Purificação e caracterização bioquímica de um inibidor de tripsina de sementes de Cassia leiandra Benth. e avaliação de sua atividade inseticida contra Aedes aegypti (Diptera: Culicidae)

Abstract

The World Health Organization (WHO) considers dengue virus infection one of the most important public health problems. Aedes aegypti (Diptera: Culicidae), a blood-sucking insect that lives and cohabits with the human population in tropical and subtropical areas, is the principal vector responsible for dengue virus transmission [3]. Ae. aegypti is also the main transmitter of Chikungunya and Zika viruses. To eliminate dengue mosquitoes, chemical control with insecticides is widely used. However, prolonged and indiscriminate application of these compounds has favored the appearance of resistant insects. This issue has driven the search for natural compounds with insecticidal activity, and plants are potential sources of such constituents. This present study reports the purification and biochemical characterization of a trypsin inhibitor (ClTI) from the seeds of Cassia leiandra Benth. (Fabaceae, Caesalpinioideae), a plant species native to the Amazon rainforest. In addition, the ClTI action on Ae. aegypti midgut proteases and its effect on the development and survival of this mosquito are assessed. ClTI was purified by DEAE-Cellulose and trypsin-Sepharose 4B chromatography, with a 15.5-fold purification and 2.4% yield. ClTI is composed of one polypeptide chain of 19,484 Da, as revealed by ESI mass spectrometry, is non-glycosylated, and comprises 35% β-sheets, 14% β-turns, and 50% disordered structures. ClTI is an uncompetitive inhibitor of bovine trypsin (IC50 of 33.81 x 10-8 M), with a Ki of 6.25 x 10-8 M, and has an amino acid sequence similar to other inhibitors of the Kunitz-type family. ClTI was stable over a broad range of pH (2.2–10.0) and temperature (30–70 °C), but DTT incubation caused a partial loss of inhibitory activity. ClTI (4.65 x 10-6 M) promoted 50% activity reduction of Ae. aegypti midgut proteases, showed a dose-dependent acute toxicity on Ae. aegypti 3rd instar larvae, with an LC50 of 2.28 x 10-2 M, and after ten days of exposure, caused a 24-h delay of larval development and 44% mortality. Our results are an important contribution to a better understanding of the insecticidal activity of protease inhibitors, suggesting that ClTI has biotechnological potential as an alternative strategy to control this multiple disease vector, which may be used alone or in combination with other insecticidal compounds to produce enhanced toxicity.