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

Author(s)

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

Affiliation

^[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

• Document type: Journal article
• Source: PARASITES & VECTORS; v. 8, OCT 6 2015.
• Web of Science Citations: 1

Abstract

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)

• FAPESP's process: 09/05405-3 - Automatic DNA sequencer update of INFAR Multi-user Laboratory, UNIFESP-EPM
• Grantee: Aparecida Sadae Tanaka
• Support Opportunities: Regular Research Grants
• FAPESP's process: 09/06003-6 - Construction of a protease inhibitor library in phage display system and its selection of specific inhibitors for digestive proteases of Aedes aegypti
• Grantee: Tatiane Sanches Soares
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 05/03514-9 - Studies of the physiological function and biotechnological potential of protease inhibitors and anti-hemostatics in hematophagous arthropods
• Grantee: Aparecida Sadae Tanaka
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 12/03657-8 - Inhibitor and proteases of ectoparasites: relationship of structure-function and identification of the role of these molecules in the interaction of diseases vector e their etiological agents
• Grantee: Aparecida Sadae Tanaka
• Support Opportunities: Research Projects - Thematic Grants