RESUMO
BACKGROUND: When feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding. METHODS: Ticks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species. RESULTS: Changes in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as "sialome switching." This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva. CONCLUSIONS: Overall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods.
Assuntos
Rhipicephalus , Animais , Feminino , Bovinos , Rhipicephalus/fisiologia , Saliva/química , Proteômica , Proteínas de Artrópodes/metabolismo , Proteínas e Peptídeos Salivares/metabolismoRESUMO
The skin is the first host tissue that the tick mouthparts, tick saliva, and a tick-borne pathogen contact during feeding. Tick salivary glands have evolved a complex and sophisticated pharmacological arsenal, consisting of bioactive molecules, to assist blood feeding and pathogen transmission. In this work, persulcatin, a multifunctional molecule that targets keratinocyte function and hemostasis, was identified from Ixodes persulcatus female ticks. The recombinant persulcatin was expressed and purified and is a 25-kDa acidic protein with 2 Kunitz-type domains. Persulcatin is a classical tight-binding competitive inhibitor of proteases, targeting plasmin (Ki: 28 nM) and thrombin (Ki: 115 nM). It blocks plasmin generation on keratinocytes and inhibits their migration and matrix protein degradation; downregulates matrix metalloproteinase 2 and matrix metalloproteinase 9; and causes a delay in blood coagulation, endothelial cell activation, and thrombin-induced fibrinocoagulation. It interacts with exosite I of thrombin and reduces thrombin-induced endothelial cell permeability by inhibiting vascular endothelial-cadherin disruption. The multifaceted roles of persulcatin as an inhibitor and modulator within the plasminogen-plasmin system and thrombin not only unveil further insights into the intricate mechanisms governing wound healing but also provide a fresh perspective on the intricate interactions between ticks and their host organisms.
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Acaricide resistance is a global problem that has impacts worldwide. Tick populations with broad resistance to all commercially available acaricides have been reported. Since resistance selection in ticks and their role in pathogen transmission to animals and humans result in important economic and public health burden, it is essential to develop new strategies for their control (i.e., novel chemical compounds, vaccines, biological control). The synganglion is the tick central nervous system and it is responsible for synthesizing and releasing signaling molecules with different physiological functions. Synganglion proteins are the targets of the majority of available acaricides. In this review we provide an overview of the mode-of-action and resistance mechanisms against neurotoxic acaricides in ticks, as well as putative target sites in synganglion, as a supporting tool to identify new target proteins and to develop new strategies for tick control.
Assuntos
Acaricidas , Doenças dos Bovinos , Ixodidae , Rhipicephalus , Infestações por Carrapato , Vacinas , Animais , Humanos , Bovinos , Acaricidas/farmacologia , Controle de Ácaros e Carrapatos , Infestações por Carrapato/prevenção & controle , Infestações por Carrapato/veterinária , Doenças dos Bovinos/prevenção & controleRESUMO
The synganglion is the central nervous system of ticks and, as such, controls tick physiology. It does so through the production and release of signaling molecules, many of which are neuropeptides. These peptides can function as neurotransmitters, neuromodulators and/or neurohormones, although in most cases their functions remain to be established. We identified and performed in silico characterization of neuropeptides present in different life stages and organs of Rhipicephalus microplus, generating transcriptomes from ovary, salivary glands, fat body, midgut and embryo. Annotation of synganglion transcripts led to the identification of 32 functional categories of proteins, of which the most abundant were: secreted, energetic metabolism and oxidant metabolism/detoxification. Neuropeptide precursors are among the sequences over-represented in R. microplus synganglion, with at least 5-fold higher transcription compared with other stages/organs. A total of 52 neuropeptide precursors were identified: ACP, achatin, allatostatins A, CC and CCC, allatotropin, bursicon A/B, calcitonin A and B, CCAP, CCHamide, CCRFamide, CCH/ITP, corazonin, DH31, DH44, eclosion hormone, EFLamide, EFLGGPamide, elevenin, ETH, FMRFamide myosuppressin-like, glycoprotein A2/B5, gonadulin, IGF, inotocin, insulin-like peptides, iPTH, leucokinin, myoinhibitory peptide, NPF 1 and 2, orcokinin, proctolin, pyrokinin/periviscerokinin, relaxin, RYamide, SIFamide, sNPF, sulfakinin, tachykinin and trissin. Several of these neuropeptides have not been previously reported in ticks, as the presence of ETH that was first clearly identified in Parasitiformes, which include ticks and mites. Prediction of the mature neuropeptides from precursor sequences was performed using available information about these peptides from other species, conserved domains and motifs. Almost all neuropeptides identified are also present in other tick species. Characterizing the role of neuropeptides and their respective receptors in tick physiology can aid the evaluation of their potential as drug targets.
