RESUMO
The present study was conducted to evaluate the effects of carvacrol and thymol on the antioxidant and detoxifying enzymes of larvae from two populations of R. microplus: Jaguar (tick population resistant to six classes of acaricides) and Porto Alegre (susceptible tick population). Carvacrol and thymol were tested at concentrations ranging from 0.14 to 5.0 mg mL-1 in both populations to determine the LC50. In addition, the LC1, LC25, and LC75 were estimated using the LC50 and HillSlope of each compound. Larvae of both populations of R. microplus were then treated with the LC1, LC25, LC50, and LC75 of each monoterpene, and those that survived were processed to evaluate the effects of the compounds on the antioxidant and detoxifying systems of larvae; these effects were assessed by determining the activity of the enzymes, glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX). Larvae from the Jaguar population treated with different lethal concentrations of carvacrol and thymol displayed a dose-dependent increase in CAT, GPX, SOD, and GST after treatment with LC25. Further, larvae treated with the LC75 had the highest levels of enzyme activity for carvacrol (1.76 mg mL-1) and thymol (1.32 mg mL-1). CAT, GPX, SOD, and GST activity in Porto Alegre population larvae treated with carvacrol and thymol also increased significantly up to the LC50 of each monoterpene. However, at the LC75 of carvacrol and thymol, a decrease in the activity of all enzymes was observed for this tick population. These findings indicate that carvacrol and thymol induced increased activity of all evaluated enzymes at different lethal concentrations in R. microplus larvae from two populations. Such findings unveil the possible mechanisms of action of these natural acaricides.
Assuntos
Acaricidas , Ixodidae , Rhipicephalus , Acaricidas/farmacologia , Animais , Antioxidantes/farmacologia , Cimenos , Larva , Timol/farmacologiaRESUMO
Ticks have developed physiological adaptations to transport, store, metabolize and secrete toxic components from the diet and environment. Different classes of enzymes are involved in these processes, however, the role of several of them is not yet characterized in Rhipicephalus microplus. In this context, this work investigated the action of antioxidant and detoxification enzymes, as well as the levels of essential cellular reductants in R. microplus partially engorged females (PEF) and fully engorged females (FEF). Results demonstrated that enzymes transcriptional levels and enzymatic activity from ovary and fat body were higher in PEF than in FEF, except for ovary Glutathione peroxidase (GPx), which was the only enzyme showing highest activity in the FEF stage. These results indicated a higher demand for antioxidant potential in these organs at the initial feeding phase than during egg-laying. In midgut, however, there was more variability in the transcriptional levels and activity of the different enzymes between the PEF and FEF phases. Similar NADPH levels were found in PEF and FEF phases, suggesting a remarkable capacity to maintain a regular supply of reducing power, despite the developmental changes and large intake of heme and iron. However, reduced glutathione (GSH) levels were variable between PEF and FEF when distinct organs were compared. Taken together, our data suggest a higher demand for reducing potential in FEF ticks. The silencing of catalase (CAT) or thioredoxin reductase (TRx) genes in females did not impair feeding, egg-laying capacity, or larvae hatching. CAT-silenced ticks had increased ovary peroxidase activity, a possible compensatory antioxidant mechanism. Altogether, the results shed light on the complexity of the antioxidant and detoxification enzyme system in ticks and its involvement in different physiological mechanisms.
Assuntos
Antioxidantes/metabolismo , Proteínas de Artrópodes/metabolismo , Rhipicephalus/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica , Rhipicephalus/enzimologiaRESUMO
To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to carry out a study of expression in (i) embryos; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v) digestive cells from partially, and (vi) fully engorged females. We obtained > 500 million Illumina reads which were assembled de novo, producing > 190,000 contigs, identifying 18,857 coding sequences (CDS). Reads from each library were mapped back into the assembled transcriptome giving a view of gene expression in different tissues. Transcriptomic expression and pathway analysis showed that several genes related in blood digestion and host-parasite interaction were overexpressed in digestive cells compared with other tissues. Furthermore, essential genes for the cell development and embryogenesis were overexpressed in ovaries. Taken altogether, these data offer novel insights into the physiology of production and role of saliva, blood digestion, energy metabolism, and development with submission of 10,932 novel tissue/cell specific CDS to the NCBI database for this important tick species.
Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Rhipicephalus/fisiologia , Animais , Bovinos , Feminino , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Especificidade de Órgãos , Ovário/química , Gravidez , Rhipicephalus/genética , Saliva/química , Análise de Sequência de RNARESUMO
Rhipicephalus appendiculatus, the brown ear tick, is an important disease vector of livestock in eastern, central and southern Africa. Rhipicephalus appendiculatus acaricide resistance requires the search for alternative methods for its control. Cystatins constitute a superfamily of cysteine peptidase inhibitors vital for tick blood feeding and development. These inhibitors were proposed as antigens in anti-tick vaccines. In this work, we applied structural and biochemical approaches to characterize a new cystatin named R. appendiculatus cystatin 2a (Racys2a). Structural modeling showed that this new protein possesses characteristic type 2 cystatin motifs, besides conservation of other structural patterns along the protein. Peptidase inhibitory assays with recombinant Racys2a showed modulation of tick and host cathepsins involved in blood digestion and immune system responses, respectively. A heterologous tick challenge with R. appendiculatus in rabbits immunized with recombinant Rhipicephalus microplus cystatin 2c (rBmcys2c) was performed to determine cross-reactivity. Histological staining showed that rBmcys2c vaccination caused damage to the gut, salivary gland and ovary tissues in R. appendiculatus. Furthermore, cystatin vaccine reduced the number of fully engorged adult females in 11.5 %. Consequently, strategies to increase the protection rate are necessary, including the selection of two or more antigens to compose a vaccine cocktail.
Assuntos
Proteínas de Artrópodes/genética , Rhipicephalus/genética , Cistatinas Salivares/genética , Vacinas/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Feminino , Filogenia , Coelhos , Rhipicephalus/metabolismo , Cistatinas Salivares/química , Cistatinas Salivares/metabolismo , Alinhamento de Sequência , Vacinas/química , Vacinas/metabolismoRESUMO
Introduction: Ticks are hematophagous ectoparasites that cause significate economical losses. Acaricide application is themain method to tick control. However, it causes environmental contamination and selects resistant ticks. The immunological control represents a suitable method to replace or complement acaricide application. During its life cycle, female ticksingest large amounts of blood, which contains toxic components able to damage biomolecules. Understanding which molecular mechanisms and proteins are involved in avoid damages caused by blood intake in ticks and other hematophagousarthropods could help to found potential candidate antigens to compose an anti-tick vaccine.Review: Hemoglobin comprises almost 20% of mammalian blood proteins, its hydrolysis during tick digestion increasestotal free heme that can potentially generate reactive oxygen species (ROS), which easily oxidize lipids, proteins and DNA,modifying they structure and function. Lipids are more susceptible to high ROS levels. It can lead to membrane instabilityand cell death. Protein modifications caused by ROS can promote the protein loss of function and cell disturbance, however,it can also work as post-translational modifications, acting in cell signaling processes. DNA presents more efficient protective mechanisms against ROS, but damages can lead to double helix rupture. Oxidative stress is defined as a disturbance inthe balance between the production and elimination of ROS, in favor of ROS production, leading to a disruption in redoxhomeostasis and/or molecular damage. Despite the well-recognized heme oxidative power and its already demonstratedcytotoxicity, ticks are able to feed on blood, controlling the redox homeostasis without causing oxidative stress. This occurs because ticks developed physiological adaptations to transport, store, metabolize and secrete toxic components fromthe diet. The strategies, such as heme compartmentalization in specialized organelles...(AU)
Assuntos
Animais , Homeostase , Carrapatos/imunologia , Estresse Oxidativo , Espécies Reativas de Oxigênio , Comportamento de Busca por HospedeiroRESUMO
Reactive oxygen species (ROS) are natural byproducts of metabolism that have toxic effects well documented in mammals. In hematophagous arthropods, however, these processes are not largely understood. Here, we describe that Rhipicephalus microplus ticks and embryonic cell line (BME26) employ an adaptive metabolic compensation mechanism that confers tolerance to hydrogen peroxide (H2O2) at concentrations too high for others organisms. Tick survival and reproduction are not affected by H2O2 exposure, while BME26 cells morphology was only mildly altered by the treatment. Furthermore, H2O2-tolerant BME26 cells maintained their proliferative capacity unchanged. We evaluated several genes involved in gluconeogenesis, glycolysis, and pentose phosphate pathway, major pathways for carbohydrate catabolism and anabolism, describing a metabolic mechanism that explains such tolerance. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by glucose uptake and energy resource availability. Transient increase in ROS levels, oxygen consumption, and ROS-scavenger enzymes, as well as decreased mitochondrial superoxide levels, were indicative of cell adaptation to high H2O2 exposure, and suggested a tolerance strategy developed by BME26 cells to cope with oxidative stress. Moreover, NADPH levels increased upon H2O2 challenge, and this phenomenon was sustained mainly by G6PDH activity. Interestingly, G6PDH knockdown in BME26 cells did not impair H2O2 tolerance, but generated an increase in NADP-ICDH transcription. In agreement with the hypothesis of a compensatory NADPH production in these cells, NADP-ICDH knockdown increased G6PDH relative transcript level. The present study unveils the first metabolic evidence of an adaptive mechanism to cope with high H2O2 exposure and maintain redox balance in ticks.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Peróxido de Hidrogênio/toxicidade , Oxidantes/toxicidade , Estresse Oxidativo/fisiologia , Rhipicephalus/metabolismo , Adaptação Fisiológica , Animais , Carboidratos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Resistência a Medicamentos , Tolerância a Medicamentos/fisiologia , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , NADP/análise , OxirreduçãoRESUMO
Cocktail vaccines are proposed as an attractive way to increase protection efficacy against specific tick species. Furthermore, such vaccines made with different tick antigens have the potential of cross-protecting against a broad range of tick species. However, there are still limitations to the selection of immunogen candidates. Acknowledging that glutathione S-transferases (GSTs) have been exploited as vaccines against ticks and other parasites, this study aimed to analyze a GST-cocktail vaccine as a potential broad-spectrum tick vaccine. To constitute the GST-cocktail vaccine, five tick species of economic importance for livestock industry were studied (Rhipicephalus appendiculatus, Rhipicephalus decoloratus, Rhipicephalus microplus, Amblyomma variegatum, and Haemaphysalis longicornis). Tick GST ORF sequences were cloned, and the recombinant GSTs were produced in Escherichia coli. rGSTs were purified and inoculated into rabbits, and the immunological response was characterized. The humoral response against rGST-Rd and rGST-Av showed a stronger cross-reactivity against heterologous rGSTs compared to rGST-Hl, rGST-Ra, and rGST-Rm. Therefore, rGST-Rd and rGST-Av were selected for constituting an experimental rGST-cocktail vaccine. Vaccination experiment in rabbits showed that rGST-cocktail caused 35% reduction in female numbers in a Rhipicephalus sanguineus infestation. This study brings forward an approach to selecting immunogens for cocktail vaccines, and the results highlight rGST-Rd and rGST-Av as potentially useful tools for the development of a broad-spectrum tick vaccine.
Assuntos
Glutationa Transferase/imunologia , Infestações por Carrapato/prevenção & controle , Carrapatos/enzimologia , Carrapatos/imunologia , Vacinas/imunologia , Animais , Reações Cruzadas/imunologia , Glutationa Transferase/química , Glutationa Transferase/genética , Fases de Leitura Aberta , Coelhos , Rhipicephalus sanguineus/enzimologia , Rhipicephalus sanguineus/imunologia , Vacinas/administração & dosagemRESUMO
Introduction: Ticks are hematophagous ectoparasites that cause significate economical losses. Acaricide application is themain method to tick control. However, it causes environmental contamination and selects resistant ticks. The immunological control represents a suitable method to replace or complement acaricide application. During its life cycle, female ticksingest large amounts of blood, which contains toxic components able to damage biomolecules. Understanding which molecular mechanisms and proteins are involved in avoid damages caused by blood intake in ticks and other hematophagousarthropods could help to found potential candidate antigens to compose an anti-tick vaccine.Review: Hemoglobin comprises almost 20% of mammalian blood proteins, its hydrolysis during tick digestion increasestotal free heme that can potentially generate reactive oxygen species (ROS), which easily oxidize lipids, proteins and DNA,modifying they structure and function. Lipids are more susceptible to high ROS levels. It can lead to membrane instabilityand cell death. Protein modifications caused by ROS can promote the protein loss of function and cell disturbance, however,it can also work as post-translational modifications, acting in cell signaling processes. DNA presents more efficient protective mechanisms against ROS, but damages can lead to double helix rupture. Oxidative stress is defined as a disturbance inthe balance between the production and elimination of ROS, in favor of ROS production, leading to a disruption in redoxhomeostasis and/or molecular damage. Despite the well-recognized heme oxidative power and its already demonstratedcytotoxicity, ticks are able to feed on blood, controlling the redox homeostasis without causing oxidative stress. This occurs because ticks developed physiological adaptations to transport, store, metabolize and secrete toxic components fromthe diet. The strategies, such as heme compartmentalization in specialized organelles...
