RESUMO
rBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential. Besides that, rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of rBmTI-A was modeled. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism spectroscopy data corroborated the secondary structure found by the homology modelling. Also, in circular dichroism data it was shown a thermostability of rBmTI-A until approximately 70 °C, corroborated by inhibitory assays toward trypsin.
Assuntos
Proteínas de Artrópodes/química , Simulação de Dinâmica Molecular , Rhipicephalus/química , Inibidores de Serina Proteinase/química , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/farmacologia , Modelos Animais de Doenças , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/metabolismo , Camundongos , Estabilidade Proteica , Enfisema Pulmonar/tratamento farmacológico , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Rhipicephalus/genética , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/farmacologiaRESUMO
Protease/anti-protease imbalance is the main pathogenic mechanism of emphysema and protease inhibitors have been recognized as potential molecules to treat the disease conditions. In this work the rBmTI-6 first domain (rBmTI-6-D1), a recombinant Kunitz-type serine proteinase inhibitor, was used to verify its effect in prevention or minimization of PPE-induced emphysema in mice. C57BL/6 mice were submitted to a PPE-induced emphysema model and treated with rBmTI-6-D1 before the emphysema development. We showed that the rBmTI-6-D1 treatment was sufficient to avoid the loss of elastic recoil, an effective decrease in alveolar enlargement and in the number of macrophages and lymphocytes in bronchoalveolar lavage fluid. Proteolytic analysis showed a significant increase in elastase activity in PPE-VE (induced emphysema) group that is controlled by rBmTI-6-D1. Kallikrein activity was decreased in the PPE-rBmTI6 (induced emphysema and inhibitor treated) group when compared to PPE-VE group. Although rBmTI-6-D1, did not present a neutrophil elastase (NE) inhibitory activity, the results show that the inhibitor interfered in the pathway of NE secretion in PPE-emphysema mice model. The role of rBmTI-6-D1 in the prevention of emphysema development in the mice model, apparently, is related with a control of inflammatory response due the trypsin/kallikrein inhibitory activity of rBmTI-6-D1.
Assuntos
Inflamação/tratamento farmacológico , Pulmão/efeitos dos fármacos , Enfisema Pulmonar/tratamento farmacológico , Inibidores de Serina Proteinase/química , Animais , Líquido da Lavagem Broncoalveolar/química , Modelos Animais de Doenças , Humanos , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Pulmão/fisiopatologia , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Polímeros/toxicidade , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/fisiopatologia , Rhipicephalus/química , Inibidores de Serina Proteinase/administração & dosagem , Inibidores de Serina Proteinase/genéticaRESUMO
The Rhipicephalus (Boophilus) microplus is an exclusive bovine ectoparasite responsible for the transmission of pathogens that decrease meat, leather and milk productions. Cattle vaccination is an alternative to control tick infestations, but the discovery of potential antigens is still a challenge for researchers. Recently, our group performed a midgut transcriptome of engorged R. microplus tick, and out of 800 ESTs sequences one cystatin-coding sequence was identified and named Rmcystatin-4. In order to understand the physiological role of Rmcystatin-4, the aim of this work was the expression, purification and functional characterization of a novel type 2 cystatin from the tick R. microplus. Rmcystatin-4 gene expression was identified mostly in tick midgut suggesting its possible role in blood digestion control. Our data showed that rRmcystatin-4 was successfully expressed in active form using Pichia pastoris system and the purified inhibitor presented high selectivity to BmCl-1 (Ki = 0.046 nM). Moreover, rRmcystatin-4 was able to impaired BmCl-1 activity towards bovine hemoglobin.