Assuntos
Ixodidae , Neuropeptídeos , Rhipicephalus , Animais , Feminino , Ixodidae/metabolismo , Neuropeptídeos/química , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Peptídeos , Rhipicephalus/genética , Rhipicephalus/metabolismo , TranscriptomaRESUMO
BACKGROUND: Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain spotted fever, a life-threatening illness. To obtain an insight into the vector-pathogen interactions, we assessed the effects of infection with R. rickettsii on the proteome cells of the tick embryonic cell line BME26. METHODS: The proteome of BME26 cells was determined by label-free high-performance liquid chromatography coupled with tandem mass spectrometry analysis. Also evaluated were the effects of infection on the activity of caspase-3, assessed by the hydrolysis of a synthetic fluorogenic substrate in enzymatic assays, and on the exposition of phosphatidyserine, evaluated by live-cell fluorescence microscopy after labeling with annexin-V. Finally, the effects of activation or inhibition of caspase-3 activity on the growth of R. rickettsii in BME26 cells was determined. RESULTS: Tick proteins of different functional classes were modulated in a time-dependent manner by R. rickettsii infection. Regarding proteins involved in apoptosis, certain negative regulators were downregulated at the initial phase of the infection (6 h) but upregulated in the middle of the exponential phase of the bacterial growth (48 h). Microorganisms are known to be able to inhibit apoptosis of the host cell to ensure their survival and proliferation. We therefore evaluated the effects of infection on classic features of apoptotic cells and observed DNA fragmentation exclusively in noninfected cells. Moreover, both caspase-3 activity and phosphatidylserine exposition were lower in infected than in noninfected cells. Importantly, while the activation of caspase-3 exerted a detrimental effect on rickettsial proliferation, its inhibition increased bacterial growth. CONCLUSIONS: Taken together, these results show that R. rickettsii modulates the proteome and exerts an inhibitory effect on apoptosis in tick cellsthat seems to be important to ensure cell colonization.
Assuntos
Apoptose , Rickettsia rickettsii/fisiologia , Carrapatos/citologia , Carrapatos/microbiologia , Animais , Caspase 3/genética , Caspase 3/metabolismo , Interações Hospedeiro-Patógeno , Carrapatos/genética , Carrapatos/metabolismoRESUMO
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
Protease inhibitors play crucial roles in parasite development and survival, modulating the immune responses of their vertebrate hosts. Members of the serpin family are irreversible inhibitors of serine proteases and regulate systems related to defence against parasites. Limited information is currently available on protease inhibitors from the liver fluke Fasciola hepatica. In this study, we characterised four serpins from F. hepatica (FhS-1-FhS-4). Biochemical characterisation revealed that recombinant FhS-2 (rFhS) inhibits the activity of human neutrophil cathepsin G, while rFhS-4 inhibits the activity of bovine pancreatic chymotrypsin and cathepsin G. Consistent with inhibitor function profiling data, rFhS-4 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner.Similar to other serpins, rFhS2 and rFhS-4 bind to heparin with high affinity. Tissue localisation demonstrated that these serpins have different spatial distributions. FhS-2 is localised in the ovary, while FhS-4 was found in gut cells. Both of them co-localised in the spines within the tegument. These findings provide the basis for study of functional roles of these proteins as part of an immune evasion mechanism in the adult fluke, and in protection of eggs to ensure parasite life cycle continuity. Further understanding of serpins from the liver fluke may lead to the discovery of novel anti-parasitic interventions.
Assuntos
Fasciola hepatica , Interações Hospedeiro-Parasita , Serpinas , Animais , Catepsina G/antagonistas & inibidores , Bovinos , Quimotripsina/antagonistas & inibidores , Fasciola hepatica/enzimologia , Feminino , HumanosRESUMO
The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.
