & VECTORS; v. 8, OCT 6 2015.
• Citações Web of Science: 1

Resumo

Background: Dengue, transmitted primarily by the bites of infected Aedes aegypti L., is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies are primarily based on controlling the vector, using insecticides, but the appearance of resistance poses new challenges. Recently, highly selective protease inhibitors by phage display were obtained for digestive enzymes of the 4th instar larvae (L4) midgut. These mutants were not confirmed as a larvicide due to the low yield of the expression of these inhibitors. In the present study, chimera molecules were constructed based on the mutations at positions P1-P4' selected previously. The T6, T23 and T149 mutants were mixed with another Kunitz inhibitor, domain 1 of the inhibitor boophilin (D1). Methods: The chimeras T6/D1, T149/D1 and T23/D1 were expressed at high levels in P. pastoris yeast, purified by ionic exchange chromatography and their homogeneity was analyzed by SDS-PAGE. The chimera inhibitors were assayed against larval trypsin, chymotrypsin and elastase using specific chromogenic substrates. The inhibitors were assayed for their larvicide potential against L4. Results: The chimeras exhibited strong inhibitory activities against the larval digestive enzymes in a dose-dependent manner. T6/D1, T149/D1 and T23/D1 exhibited strong larvicidal activity against L4 of Ae. aegypti with inhibitor concentrations in the mu M range. A synergistic increase in mortality was observed when a mixture of the three chimeric inhibitors was tested. Conclusions: The strategy for constructing the chimeric inhibitors was successful. The chimeras showed strong larvicidal activity against Ae. aegypti. In the future, our findings can be used to design synthetic inhibitors for larvae digestive enzymes as an alternative method to control the dengue vector. (AU)

• Processo FAPESP: 09/05405-3 - Atualização do sequenciador automático de DNA da Unidade Multi-usuário do INFAR, UNIFESP-EPM
• Beneficiário: Aparecida Sadae Tanaka
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 09/06003-6 - Construção de uma biblioteca de inibidores de proteases em Sistema Phage Display e seleção de inibidores específicos para proteases digestivas de larvas de Aedes aegypti
• Beneficiário: Tatiane Sanches Soares
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 05/03514-9 - Estudos da função fisiológica e potencial biotecnológico de inibidores de proteases e anti-hemostáticos presentes em artrópodes hematófagos
• Beneficiário: Aparecida Sadae Tanaka
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 12/03657-8 - Inibidores e proteases de ectoparasitas: relação de estrutura-função e identificação do papel dessas moléculas na interação de vetores de doenças e seus agentes etiológicos
• Beneficiário: Aparecida Sadae Tanaka
• Modalidade de apoio: Auxílio à Pesquisa - Temático