Assuntos
Proteínas de Artrópodes , Mucosa Intestinal/metabolismo , Rhipicephalus , Cistatinas Salivares , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/isolamento & purificação , Bovinos , Expressão Gênica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Rhipicephalus/química , Rhipicephalus/genética , Rhipicephalus/metabolismo , Cistatinas Salivares/biossíntese , Cistatinas Salivares/química , Cistatinas Salivares/genética , Cistatinas Salivares/isolamento & purificaçãoRESUMO
This study evaluated a recombinant aquaporin 1 protein of Rhipicephalus (Boophilus) microplus (RmAQP1) as antigen in a vaccine against R. sanguineus. Five dogs were immunized with RmAQP1 (10 µg) + adjuvant (Montanide) (G1), and five were inoculated with adjuvant only (G2), three times. Twenty-one days after the last immunization, animals of both groups were challenged with R. sanguineus larvae, nymphs and adults, and their biotic potential was compared. Blood samples were collected before each immunization and every 28 days after the last immunization for 10 weeks. Serum antibody titers (IgG) were assessed by ELISA. We observed that: engorgement period of adult females from G1 was 12% shorter than G2; larvae from G1 had 8.7% longer engorgement period than G2 and weighed 7.2% less; nymphs from G1 had 4.5% shorter engorgement period than G2 and weighed 3.6% less; although the antibody titers increased following the second immunization, they rapidly decreased after the third immunization. Results indicated low immunoprotection of RmAQP1 against adult R. sanguineus ticks, and possible efficacy on larvae and nymphs fed on immunized dogs. Further studies should be performed for a full evaluation of the immunoprotection of RmAQP1 against R. sanguineus infestations in dogs.(AU)
Este estudo avaliou a proteína recombinante (aquaporina) do carrapato Rhipicephalus (Boophilus) microplus como antígeno em vacina contra Rhipicephalus sanguineus. Cinco cães foram imunizados com RmAQP1 (10 µg) + adjuvante (G1) e cinco foram inoculados apenas com adjuvante (G2), três vezes. 21 dias após a última imunização todos os animais foram desafiados com larvas, ninfas e adultos de R. sanguineus, e potencial biótico dos carrapatos foi comparado. Amostras de sangue foram coletadas antes de cada imunização e a cada 28 dias após a última imunização, durante 10 semanas. Títulos de anticorpos dos soros dos cães foram avaliados por ELISA. Resultados: o período de ingurgitamento das fêmeas do G1 foi 12% mais curto que o período de ingurgitamento de G2; o período de ingurgitamento das larvas do G1 8,7% foi mais longo e o peso 7,2% menor que no caso de G2; o período de ingurgitamento das ninfas do G1 4,5% foi mais curto e peso 3,6% menor que no caso do G2; aumento dos títulos de anticorpos do G1 após a segunda imunização e declínio após a terceira imunização. Os resultados indicaram baixo potencial de imunoproteção de RmAQP1 contra R. sanguineus adultos, e possível eficácia contra larvas e ninfas, na dose testada. Sugere-se desenvolver novos estudos para melhor avaliação da eficácia de RmAQP1 contra R. sanguineus em cães.(AU)
Assuntos
Animais , Cães , Imunização/veterinária , Aquaporina 1/administração & dosagem , Aquaporina 1/imunologia , Rhipicephalus/química , Rhipicephalus sanguineus/imunologia , Antígenos/administração & dosagem , Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterináriaRESUMO
The aim of this study was to compare Larval Immersion Test in syringes, originally proposed by Souza et al. (2008), with two modified bioassays proposed by Silva et al. (2011) and Sindhu et al. (2012). Rhipicephalus (Boophilus) microplus larvae were exposed to nine dilutions of commercial acaricide. After 24 hours, the number of dead larvae was counted. The LC50 for the three methods were 0.186%, 0.125% and 0.153%, respectively. Mortality of larvae exposed to chemical dilutions on Silva et al. (2011) were significantly higher than other methods at lower concentrations, probably in consequence of longer exposure time of larvae to solutions. However, modifications proposed by Sindhu et al. (2012) make technic execution more practical and safer.(AU)
O objetivo deste trabalho foi comparar a técnica de imersão de larvas em seringas proposta originalmente por Souza et al. (2008), com duas modificações propostas por Silva et al. (2011) e Sindhu et al. (2012). Larvas de Rhipicephalus (Boophilus) microplus foram expostas a nove diluições de acaricida comercial. Após 24 horas, contabilizou-se o número de larvas mortas. A CL50 nos três métodos foi de 0,186%, 0,125% e 0,153%, respectivamente. Houve maior mortalidade de larvas no método de Silva et al. (2011), nas menores concentrações, quando comparado aos demais métodos, provavelmente devido ao maior tempo de exposição das larvas ao composto químico. Entretanto, as modificações propostas por Sindhu et al. (2012) proporcionam maior praticidade e segurança na execução da técnica.(AU)
Assuntos
Imersão , Rhipicephalus/química , Técnicas In Vitro , SeringasRESUMO
The aim of this study was to compare Larval Immersion Test in syringes, originally proposed by Souza et al. (2008), with two modified bioassays proposed by Silva et al. (2011) and Sindhu et al. (2012). Rhipicephalus (Boophilus) microplus larvae were exposed to nine dilutions of commercial acaricide. After 24 hours, the number of dead larvae was counted. The LC50 for the three methods were 0.186%, 0.125% and 0.153%, respectively. Mortality of larvae exposed to chemical dilutions on Silva et al. (2011) were significantly higher than other methods at lower concentrations, probably in consequence of longer exposure time of larvae to solutions. However, modifications proposed by Sindhu et al. (2012) make technic execution more practical and safer.