Assuntos
Anoxybacillus/metabolismo , Extremófilos/metabolismo , Peptídeo Hidrolases/metabolismo , Anoxybacillus/classificação , Anoxybacillus/isolamento & purificação , Anoxybacillus/fisiologia , Brasil , Estabilidade Enzimática , Extremófilos/classificação , Extremófilos/isolamento & purificação , Extremófilos/fisiologia , Concentração de Íons de Hidrogênio , Queratinas/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Temperatura , Termolisina/química , Termolisina/metabolismo , TermotolerânciaRESUMO
Here we present the proteomic profile datasets of two Fasciola hepatica NEJ isolates derived from different snail hosts: Lymnaea viatrix and Pseudosuccinea columella. The data used in the analysis are related to the article 'A proteomic comparison of excretion/secretion products in Fasciola hepatica newly excysted juveniles (NEJ) derived from Lymnaea viatrix or Pseudosuccinea columella' (Di Maggio et al., 2019).
RESUMO
In parasites, cathepsins are implicated in mechanisms related to organism surveillance and host evasion. Some parasite cathepsins have fibrinogenolytic and fibrinolytic activity, suggesting that they may contribute to maintain blood meal fluidity for extended feeding periods. Here, it is shown that BmGTI (Rhipicephalus [Boophilus] microplus Gut Thrombin Inhibitor), a protein previously described as an inhibitor of fibrinogen hydrolysis and platelet aggregation by thrombin, and BmCL1 (Rhipicephalus [Boophilus] microplus Cathepsin-L like 1) are the same protein, hereinafter referred to using the earliest name (BmCL1). To further characterize BmCL1, Rhipicephalus microplus native and recombinant (rBmCL1) proteins were obtained. Native BmCL1 was isolated using thrombin-affinity chromatography, and it displays thrombin inhibition activity. We subsequently investigated rBmCL1 interaction with thrombin. We show that rBmCL1 and thrombin have a dissociation constant (ΚD) of 130.2⯱â¯11.2â¯nM, and this interaction likely occurs due to a more electronegative surface of BmCL1 at pH 7.5 than at pH 5.0, which may favor an electrostatic binding to positively charged thrombin exosites. During BmCL1-thrombin interaction, thrombin is not degraded or inhibited. rBmCL1 impairs thrombin-induced fibrinogen clotting via a fibrinogenolytic activity. Fibrinogen degradation by BmCL1 occurs by the hydrolysis of Aα- and Bß-chains, generating products similar to those produced by fibrinogenolytic cathepsins from other organisms. In conclusion, BmCL1 likely has an additional role in R. microplus blood digestion, besides its role in hemoglobin degradation at acid pH. BmCL1 fibrinogenolytic activity indicates a proteolytic activity in the neutral lumen of tick midgut, contributing to maintain the fluidity of the ingested blood, which remains to be confirmed in vivo.
Assuntos
Catepsina L/metabolismo , Rhipicephalus/enzimologia , Trombina/metabolismo , Sequência de Aminoácidos , Animais , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Anticoagulantes/metabolismo , Catepsina L/química , Catepsina L/isolamento & purificação , Bovinos , Cinética , Modelos Moleculares , ProteóliseRESUMO
The characteristics of parasitic infections are often tied to host behavior. Although most studies have investigated definitive hosts, intermediate hosts can also play a role in shaping the distribution and accumulation of parasites. This is particularly relevant in larval stages, where intermediate host's behavior could potentially interfere in the molecules secreted by the parasite into the next host during infection. To investigate this hypothesis, we used a proteomic approach to analyze excretion/secretion products (ESP) from Fasciola hepatica newly excysted juveniles (NEJ) derived from two intermediate host species, Lymnaea viatrix and Pseudosuccinea columella. The two analyzed proteomes showed differences in identity, abundance, and functional classification of the proteins. This observation could be due to differences in the biological cycle of the parasite in the host, environmental aspects, and/or host-dependent factors. Categories such as protein modification machinery, protease inhibitors, signal transduction, and cysteine-rich proteins showed different abundance between samples. More specifically, differences in abundance of individual proteins such as peptidyl-prolyl cis-trans isomerase, thioredoxin, cathepsin B, cathepsin L, and Kunitz-type inhibitors were identified. Based on the differences identified between NEJ ESP samples, we can conclude that the intermediate host is a factor influencing the proteomic profile of ESP in F. hepatica.
