RESUMO
The indiscriminate use of acaricides is a problem worldwide and has increased the selection of acaricide-resistant tick populations. The goal of this study was to evaluate the acaricide effects of two essential oils (from Schinus molle and Bulnesia sarmientoi) using the larval immersion test on three Rhipicephalus tick species. Rhipicephalus evertsi, Rhipicephalus appendiculatus and Rhipicephalus pulchelus ticks collected in Kenya, without history of acaricide exposure, were tested, as well as individuals from two populations of Rhipicephalus microplus (with or without history of acaricide exposure), for comparison. The sample most resistant to the treatments was a population of R. microplus with previous acaricide exposure, whereas the least tolerant sample was a strain of the same species that never had contact with acaricides (Porto Alegre strain). Interestingly, the field tick samples without previous acaricide exposure responded to essential oils with a mortality profile resembling that observed in the acaricide-resistant R. microplus field population, and not the susceptible Porto Alegre strain. The essential oil of B. sarmientoi and its two components tested (guaiol and bulnesol) caused the highest mortality rates in the tested species and are potential molecules for future studies on control methods against these species.
Assuntos
Acaricidas , Óleos Voláteis , Rhipicephalus , Infestações por Carrapato , Acaricidas/farmacologia , Animais , Quênia , Óleos Voláteis/farmacologiaRESUMO
Cystatins are cysteine peptidase inhibitors that in ticks mediate processes such as blood feeding and digestion. The ixodid tick Ixodes persulcatus is endemic to the Eurasia, where it is the principal vector of Lyme borreliosis. To date, no I. persulcatus cystatin has been characterized. In the present work, we describe three novel cystatins from I. persulcatus, named JpIpcys2a, JpIpcys2b and JpIpcys2c. In addition, the potential of tick cystatins as cross-protective antigens was evaluated by vaccination of hamsters using BrBmcys2c, a cystatin from Rhipicephalus microplus, against I. persulcatus infestation. Sequence analysis showed that motifs that are characteristic of cystatins type 2 are fully conserved in JpIpcys2b, while mutations are present in both JpIpcys2a and JpIpcys2c. Protein-protein docking simulations further revealed that JpIpcys2a, JpIpcys2b and JpIpcys2c showed conserved binding sites to human cathepsins L, all of them covering the active site cleft. Cystatin transcripts were detected in different I. persulcatus tissues and instars, showing their ubiquitous expression during I. persulcatus development. Serological analysis showed that although hamsters immunized with BrBmcys2c developed a humoral immune response, this response was not adequate to protect against a heterologous challenge with I. persulcatus adult ticks. The lack of cross-protection provided by BrBmcys2c immunization is perhaps linked to the fact that cystatins cluster into multigene protein families that are expressed differentially and exhibit functional redundancy. How to target such small proteins that are secreted in low quantities remains a challenge in the development of suitable anti-tick vaccine antigens.
Assuntos
Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Cistatinas/química , Cistatinas/genética , Ixodes/metabolismo , Infestações por Carrapato/prevenção & controle , Animais , Anticorpos/sangue , Anticorpos/imunologia , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/isolamento & purificação , Sítios de Ligação , Catepsina L/química , Cricetinae , Humanos , Imunidade Humoral , Ixodes/imunologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Família Multigênica , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Rhipicephalus/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
BACKGROUND: Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase from R. microplus embryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status. METHODS: In this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface. RESULTS: Cysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PPi), probably by inducing disulfide bridge formation. CONCLUSIONS: Our results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface. GENERAL SIGNIFICANCE: This study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status.
Assuntos
Pirofosfatase Inorgânica/metabolismo , Multimerização Proteica , Compostos de Sulfidrila/metabolismo , Carrapatos/enzimologia , Aminoácidos/metabolismo , Animais , Cálcio/farmacologia , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Fluoretos/farmacologia , Dissulfeto de Glutationa/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/metabolismo , Oxidantes/farmacologia , Oxirredução , Multimerização Proteica/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Substâncias Redutoras/farmacologiaRESUMO
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
As blood-sucking parasites, ticks inflict great damage to animals and humans in many parts of the world. The continued use of chemical acaricides is not sustainable due to increasing tick resistance, growing public concern over drug residues in food and in the environment, and the high cost of developing new acaricides. Therefore, an alternative control strategy is urgently needed. Vaccines against ticks have been shown to be functionally feasible, as highlighted by the success of Bm86 vaccines against Rhipicephalus (Boophilus) microplus and closely related tick species. However, a limited number of tick antigens with cross-protective epitopes have been characterized so far, limiting widespread deployment of the available vaccines, including those derived from Bm86. Therefore, identifying tick antigens with potential broad-spectrum protection against multiple tick species is subject of vigorous research at present. In this paper, progress towards effective anti-tick vaccines is reviewed in the light of emerging data from studies including heterologous tick challenge. Taken together, these studies indicate that the decades-long search for a universal tick vaccine is making progress, with such a vaccine likely to be based on multiple cross-reactive antigens.