O objetivo deste trabalho foi comparar a técnica de imersão de larvas em seringas proposta originalmente por Souza et al. (2008), com duas modificações propostas por Silva et al. (2011) e Sindhu et al. (2012). Larvas de Rhipicephalus (Boophilus) microplus foram expostas a nove diluições de acaricida comercial. Após 24 horas, contabilizou-se o número de larvas mortas. A CL50 nos três métodos foi de 0,186%, 0,125% e 0,153%, respectivamente. Houve maior mortalidade de larvas no método de Silva et al. (2011), nas menores concentrações, quando comparado aos demais métodos, provavelmente devido ao maior tempo de exposição das larvas ao composto químico. Entretanto, as modificações propostas por Sindhu et al. (2012) proporcionam maior praticidade e segurança na execução da técnica.
Assuntos
Imersão , Rhipicephalus/química , Seringas , Técnicas In VitroRESUMO
Abstract The Rhipicephalus (Boophilus) microplus complex currently consists of five taxa, namely R. australis, R. annulatus, R. (B.) microplus clade A sensu, R. microplus clade B sensu, and R. (B.) microplus clade C sensu. Mitochondrial DNA-based methods help taxonomists when they are facing the morpho-taxonomic problem of distinguishing members of the R. (B.) microplus complex. The purpose of this study was to perform molecular characterization of ticks in all five regions of Brazil and infer their phylogenetic relationships. Molecular analysis characterized 10 haplotypes of the COX-1 gene. Molecular network analysis revealed that haplotype H-2 was the most dispersed of the studied populations (n = 11). Haplotype H-3 (n = 2) had the greatest genetic differentiation when compared to other Brazilian populations. A Bayesian phylogenetic tree of the COX-1 gene obtained strong support. In addition, it was observed that the population of R. (B.) microplus haplotype H-3 exhibited diverging branches among the other Brazilian populations in the study. The study concludes that the different regions of Brazil have R. (B.) microplus tick populations with distinct haplotypes.(AU)
Resumo Carrapatos do complexo R. (B.) microplus se distribuem em cinco taxa: R. australis, R. annulatus, R. (B.) microplus clado A sensu R. microplus clado B sensue e R. (B.) microplus clado C sensu. Métodos baseados no DNA mitocondrial podem auxiliar taxonomistas quando há dificuldades em estabelecer diferenças morfológicas para distinguir membros do complexo R. (B.) microplus. O objetivo deste estudo foi a caracterização molecular e a inferência de relações filogenéticas em carrapatos de todas as cinco regiões geográficas do Brasil. Para o gene COX-1, a análise molecular caracterizou 10 haplótipos. Na análise molecular em rede foi observado que o haplótipo H-2 é o mais disperso entre as populações (n=11). O haplótipo H-3 (n=2) foi o que obteve maior diferenciação genética ao ser comparado com outras populações brasileiras. A árvore filogenética Bayesiana de gene COX-1 gerou suporte robusto e foi observado que a população de R. (B.) microplus haplótipo H-3 apresentou ramificação com divergência entre as outras populações brasileiras apresentadas neste estudo. Conclui-se que as populações brasileiras possuem diversidade haplotípica com divergência entre as diversas populações de R. (B.) microplus no Brasil.(AU)
Assuntos
Rhipicephalus/química , Rhipicephalus/classificação , Rhipicephalus/genéticaRESUMO