Assuntos
Fasciola hepatica/metabolismo , Proteínas de Helminto/metabolismo , Lymnaea/parasitologia , Proteômica , Caramujos/parasitologia , Animais , Anidrases Carbônicas/classificação , Anidrases Carbônicas/metabolismo , Proteínas de Helminto/classificação , Larva/metabolismo , Peptídeo Hidrolases/classificação , Peptídeo Hidrolases/metabolismo , Peroxirredoxinas/classificação , Peroxirredoxinas/metabolismo , Inibidores de Proteases/classificação , Inibidores de Proteases/metabolismo , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/metabolismoRESUMO
Lipids play key roles in arthropod metabolism. In ticks, these biomolecules are transported from fat body to other organs, such as ovary and Gené's organ. Gené's organ, an apparatus found exclusively in female ticks, secretes a protective wax coat onto the egg surface, increasing egg viability in the environment due to waterproof, cohesive, and antimicrobial properties. In this work, a combined transcriptomic and proteomic approach shows that Gené's organ not solely secrets compounds taken up from the hemolymph, but is actively engaged in synthesis, modification, and oxidation of lipids. Gené's organ was analyzed at two distinct stages: 1) when ticks detach from host by the end of hematophagous phase, and 2) during egg-laying. Data show that Gené's organ undergoes a maturation process before the onset of oviposition, in preparation for its role during egg-laying. Because it deals with a wax-secreting organ, the study focused on lipid metabolism, examining a full machinery to synthesize, modify, and oxidize fatty acids. Proteins involved in sterol modification, transport, and degradation were also addressed. In addition to highlighting Gené's organ importance in tick reproductive physiology, the results reveal proteins and pathways crucial to egg wax secretion, and consequently, egg development in the environment. Tools targeting these molecules and pathways would impair egg viability in the environment, and therefore have the potential to be developed into novel tick control methods.
Assuntos
Metabolismo dos Lipídeos , Proteoma , Carrapatos/anatomia & histologia , Carrapatos/metabolismo , Transcriptoma , Animais , Feminino , Perfilação da Expressão Gênica , Oviposição , Óvulo , Proteômica , Controle de Ácaros e Carrapatos , Carrapatos/genética , CerasRESUMO
Ticks are arthropod ectoparasites of importance for public and veterinary health. The understanding of tick oogenesis and embryogenesis could contribute to the development of novel control methods. However, to date, studies on the temporal dynamics of proteins during ovary development were not reported. In the present study we followed protein profile during ovary maturation. Proteomic analysis of ovary extracts was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using shotgun strategy, in addition to dimethyl labelling-based protein quantification. A total of 3,756 proteins were identified, which were functionally annotated into 30 categories. Circa 80% of the annotated proteins belong to categories related to basal metabolism, such as protein synthesis and modification machineries, nuclear regulation, cytoskeleton, proteasome machinery, transcriptional machinery, energetic metabolism, extracellular matrix/cell adhesion, immunity, oxidation/detoxification metabolism, signal transduction, and storage. The abundance of selected proteins involved in yolk uptake and degradation, as well as vitellin accumulation during ovary maturation, was assessed using dimethyl-labelling quantification. In conclusion, proteins identified in this study provide a framework for future studies to elucidate tick development and validate candidate targets for novel control methods.
Assuntos
Proteínas de Artrópodes/metabolismo , Ovário/crescimento & desenvolvimento , Proteoma/análise , Carrapatos/crescimento & desenvolvimento , Vitelogênese , Animais , Feminino , Ovário/metabolismo , Proteoma/metabolismo , Carrapatos/metabolismoRESUMO
The cattle tick Rhipicephalus microplus is a hematophagous ectoparasite that causes important economic losses in livestock. Different species of ticks harbor a symbiont bacterium of the genus Coxiella. It was showed that a Coxiella endosymbiont from R. microplus (CERM) is a vertically transmitted mutualist symbiont, comprising 98% of the 16S rRNA sequences in both eggs and larvae. Sequencing of the bacterial genome revealed genes for biosynthetic pathways for several vitamins and key metabolic cofactors that may provide a nutritional complement to the tick host. The CERM was abundant in ovary and Malpighian tubule of fully engorged female. Tetracycline treatment of either the tick or the vertebrate host reduced levels of bacteria in progeny in 74% for eggs and 90% for larvae without major impact neither on the reproductive fitness of the adult female or on embryo development. However, CERM proved to be essential for the tick to reach the adult life stage, as under antibiotic treatment no tick was able to progress beyond the metanymph stage. Data presented here suggest that interference in the symbiotic CERM-R. microplus relationship may be useful to the development of alternative control methods, highlighting the interdependence between ticks and their endosymbionts.
