RESUMO
The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.
Assuntos
Ixodes , Saliva , Animais , Humanos , Saliva/metabolismo , Cisteína , Glicosaminoglicanos , Catepsinas/metabolismo , Ixodes/metabolismo , Espectroscopia de Ressonância MagnéticaRESUMO
Ticks are blood-feeding arachnids that are known to transmit various pathogenic microorganisms to their hosts. During blood feeding, ticks activate their metabolism and immune system to efficiently utilise nutrients from the host's blood and complete the feeding process. In contrast to insects, in which the fat body is known to be a central organ that controls essential metabolic processes and immune defense mechanisms, the function of the fat body in tick physiology is still relatively unexplored. To fill this gap, we sought to uncover the repertoire of genes expressed in the fat body associated with trachea (FB/Tr) by analyzing the transcriptome of individual, partially fed (previtellogenic) Ixodes ricinus females. The resulting catalog of individual mRNA sequences reveals a broad repertoire of transcripts encoding proteins involved in nutrient storage and distribution, as well as components of the tick immune system. To gain a detailed insight into the secretory products of FB/Tr specifically involved in inter-tissue transport and humoral immunity, the transcriptomic data were complemented with the proteome of soluble proteins in the hemolymph of partially fed female ticks. Among these proteins, the hemolipoglyco-carrier proteins were predominant. When comparing immune peptides and proteins from the fat body with those produced by hemocytes, we found that the fat body serves as a unique producer of certain immune components. Finally, time-resolved transcriptional regulation of selected immune transcripts from the FB/Tr was examined in response to experimental challenges with model microbes and analyzed by RT-qPCR. Overall, our data show that the fat body of ticks, similar to insects, is an important metabolic tissue that also plays a remarkable role in immune defense against invading microbes. These findings improve our understanding of tick biology and its impact on the transmission of tick-borne pathogens.
Assuntos
Hemolinfa , Ixodes , Feminino , Animais , Proteômica , Corpo Adiposo/metabolismo , Ixodes/genética , Ixodes/metabolismo , Perfilação da Expressão Gênica , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismoRESUMO
The control of ticks through vaccination offers a sustainable alternative to the use of chemicals that cause contamination and the selection of resistant tick strains. However, only a limited number of anti-tick vaccines have reached commercial realization. In this sense, an antigen effective against different tick species is a desirable target for developing such vaccines. A peptide derived from the tick P0 protein (pP0) conjugated to a carrier protein has been demonstrated to be effective against the Rhipicephalus microplus, Rhipicephalus sanguineus, and Amblyomma mixtum tick species. The aim of this work was to assess the efficacy of this peptide when conjugated to the Bm86 protein against Dermacentor nitens and Ixodes ricinus ticks. An RNAi experiment using P0 dsRNA from I. ricinus showed a dramatic reduction in the feeding of injected female ticks on guinea pigs. In the follow-up vaccination experiments, rabbits were immunized with the pP0-Bm86 conjugate and challenged simultaneously with larvae, nymphs, and the adults of I. ricinus ticks. In the same way, horses were immunized with the pP0-Bm86 conjugate and challenged with D. nitens larva. The pP0-Bm86 conjugate showed efficacies of 63% and 55% against I. ricinus and D. nitens ticks, respectively. These results, combined with previous reports of efficacy for this conjugate, show the promising potential for its development as a broad-spectrum anti-tick vaccine.
RESUMO
Ticks are ectoparasites that feed on blood and have an impressive ability to consume and process enormous amounts of host blood, allowing extremely long periods of starvation between blood meals. The central role in the parasitic lifestyle of ticks is played by the midgut. This organ efficiently stores and digests ingested blood and serves as the primary interface for the transmission of tick-borne pathogens. In this study, we used a label-free quantitative approach to perform a novel dynamic proteomic analysis of the midgut of Ixodesricinus nymphs, covering their development from unfed to pre-molt stages. We identified 1534 I. ricinus-specific proteins with a relatively low proportion of host proteins. This proteome dataset, which was carefully examined by manual scrutiny, allowed precise annotation of proteins important for blood meal processing and their dynamic changes during nymphal ontogeny. We focused on midgut molecules related to lipid hydrolysis, storage, and transport, opening a yet unexplored avenue for studying lipid metabolism in ticks. Further dynamic profiling of the tick's multi-enzyme digestive network, protease inhibitors, enzymes involved in redox homeostasis and detoxification, antimicrobial peptides, and proteins responsible for midgut colonization by Borrelia spirochetes promises to uncover new targets for targeting tick nymphs, the most critical life stage for transmission the pathogens that cause tick-borne diseases.