The aim of this study was to assess the activity of aqueous (AE) and ethanolic extracts (EE) and pilocarpine hydrochloride, which were extracted and isolated from Pilocarpus microphyllus (Jaborandi), respectively, on Rhipicephalus (Boophilus) microplus. High performance liquid chromatography (HPLC) was performed to quantify these compounds. Larval packet and adult immersion tests were conducted with different concentrations. Five AE and EE concentrations, ranging from 6.2 to 100.0 mg mL1, and six concentrations of pilocarpine hydrochloride, ranging from 0.7 to 24.0 mg mL1, were tested. The lethal concentration (LC50) of each extract for larvae and engorged females was calculated through Probit analysis. The concentration of pilocarpine hydrochloride obtained from the EE and the AE was 1.3 and 0.3% (m/m), respectively. Pilocarpine hydrochloride presented the highest acaricidal activity on larvae (LC50 2.6 mg mL1) and engorged females (LC50 11.8 mg mL1) of R.(B.) microplus, followed by the EE which presented LC50 of 56.4 and 15.9 mg mL1, for larvae and engorged females, respectively. Such results indicate that pilocarpine hydrochloride has acaricidal activity, and may be the primary compound responsible for this activity by P. microphyllus EE.(AU)
O objetivo desse estudo foi avaliar a atividade dos extratos aquoso (AE) e etanólico (EE) e do cloridrato de pilocarpina, que foram, respectivamente, extraídos e isolado de Pilocarpus microphyllus (Jaborandi), sobre Rhipicephalus (Boophilus) microplus. Cromatografia líquida de alta eficiência foi realizada para quantificação dos compostos. Testes de pacote de larvas e de imersão de adultos foram realizados com diferentes concentrações. Cinco concentrações do AE e EE variando de 6,2 a 100,0 mg mL1 e seis concentrações do cloridrato de pilocarpina variando de 0,7 a 24,0 mg mL1 foram testadas. A concentração letal (CL50) de cada extrato para larvas e fêmeas ingurgitadas foi estimada por meio da análise Probit. A concentração de cloridrato de pilocarpina obtida do EE e AE foi de 1,3 e 0,3% (m/m), respectivamente. O cloridrato de pilocarpina apresentou a maior atividade carrapaticida sobre larvas (CL50 2,6 mg mL1) e fêmeas ingurgitadas (CL50 11,8 mg mL1) de R. (B.) microplus, seguido do EE que apresentou CL50 de 56,4 e 15,9 mg mL1, para larvas e fêmeas ingurgitadas, respectivamente. Tais resultados indicam que o cloridrato de pilocarpina apresenta atividade carrapaticida e pode ser o principal responsável pela atividade acaricida do EE de P. microphyllus.(AU)
Assuntos
Rhipicephalus/química , Rhipicephalus/classificação , JaborandiRESUMO
In the present study, lab-on-a-chip electrophoresis (LoaC) was suggested as an alternative method to the conventional polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) to analyze raw cell-free tick hemolymph. Rhipicephalus microplus females were exposed to the entomopathogenic fungus Metarhizium anisopliae senso latu IBCB 116 strain and/or to the entomopathogenic nematode Heterorhabditis indica LPP1 strain. Hemolymph from not exposed or exposed ticks was collected 16 and 24 h after exposure and analyze by SDS-PAGE or LoaC. SDS-PAGE yielded 15 bands and LoaC electrophoresis 17 bands. Despite the differences in the number of bands, when the hemolymph protein profiles of exposed or unexposed ticks were compared in the same method, no suppressing or additional bands were detected among the treatments regardless the method (i.e., SDS-PAGE or chip electrophoresis using the Protein 230 Kit®). The potential of LoaC electrophoresis to detect protein bands from tick hemolymph was considered more efficient in comparison to the detection obtained using the traditional SDS-PAGE method, especially when it comes to protein subunits heavier than 100 KDa. LoaC electrophoresis provided a very good reproducibility, and is much faster than the conventional SDS-PAGE method, which requires several hours for one analysis. Despite both methods can be used to analyze tick hemolymph composition, LoaC was considered more suitable for cell-free hemolymph protein separation and detection. LoaC hemolymph band percent data reported changes in key proteins (i.e., HeLp and vitellogenin) exceptionally important for tick embryogenesis. This study reported, for the first time, tick hemolymph protein profile using LoaC.