Assuntos
Animais , Carrapatos/imunologia , Estresse Oxidativo , Homeostase , Comportamento de Busca por Hospedeiro , Espécies Reativas de OxigênioRESUMO
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
Probiotics are live microorganisms which are beneficial for the host when ingested at high enough concentrations. The methylotrophic yeast Pichia pastoris is widely used as heterologous protein production platform. However, its use as probiotic is poorly studied. The objective of this study was to evaluate some probiotic properties of the P. pastoris strain X-33 wild type. The resistance to in vitro and in vivo gastrointestinal conditions, stability in feed, safety, and antibacterial activity against Salmonella Typhimurium were evaluated. The yeast remained viable and persisted at appropriate concentration in the diet for at least 2 months, survived the stresses of the gastrointestinal tract in vitro and in vivo, caused no behavioral changes or lesions when administered to mice, inhibited the growth of S. Typhimurium in culture media, and reduced adhesion of the bacteria to the intestinal cells HCT-116. In the challenge experiment with a LD50 of virulent S. Typhimurium strain, mice supplemented with the yeast had a higher survival rate (50 % when administered by gavage and 80 % via the diet, compared with 20 and 50 %, respectively, in the control group). In addition, the S. Typhimurium concentration in the intestine of the surviving mice was lower; the score of intestinal lesions, lower; and the pathogen, not detected in the liver, spleen, and feces when compared to the control group (p < 0.05). It was concluded that the yeast Pichia pastoris X-33 has probiotic properties with remarkable antibacterial activity against S. Typhimurium.
Assuntos
Antibiose , Pichia/fisiologia , Probióticos/análise , Salmonella typhimurium/crescimento & desenvolvimento , Animais , Aderência Bacteriana , Trato Gastrointestinal/microbiologia , Camundongos , Salmonella typhimurium/fisiologiaRESUMO
BACKGROUND: Cystatins are a group of cysteine protease inhibitors responsible for physiological proteolysis regulation and present in a wide range of organisms. Studies about this class of inhibitors in parasites have contributed to clarify their roles in important physiological processes, like blood digestion and modulation of host immune response during blood feeding. Thus, cystatins are a subject of research on the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to find potential targets in multi-species control methods. However, cystatin functions in ticks remain undetermined, especially in Rhipicephalus microplus and Ixodes ovatus, two species that affect livestock and human health, respectively. METHODS: Here we report the inhibitory profile of two R. microplus (BrBmcys2b and BrBmcys2c) and one I. ovatus (JpIocys2a) cystatins to commercial cathepsins B, C, and L. The presence of native cystatins in R. microplus tissues was analyzed using sera against recombinant BrBmcys2b and BrBmcys2c. Also, a peptide from JpIocys2a was synthesized for rabbit immunization, and this serum was used to analyze the cross antigenicity between R. microplus and I. ovatus cystatins. RESULTS: Enzymatic inhibition profile of tick cystatins shows a distinct modulation for cathepsins related to tick blood digestion and evasion of host immune response. Furthermore, BrBmcys2b was detected in saliva and different tissues along tick stages, while BrBmcys2c was detected mainly in gut from partially engorged R. microplus females, demonstrating a distinct pattern of cystatin expression, secretion and traffic between tick tissues. Moreover, phylogenetic analysis suggests that JpIocys2a belongs to the group of tick gut secreted cystatins. Finally, cross-antigenicity assays revealed that antibodies against the JpIocys2a peptide recognize native and recombinant R. microplus cystatins. CONCLUSION: The presence of these proteins in different tissues and their ability to differentially inhibit cathepsins suggest distinct roles for JpIocys2a, BrBmcys2b, and BrBmcys2c in blood digestion, egg and larvae development, and modulation of host immune response in tick physiology. The cross-antigenicity between native and recombinant cystatins supports further experiments using JpIocys2a, BrBmcys2b, and BrBmcys2c as vaccine antigens.