Assuntos
Ixodes , Animais , Ixodes/parasitologia , Proteoma , Proteômica , Sistema DigestórioRESUMO
Ticks are blood-feeding ectoparasites that devastate cattle farming and are an omnipresent nuisance to pets and humans, posing a threat of pathogen transmission. Laboratory experimental models can be instrumental in the search for molecular targets of novel acaricides or vaccines. Mainly, though, the experimental models represent invaluable tools for broadening our basic understanding of key processes of tick blood-feeding physiology and vector competence. In order to understand the function of a single component within the full complexity of a feeding tick, genetic or biochemical interventions are used for systemic phenotypisation. In this work, we summarise current experimental modalities that represent powerful approaches for determining biological functions of tick molecular components.
Assuntos
Doenças dos Bovinos , Doenças Transmitidas por Carrapatos , Carrapatos , Vacinas , Animais , Humanos , Bovinos , GenômicaRESUMO
BACKGROUND: Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate. METHODS: Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA). RESULTS: Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles. CONCLUSIONS: Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate.
Assuntos
Hipersensibilidade Alimentar , Ixodes , Picadas de Carrapatos , Animais , Humanos , Peixe-Zebra/metabolismo , Saliva , Galactose , Imunoglobulina E , Hipersensibilidade Alimentar/etiologia , Proteínas de Artrópodes , Imunoglobulina M , MamíferosRESUMO
Dermanyssus gallinae is a blood-feeding mite that parasitises wild birds and farmed poultry. Its remarkably swift processing of blood, together with the capacity to blood-feed during most developmental stages, makes this mite a highly debilitating pest. To identify specific adaptations to digestion of a haemoglobin-rich diet, we constructed and compared transcriptomes from starved and blood-fed stages of the parasite and identified midgut-enriched transcripts. We noted that midgut transcripts encoding cysteine proteases were upregulated with a blood meal. Mapping the full proteolytic apparatus, we noted a reduction in the suite of cysteine proteases, missing homologues for Cathepsin B and C. We have further identified and phylogenetically analysed three distinct transcripts encoding vitellogenins that facilitate the reproductive capacity of the mites. We also fully mapped transcripts for haem biosynthesis and the ferritin-based system of iron storage and inter-tissue trafficking. Additionally, we identified transcripts encoding proteins implicated in immune signalling (Toll and IMD pathways) and activity (defensins and thioester-containing proteins), RNAi, and ion channelling (with targets for commercial acaricides such as Fluralaner, Fipronil, and Ivermectin). Viral sequences were filtered from the Illumina reads and we described, in part, the RNA-virome of D. gallinae with identification of a novel virus, Red mite quaranjavirus 1.
Assuntos
Infestações por Ácaros , Ácaros , Doenças das Aves Domésticas , Animais , Aves Domésticas , Infestações por Ácaros/veterinária , Infestações por Ácaros/parasitologia , RNA-Seq , Viroma , Galinhas , Ácaros/genéticaRESUMO
Genomes of ticks display reductions, to various extents, in genetic coding for enzymes of the haem biosynthetic pathway. Here, we mined available transcriptomes of soft tick species and identified transcripts encoding only half of the enzymes involved in haem biosynthesis. Transcripts identified across most species examined were those coding for porphobilinogen synthase, coproporphyrinogen oxidase, protoporphyrinogen oxidase, and ferrochelatase. Genomic retention of porphobilinogen synthase seems to be soft tick-restricted as no such homologue has been identified in any hard tick species. Bioinformatic mining is thus strongly indicative of the lack of biochemical capacity for de novo haem biosynthesis, suggesting a requirement for dietary haem. In the hard tick Ixodes ricinus, depletion of dietary haem, i.e. serum feeding, leads to oviposition of haem-free eggs, with no apparent embryogenesis and larvae formation. In this work, we show that serum-fed Ornithodoros moubata females, unlike those of I. ricinus, laid haem-containing eggs similarly to blood-fed controls, but only by a small proportion of the serum-fed females. To enhance the effect of dietary haem depletion, O. moubata ticks were serum-fed consecutively as last nymphal instars and females. These females laid eggs with profoundly reduced haem deposits, confirming the host origin of the haem. These data confirm the ability of soft ticks to take up and allocate host haem to their eggs in order to drive reproduction of the ticks.