Assuntos
Proteínas de Artrópodes/química , Eletroforese em Gel de Poliacrilamida/métodos , Fungos/fisiologia , Rhipicephalus/química , Rhipicephalus/microbiologia , Rhipicephalus/parasitologia , Animais , Proteínas de Artrópodes/metabolismo , Eletroforese em Gel de Poliacrilamida/instrumentação , Feminino , Fungos/classificação , Fungos/genética , Fungos/isolamento & purificação , Hemolinfa/química , Hemolinfa/metabolismo , Dispositivos Lab-On-A-Chip , Nematoides/classificação , Nematoides/genética , Nematoides/isolamento & purificação , Nematoides/fisiologia , Reprodutibilidade dos Testes , Rhipicephalus/metabolismoRESUMO
Aiming to characterize the potential off-target effects of fluazuron on ticks, biochemical analyses were conducted to evaluate changes in the carbohydrate metabolism of Rhipicephalus (Boophilus) microplus ticks after exposure to fluazuron. Hemolymph and fat body were collected from female ticks before and after (4, 8 and 15 days) exposure to fluazuron. Spectrophotometric analyses were done to quantify glucose concentration and lactate dehydrogenase (LDH) activity in the hemolymph and the concentration of glycogen in the ticks fat body. High Performance Liquid Chromatography (HPLC) was employed to determine the concentration of carboxylic acids in the hemolymph and to evaluate changes in intermediary metabolic processes requiring oxygen consumption. Increases in the levels of LDH activity and lactic acid concentration indicated that fluazuron enhanced fermentative metabolism in ticks. Exposure to fluazuron was also found to increase glucose concentrations in the hemolymph over time, although no significant differences were noted daily. In addition to expanding the body of knowledge about the mode of action of fluazuron, investigations into these mechanisms may also be useful in discovering new and as yet unexplored secondary effects.(AU)
Com o objetivo de caracterizar os efeitos não-alvo da ação do fluazuron, foram realizados testes bioquímicos para analisar possíveis alterações no metabolismo de carboidratos em carrapatos Rhipicephalus (Boophilus) microplus após sua exposição ao composto. Foram coletados hemolinfa e corpo gorduroso de fêmeas ingurgitadas antes e após (4, 8 e 15 dias) a exposição ao fluazuron. Análises espectrofotométricas foram usadas para quantificar a concentração de glicose e a atividade da lactato desidrogenase (LDH) na hemolinfa e concentração de glicogênio no corpo gorduroso. Cromatografia Líquida de Alta Eficiência (CLAE) foi usada para determinação das concentrações de ácidos carboxílicos na hemolinfa e avaliar possíveis alterações em metabolismo intermediário em relação ao consumo de oxigênio. Aumento na atividade de LDH e concentração de ácido lático indicaram que o fluazuron pode regular o metabolismo fermentativo em carrapatos. A exposição ao fluazuron também aumentou a concentração de glicose na hemolinfa, apesar de não ter havido diferença significativa na comparação entre as médias no mesmo dia de avaliação. Além de aumentar o conhecimento sobre o modo de ação do fluazuron, investigações sobre tais mecanismos também são úteis no descobrimento de novos efeitos secundários ainda não explorados.(AU)
Assuntos
Animais , Feminino , Rhipicephalus/química , Rhipicephalus/metabolismo , Rhipicephalus/fisiologia , Reguladores de Crescimento de Plantas/análiseRESUMO
Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.
Assuntos
Rhipicephalus/química , Inibidores de Serina Proteinase/isolamento & purificação , Animais , Larva/química , ProteínasRESUMO
Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.
Carrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.
Assuntos
Animais , Rhipicephalus/química , Inibidores de Serina Proteinase/isolamento & purificação , Larva/química , ProteínasRESUMO
Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.
Carrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.
Assuntos
Animais , Rhipicephalus/química , Inibidores de Serina Proteinase/isolamento & purificação , Larva/química , ProteínasRESUMO
Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential roles in many organisms. In arthropods these proteins are involved in innate immune system, morphogenesis and development. In mammals serpins regulate pathways that are essential to life such as blood coagulation, fibrinolysis, inflammation and complement activation, some of which are considered the host's first line of defense to hematophagous and/or blood dueling parasites. Thus, it is hypothesized that ticks use serpins to evade host defense, facilitating parasitism. This study describes eighteen full-length cDNA sequences encoding serpins identified in Rhipicephalus (Boophilus) microplus, here named RmS 1-18 (R. microplus serpin). Spatial and temporal transcriptional profiling demonstrated that R. microplus serpins are transcribed during feeding, suggesting their participation in tick physiology regulation. We speculate that the majority of R. microplus serpins are conserved in other ticks, as indicated by phylogeny analysis. Over half of the 18 RmSs are putatively functional in the extracellular environment, as indicated by putative signal peptides on 11 of 18 serpins. Comparative modeling and structural-based alignment revealed that R. microplus serpins in this study retain the consensus secondary of typical serpins. This descriptive study enlarges the knowledge on the molecular biology of R. microplus, an important tick species.