Assuntos
Coxiella/fisiologia , Rhipicephalus/microbiologia , Simbiose , Animais , Coxiella/efeitos dos fármacos , Coxiella/genética , Feminino , Genoma Bacteriano/genética , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/microbiologia , Ninfa/efeitos dos fármacos , Ninfa/crescimento & desenvolvimento , Ninfa/microbiologia , Óvulo/efeitos dos fármacos , Óvulo/crescimento & desenvolvimento , Óvulo/microbiologia , Rhipicephalus/crescimento & desenvolvimento , Simbiose/efeitos dos fármacos , Tetraciclina/farmacologiaRESUMO
Fasciola hepatica is the agent of fasciolosis, a foodborne zoonosis that affects livestock production and human health. Although flukicidal drugs are available, re-infection and expanding resistance to triclabendazole demand new control strategies. Understanding the molecular mechanisms underlying the complex interaction with the mammalian host could provide relevant clues, aiding the search for novel targets in diagnosis and control of fasciolosis. Parasite survival in the mammalian host is mediated by parasite compounds released during infection, known as excretory/secretory (E/S) products. E/S products are thought to protect parasites from host responses, allowing them to survive for a long period in the vertebrate host. This work provides in-depth proteomic analysis of F. hepatica intra-mammalian stages, and represents the largest number of proteins identified to date for this species. Functional classification revealed the presence of proteins involved in different biological processes, many of which represent original findings for this organism and are important for parasite survival within the host. These results could lead to a better comprehension of host-parasite relationships, and contribute to the development of drugs or vaccines against this parasite.
Assuntos
Fasciola hepatica/crescimento & desenvolvimento , Proteínas de Helminto/metabolismo , Fígado/parasitologia , Proteômica/métodos , Animais , Fasciola hepatica/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Interações Hospedeiro-ParasitaRESUMO
BACKGROUND: Haemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods. METHODS: H. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences. RESULTS: A total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products. CONCLUSIONS: This study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis.
Assuntos
Proteínas de Artrópodes/química , Ixodidae/crescimento & desenvolvimento , Ixodidae/metabolismo , Proteoma/química , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Feminino , Ixodidae/química , Ixodidae/genética , Masculino , Ninfa/química , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica , Coelhos , Saliva/química , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismoRESUMO
Metalloproteases (MPs) have been considered essential for blood feeding and other physiological functions in several hematophagous animals, including ticks. We report the characterization of MP sequences of three important ticks from Asia, Africa and America: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) and R. microplus (BrRm-MPs). Amino acid sequence identity between R. microplus and R. sanguineus MPs ranged from 76 to 100 %, and identities among I. persulcatus, I. ricinus and I. scapularis MP sequences ranged from 88 to 97 %. This high sequence identity and typical functional motifs show that all sequences are MPs. The presence of a zinc binding site, a Met-turn and cysteine rich domain at the C-terminal region indicates that these proteins belong to the reproplysin family of MPs. Differences in amino acid sequences of BrRm-MP1, BrRm-MP2, BrRm-MP4 and BrRm-MP5 (from Porto Alegre strain ticks) were 6, 2, 7 and 5 %, respectively, when compared with sequences deposited in GenBank for the same genes from other R. microplus isolates. Analyses of MPs predicted that they have various highly antigenic regions. Semi-quantitative RT-PCR analysis revealed the presence of transcripts in salivary glands of partially and fully fed female ticks. None of these transcripts were observed in males (except BrRm-MP4) and eggs. These enzymes may be functional components required during tick feeding to manipulate host defenses and support tick hematophagy.
Assuntos
Ixodidae/enzimologia , Metaloproteases/genética , Filogenia , Glândulas Salivares/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Ixodidae/genética , Masculino , Dados de Sequência Molecular , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
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.