Characterization of thrombin inhibitory mechanism of rAaTI, a Kazal-type inhibitor from Aedes aegypti with anticoagulant activity

Saliva of blood-sucking arthropods contains a complex mixture of anti-haemostatic, anti-inflammatory and immune-modulator compounds. Among anti-haemostatic factors, there are anticoagulants, vasodilators and platelet aggregation inhibitors. Previous analyses of the sialotranscriptome of Aedes aegypti showed the potential presence of a Kazal-type serine protease inhibitor in the female salivary glands, carcass and also in the whole male, which inhibitor we named AaTI (A. aegypti thrombin inhibitor). Recently, we expressed and characterized rAaTI as a trypsin inhibitor, and its anticoagulant activity [1]. in this work we characterized the thrombin inhibition mechanism of rAaTI. Recombinant AaTI was able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. in contrast, AaTI Delta (rAaTI truncated form) and C-terminal AaTI acidic tail prolong only thrombin time. in the competition assay, rAaTI, AaTI Delta or C-terminal AaTI acidic tail thrombin interactions seem to be affected by heparin but not by hirudin, suggesting that rAaTI binds to thrombin exosite 2. Finally, the thrombin inhibition assay of rAaTI showed an uncompetitive inhibition mechanism. in conclusion, rAaTI can probably inhibit thrombin by interacting with thrombin exosite 2, and the interaction is not mediated by the AaTI C-terminal region, since the truncated AaTI Delta form also prolongs thrombin time. (C) 2010 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilInst Butantan, Lab Fisiopatol, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilFAPESP: 05/03514-9FAPESP: 07/56614-6CNPq: 470070/2004-8CNPq: 575829/2008-7Web of Scienc

Comparative proteomic profiling and functional characterization of venom pooled from captive Crotalus durissus terrificus specimens and the Brazilian crotalic reference venom

10 págs, 5 figuras. Información suplementaria en: https://doi.org/10.1016/j.toxicon.2020.07.001The South American rattlesnake Crotalus durissus spp has a wide geographic distribution in Brazil. Although responsible for only a low proportion of ophidian accidents, it is considered one of the most medically important species of venomous snakes due to the high mortality rate (1.87%). Snake venom is a complex phenotype commonly subjected to individual intraspecific, ontogenetic and geographic variability. Compositional differences in pooled venom used in the immunization process may impact the efficacy of the antivenom. In order to assure standardized high-quality antivenom, the potency of each Brazilian crotalic antivenom batch is determined against the 'Brazilian Crotalic Reference Venom' (BCRV). BCRV is produced by Instituto Butantan using venom obtained from the first milking of recently wild-caught C. d. terrificus specimens brought to the Institute. The decrease in the number of snake donations experienced in recent years can become a threat to the production of future batches of BCRV. To evaluate the feasibility of using venom from long-term captive animals in the formulation of BCRV, we have compared the proteomic, biochemical and biological profiles of C. d. terrificus venom pooled from captive specimens (CVP- captive venom pool) and BCRV. Electrophoretic and venomics analyses revealed a very similar venom composition profile, but also certain differences in toxins abundance, with some low abundant protein families found only in BCRV. Enzymatic (L-amino acid oxidase, phospholipase A2 and proteolytic) and biological (myotoxic and coagulant) activities showed higher values in CVP than in BCRV. CVP also possessed slightly higher lethal effect, although the Instituto Butantan crotalic antivenom showed equivalent potency neutralizing BCRV and CVP. Our results strongly suggest that venom from long-term captive C. d. terrificus might be a valid alternative to generate an immunization mixture of equivalent quality to the currently in use reference venom.This work was financially supported by Fundaç~ao de Amparo �a Pesquisa do Estado de S~ao Paulo – FAPESP (grant numbers: 2017/ 01890–0, 2018/25786–0; 2018/25899–0, 2017/26533–6 and 2017/ 16908–2), Conselho Nacional de Desenvolvimento Científico e Tec-nol�ogico (CNPq) (405399/2018–9) and partly funded by grant BFU2017-89103-P (Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain).Peer reviewe

Differential expression profiles in the midgut of Triatoma infestans infected with Trypanosoma cruzi.

Chagas disease, or American trypanosomiasis, is a parasitic disease caused by the protozoan Trypanosoma cruzi and is transmitted by insects from the Triatominae subfamily. To identify components involved in the protozoan-vector relationship, we constructed and analyzed cDNA libraries from RNA isolated from the midguts of uninfected and T. cruzi-infected Triatoma infestans, which are major vectors of Chagas disease. We generated approximately 440 high-quality Expressed Sequence Tags (ESTs) from each T. infestans midgut cDNA library. The sequences were grouped in 380 clusters, representing an average length of 664.78 base pairs (bp). Many clusters were not classified functionally, representing unknown transcripts. Several transcripts involved in different processes (e.g., detoxification) showed differential expression in response to T. cruzi infection. Lysozyme, cathepsin D, a nitrophorin-like protein and a putative 14 kDa protein were significantly upregulated upon infection, whereas thioredoxin reductase was downregulated. In addition, we identified several transcripts related to metabolic processes or immunity with unchanged expressions, including infestin, lipocalins and defensins. We also detected ESTs encoding juvenile hormone binding protein (JHBP), which seems to be involved in insect development and could be a target in control strategies for the vector. This work demonstrates differential gene expression upon T. cruzi infection in the midgut of T. infestans. These data expand the current knowledge regarding vector-parasite interactions for Chagas disease

A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito`s development. (C) 2010 Elsevier Masson SAS. All rights reserved.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[05/03514-9]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq Conselho Nacional de Desenvolvimento Cientifico e Tecnologico[470070/2004-8]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq Conselho Nacional de Desenvolvimento Cientifico e Tecnologico[575829/2008-7]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP[07/56614-6

qRT-PCR of infestin.

<p>Adult insects infected with <i>T. cruzi</i> and uninfected <i>T. infestans</i> were used for analysis (three biological samples were used for both the uninfected and infected groups). All data were normalized to 18S ribosomal RNA, representing the mean (n = 3) of identical triplicates ± standard deviation. An unpaired <i>t</i> test was performed for statistical analysis.</p

Functional classification of transcripts that originated from upregulated contigs.

<p>Functional classification of transcripts that originated from upregulated contigs.</p

A pie chart showing the species distribution of BLASTx hits of the <i>T. infestans</i> clusters for several organisms.

<p>A pie chart showing the species distribution of BLASTx hits of the <i>T. infestans</i> clusters for several organisms.</p

Functional classification of transcripts that originated from downregulated contigs.

<p>Functional classification of transcripts that originated from downregulated contigs.</p