Assuntos
Rhipicephalus/química , Serpinas/análise , Sequência de Aminoácidos , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Sequência Consenso , DNA Complementar/química , Feminino , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , RNA/química , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Rhipicephalus/classificação , Alinhamento de Sequência/veterinária , Serpinas/química , Espectrofotometria/veterinária , Infestações por Carrapato/parasitologia , Infestações por Carrapato/veterináriaRESUMO
The Rhipicephalus (Boophilus) microplus is the most important cattle tick in Brazil. For the ticks control, various studies on plants have been developed in an attempt to find extracts with acaricidal properties. This research was aimed at evaluating in vitro and in vivo effects of citronella (Cymbopogon winterianus Jowitt) oil on cattle ticks. Negative control group and eight concentrations of citronella oil and mineral oil (0.5; 1; 2; 5; 10; 20; 50; 100%), were used on in vitro trials with engorged female ticks. The efficacy against cattle ticks was 0; 34.5; 43; 48; 82; 99; 99; 100 and 100%, respectively, for citronella oil. For mineral oil contradictory results of acaricidal efficacy were observed. At the in vivo trial eighteen Holstein cows were allocated to three groups: negative control; citronella oil at 8.6% - level estimated by regression analysis, accounting for 95% efficacy against ticks on in vitro trial and amitraz at 0.025%. Engorged female ticks were counted before, mean of days -3, -2, -1, and after treatment, 1, 2, 3, 5, 7, 10, 14 and 21 days. On the 1 st and 2nd milked after treatments, physiologic variables were evaluate (negative control x phytotherapic treatment). The efficacy against tick was 0; 91.3 and 60%, respectively, at 21 days after treatment. Similar results were found with physiologic variables between treatments.
O Rhipicephalus (Boophilus) microplus é o carrapato bovino mais importante no Brasil. Para o controle do carrapato, vários estudos com plantas têm sido desenvolvidos numa tentativa de encontrar extratos com propriedades acaricidas. Esta pesquisa foi conduzida com o objetivo de avaliar o efeito in vitro e in vivo do óleo de citronela (Cymbopogon winterianus Jowitt) sobre o carrapato bovino. Na experimentação in vitro foi utilizado o grupo controle negativo e oito concentrações de óleo de citronela e óleo mineral (0,5; 1; 2; 5; 10; 20; 50; 100%), em fêmeas ingurgitadas de carrapatos. A eficácia carrapaticida para o óleo de citronela foi de 0; 34,5; 43; 48; 82; 99; 99; 100 e 100%, respectivamente; para o óleo mineral foram observados resultados irregulares na eficácia acaricida. Para a experimentação in vivo foram constituídos três grupos: controle negativo; óleo de citronela a 8,6% nível estimado mediante análise de regressão, correspondendo a 95% de eficácia acaricida da pesquisa in vitro e amitraz a 0,025%, com dezoito vacas da raça Holandesa. Antes, média dos dias -3, -2, -1 e após a aplicação do produto, 1, 2, 3, 5, 7, 10, 14 e 21 dias, foram contadas fêmeas ingurgitadas de carrapato. Na 1ª e na 2ª ordenha após a aplicação dos tratamentos, foram avaliadas variáveis fisiológicas (controle negativo x tratamento fitoterápico). A eficácia carrapaticida foi de 0; 91,3 e 60%
Assuntos
Animais , Fitoterapia , Rhipicephalus/fisiologia , Rhipicephalus/parasitologia , Rhipicephalus/química , Cymbopogon/efeitos adversos , Cymbopogon/químicaRESUMO
The Rhipicephalus (Boophilus) microplus is the most important cattle tick in Brazil. For the ticks control, various studies on plants have been developed in an attempt to find extracts with acaricidal properties. This research was aimed at evaluating in vitro and in vivo effects of citronella (Cymbopogon winterianus Jowitt) oil on cattle ticks. Negative control group and eight concentrations of citronella oil and mineral oil (0.5; 1; 2; 5; 10; 20; 50; 100%), were used on in vitro trials with engorged female ticks. The efficacy against cattle ticks was 0; 34.5; 43; 48; 82; 99; 99; 100 and 100%, respectively, for citronella oil. For mineral oil contradictory results of acaricidal efficacy were observed. At the in vivo trial eighteen Holstein cows were allocated to three groups: negative control; citronella oil at 8.6% - level estimated by regression analysis, accounting for 95% efficacy against ticks on in vitro trial and amitraz at 0.025%. Engorged female ticks were counted before, mean of days -3, -2, -1, and after treatment, 1, 2, 3, 5, 7, 10, 14 and 21 days. On the 1 st and 2nd milked after treatments, physiologic variables were evaluate (negative control x phytotherapic treatment). The efficacy against tick was 0; 91.3 and 60%, respectively, at 21 days after treatment. Similar results were found with physiologic variables between treatments.(AU)
