Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae
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 resis...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2015-10-01
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| Series: | Parasites & Vectors |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13071-015-1127-4 |
| _version_ | 1797811884716457984 |
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| author | Tatiane Sanches Soares Ricardo José Soares Torquato Yamile Gonzalez Gonzalez Francisco Jose Alves Lemos Aparecida Sadae Tanaka |
| author_facet | Tatiane Sanches Soares Ricardo José Soares Torquato Yamile Gonzalez Gonzalez Francisco Jose Alves Lemos Aparecida Sadae Tanaka |
| author_sort | Tatiane Sanches Soares |
| collection | DOAJ |
| description | 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 μM 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. |
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| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-03-13T07:29:21Z |
| publishDate | 2015-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Parasites & Vectors |
| spelling | doaj.art-527ae683169c4ad8b01e424757e33c8f2023-06-04T11:10:12ZengBMCParasites & Vectors1756-33052015-10-01811810.1186/s13071-015-1127-4Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvaeTatiane Sanches Soares0Ricardo José Soares Torquato1Yamile Gonzalez Gonzalez2Francisco Jose Alves Lemos3Aparecida Sadae Tanaka4Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São PauloDepartamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São PauloDepartamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São PauloLaboratório de Biotecnologia, Universidade Estadual do Norte FluminenseDepartamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São PauloAbstract 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 μM 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.https://doi.org/10.1186/s13071-015-1127-4MosquitoSerine protease inhibitorLarvaeAedes aegyptiLarvicide |
| spellingShingle | Tatiane Sanches Soares Ricardo José Soares Torquato Yamile Gonzalez Gonzalez Francisco Jose Alves Lemos Aparecida Sadae Tanaka Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae Parasites & Vectors Mosquito Serine protease inhibitor Larvae Aedes aegypti Larvicide |
| title | Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae |
| title_full | Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae |
| title_fullStr | Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae |
| title_full_unstemmed | Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae |
| title_short | Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae |
| title_sort | production of serine protease inhibitors by mutagenesis and their effects on the mortality of aedes aegypti l larvae |
| topic | Mosquito Serine protease inhibitor Larvae Aedes aegypti Larvicide |
| url | https://doi.org/10.1186/s13071-015-1127-4 |
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