O Rhipicephalus (Boophilus) microplus é o carrapato bovino mais importante no Brasil. Para o controle do carrapato, vários estudos com plantas têm sido desenvolvidos numa tentativa de encontrar extratos com propriedades acaricidas. Esta pesquisa foi conduzida com o objetivo de avaliar o efeito in vitro e in vivo do óleo de citronela (Cymbopogon winterianus Jowitt) sobre o carrapato bovino. Na experimentação in vitro foi utilizado o grupo controle negativo e oito concentrações de óleo de citronela e óleo mineral (0,5; 1; 2; 5; 10; 20; 50; 100%), em fêmeas ingurgitadas de carrapatos. A eficácia carrapaticida para o óleo de citronela foi de 0; 34,5; 43; 48; 82; 99; 99; 100 e 100%, respectivamente; para o óleo mineral foram observados resultados irregulares na eficácia acaricida. Para a experimentação in vivo foram constituídos três grupos: controle negativo; óleo de citronela a 8,6% nível estimado mediante análise de regressão, correspondendo a 95% de eficácia acaricida da pesquisa in vitro e amitraz a 0,025%, com dezoito vacas da raça Holandesa. Antes, média dos dias -3, -2, -1 e após a aplicação do produto, 1, 2, 3, 5, 7, 10, 14 e 21 dias, foram contadas fêmeas ingurgitadas de carrapato. Na 1ª e na 2ª ordenha após a aplicação dos tratamentos, foram avaliadas variáveis fisiológicas (controle negativo x tratamento fitoterápico). A eficácia carrapaticida foi de 0; 91,3 e 60%(AU)
Assuntos
Animais , Rhipicephalus/química , Rhipicephalus/fisiologia , Rhipicephalus/parasitologia , Fitoterapia , Cymbopogon/efeitos adversos , Cymbopogon/químicaRESUMO
Various classes of endopeptidases and their inhibitors facilitate blood feeding and digestion in ticks. Cystatins, a family of tight-binding and reversible inhibitors of cysteine endopeptidases, have recently been found in several tick tissues. Moreover, vaccine trials using tick cystatins have been found to induce protective immune responses against tick infestation. However, the mode of action of tick cystatins is still poorly understood, limiting the elucidation of their physiological role. Against this background, we have investigated sequence characteristics and immunogenic properties of 5 putative cystatins from Rhipicephalus (Boophilus) microplus from Brazil and Uruguay. The similarity of the deduced amino acid sequences among cystatins from the Brazilian tick strain was 27-42%, all of which had a secretory signal peptide. The cystatin motif (QxVxG), a glycine in the N-terminal region, and the PW motif in the second hairpin loop in the C-terminal region are highly conserved in all 5 cystatins identified in this study. Four cysteine residues in the C terminus characteristic of type 2 cystatins are also present. qRT-PCR revealed differential expression patterns among the 5 cystatins identified, as well as variation in mRNA transcripts present in egg, larva, gut, salivary glands, ovary, and fat body tissues. One R. microplus cystatin showed 97-100% amino acid similarity between Brazilian and Uruguayan isolates. Furthermore, by in silico analysis, antigenic amino acid regions from R. microplus cystatins showed high degrees of homology (54-92%) among Rhipicephalus spp. cystatins. Three Brazilian R. microplus cystatins were expressed in Escherichia coli, and immunogenicity of the recombinant proteins were determined by vaccinating mice. Western blotting using mice sera indicated cross-reactivity between the cystatins, suggesting shared epitopes. The present characterization of Rhipicephalus spp. cystatins represents an empirical approach in an effort to evaluate the physiological role of cystatins in a larger context of targeting them for use in future tick control strategies.
Assuntos
Doenças dos Bovinos/parasitologia , Cistatinas/química , Cistatinas/imunologia , Rhipicephalus/imunologia , Infestações por Carrapato/veterinária , Sequência de Aminoácidos , Animais , Brasil , Bovinos , Doenças dos Bovinos/imunologia , Biologia Computacional , Cistatinas/genética , DNA Complementar/química , DNA Complementar/genética , Feminino , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rhipicephalus/química , Rhipicephalus/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Infestações por Carrapato/imunologia , Infestações por Carrapato/parasitologiaRESUMO
Rhipicephalus (Boophilus) microplus is constantly challenged during its life cycle by microorganisms present in their hosts or in the environment. Tick eggs may be especially vulnerable to environmental conditions because they are exposed to a rich and diverse microflora in the soil. Despite being oviposited in such hostile sites, tick eggs remain viable, suggesting that the egg surface has defense mechanisms against opportunistic and/or pathogenic organisms. R. microplus engorged females deposit a superficial wax layer onto their eggs during oviposition. This egg wax is essential for preventing desiccation as well as acting as a barrier against attack by microorganisms. In this study, we report the detection of anti-biofilm activity of R. microplus egg wax against Pseudomonas aeruginosa PA14. Genes involved in the functions of production and maintenance of the biofilm extracellular matrix, pelA and cdrA, respectively, were markedly downregulated by a tick egg-wax extract. Moreover, this extract strongly inhibited fliC gene expression. Instead of a compact extracellular matrix, P. aeruginosa PA14 treated with egg-wax extract produces a fragile one. Also, the colony morphology of cells treated with egg-wax extract appears much paler and brownish, instead of the bright purple characteristic of normal colonies. Swarming motility was also inhibited by treatment with the egg-wax extract. The inhibition of P. aeruginosa biofilm does not seem to depend on inhibition of the quorum sensing system since mRNA levels of the 3 regulators of this system were not inhibited by egg-wax extract.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Óvulo/química , Pseudomonas aeruginosa/efeitos dos fármacos , Rhipicephalus/química , Adesinas Bacterianas/efeitos dos fármacos , Adesinas Bacterianas/genética , Animais , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Bovinos , Regulação para Baixo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Microscopia Eletrônica de Varredura , Óvulo/microbiologia , Estresse Oxidativo , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/fisiologia , RNA Bacteriano/genética , Rhipicephalus/microbiologia , VirulênciaRESUMO
The cattle tick Rhipicephalus (Boophilus) microplus lays eggs in the soil near the roots of grass, or in similar highly moist environments that are prone to biofilm formation. Tick eggs have a protective wax coating that may be a source of nutrients for microorganisms. However, as the eggs remain viable and show no visible signs of microbial colonization, we hypothesized that the coating might have anti-biofilm properties. We show here that the coating inhibits biofilm formation by both Gram-negative and Gram-positive bacteria, though by different mechanisms. We have identified the anti-biofilm molecule as N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucine (boophiline), and we show that it inhibits the expression of fliC (flagellin) and cdrA (biofilm scaffold), whose products are necessary for biofilm formation in Pseudomonas aeruginosa. Boophiline is a novel biofilm inhibitor being also effective against Staphylococcus epidermidis biofilm. In our study we show evidences of the boophiline mode of action in the protection of arthropod eggs against biofilm colonization.
Assuntos
Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Colestenos/farmacologia , Isoleucina/análogos & derivados , Rhipicephalus/química , Rhipicephalus/microbiologia , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Colestenos/isolamento & purificação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Isoleucina/isolamento & purificação , Isoleucina/farmacologia , Óvulo/química , Óvulo/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
ATP-binding cassette (ABC) transporters are efflux transporters found in all organisms. These proteins are responsible for pumping xenobiotic and endogenous metabolites through extra- and intracellular membranes, thereby reducing cellular concentrations of toxic compounds. ABC transporters have been associated with drug resistance in several nematodes and parasitic arthropods. Here, the ability of ABC transporter inhibitors to enhance ivermectin (IVM) sensitivity was tested in larvae and adult females of Rhipicephalus (Boophilus) microplus. Larvae of susceptible and IVM-resistant tick populations were pre-exposed to sub-lethal doses of the ABC transporter inhibitors Cyclosporin A (CsA) and MK571, and subsequently treated with IVM in a Larval Packet Test (LPT). ABC transporter inhibition by both drugs significantly reduced the concentration for 50% lethality (LC(50)) values of four IVM-resistant populations but IVM sensitivity of a susceptible population remained unchanged. IVM sensitivity in adults was assessed through an artificial feeding assay. The addition of CsA to a blood meal substantially affected IVM toxicity in adult female ticks from a resistant population by reducing oviposition and egg viability, although it did not alter IVM toxicity in susceptible females. Three partial nucleotide sequences with similarity to ABC transporters were retrieved from the DFCI Boophilus microplus Gene Index (http://compbio.dfci.harvard.edu/index.html). Their transcriptional levels in the midgut of resistant and susceptible females were determined by quantitative PCR, showing that one of these sequences was significantly up-regulated in IVM-resistant females and suggesting its participation in IVM detoxification. We believe this work reports the first known evidence for the participation of ABC transporters in IVM resistance in R. microplus.