Assuntos
Argasidae , Ixodidae , Ornithodoros , Animais , Feminino , Heme , Sintase do PorfobilinogênioRESUMO
Anaplasma phagocytophilum is the causative agent of tick-borne fever (TBF) and human granulocytic anaplasmosis (HGA) and is currently considered an emerging disease in the USA, Europe, and Asia. The increased prevalence of A. phagocytophilum as a human pathogen requires the detailed characterization of human isolates and the implementation of appropriate animal models. In this study, we demonstrated that the dynamics of infection with the human isolate of A. phagocytophilum NY-18 was variable in three different strains of mice (SCID, C3H/HeN, BALB/c). We further evaluated the ability of Ixodes ricinus to acquire and transmit A. phagocytophilum NY-18 and compared it with Ixodes scapularis. Larvae of both tick species effectively acquired the pathogen while feeding on infected mice. The infection rates then decreased during the development to nymphs. Interestingly, molted I. ricinus nymphs were unable to transmit the pathogen to naïve mice, which contrasted with I. scapularis. The results of our study suggest that I. ricinus is not a competent vector for the American human Anaplasma isolate. Further studies are needed to establish reliable transmission models for I. ricinus and European human isolate(s) of A. phagocytophilum.
RESUMO
BACKGROUND: Ticks are obligate hematophagous arthropods transmitting a wide range of pathogens to humans and animals. They also harbor a non-pathogenic microbiota, primarily in the ovaries and the midgut. In the previous study on Ixodes ricinus, we used a culture-independent approach and showed a diverse but quantitatively poor midgut bacterial microbiome. Our analysis also revealed the absence of a core microbiome, suggesting an environmental origin of the tick midgut microbiota. METHODS: A bacterial analysis of the midgut of adult females collected by flagging from two localities in the Czech Republic was performed. Using the culture-independent approach, we tested the hypothesis that the midgut microbiome is of the environmental origin. We also cultured indigenous bacteria from the tick midgut and used these to feed ticks artificially in an attempt to manipulate the midgut microbiome. RESULTS: The midgut showed a very low prevalence and abundance of culturable bacteria, with only 37% of ticks positive for bacteria. The culture-independent approach revealed the presence of Borrelia sp., Spiroplasma sp., Rickettsia sp., Midichloria sp. and various mainly environmental Gram-positive bacterial taxa. The comparison of ticks from two regions revealed that the habitat influenced the midgut bacterial diversity. In addition, the midgut of ticks capillary fed with the indigenous Micrococcus luteus (Gram-positive) and Pantoea sp. (Gram-negative) could not be colonized due to rapid and effective clearance of both bacterial taxa. CONCLUSIONS: The midgut microbiome of I. ricinus is diverse but low in abundance, with the exception of tick-borne pathogens and symbionts. The environment impacts the diversity of the tick midgut microbiome. Ingested extracellular environmental bacteria are rapidly eliminated and are not able to colonize the gut. We hypothesize that bacterial elimination triggered in the midgut of unfed adult females is critical to maintain low microbial levels during blood-feeding.
Assuntos
Borrelia , Ixodes , Microbiota , Rickettsia , Animais , República Tcheca/epidemiologia , Feminino , Ixodes/microbiologiaRESUMO
Ticks are blood-feeding ectoparasites with distinct genomic reductions, inevitably linking them to a parasitic lifestyle. Ticks have lost the genomic coding and, thus, biochemical capacity to synthesize heme, an essential metabolic cofactor, de novo. Instead, they are equipped with acquisition and distribution pathways for reuse of host heme. Unlike insects or mammals, ticks and mites cannot cleave the porphyrin ring of heme to release iron. Bioavailable iron is thus acquired by ticks from the host serum transferrin. Somatic trafficking of iron, however, is independent of heme and is mediated by a secretory type of ferritin. Heme and iron systemic homeostasis in ticks represents, therefore, key adaptive traits enabling successful feeding and reproduction.
Assuntos
Ácaros , Carrapatos , Animais , Heme/metabolismo , Homeostase , Ferro/metabolismo , MamíferosRESUMO
It has been demonstrated that impairing protein synthesis using drugs targeted against tRNA amino acid synthetases presents a promising strategy for the treatment of a wide variety of parasitic diseases, including malaria and toxoplasmosis. This is the first study evaluating tRNA synthetases as potential drug targets in ticks. RNAi knock-down of all tested tRNA synthetases had a strong deleterious phenotype on Ixodes ricinus feeding. Our data indicate that tRNA synthetases represent attractive, anti-tick targets warranting the design of selective inhibitors. Further, we tested whether these severely impaired ticks were capable of transmitting Borrelia afzelii spirochaetes. Interestingly, biologically handicapped I. ricinus nymphs transmitted B. afzelii in a manner quantitatively sufficient to develop a systemic infection in mice. These data suggest that initial blood-feeding, despite the incapability of ticks to fully feed and salivate, is sufficient for activating B. afzelii from a dormant to an infectious mode, enabling transmission and dissemination in host tissues.
Assuntos
Acaricidas/farmacologia , Doença de Lyme/transmissão , Carrapatos/efeitos dos fármacos , Carrapatos/microbiologia , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/genética , Animais , Grupo Borrelia Burgdorferi , Desenvolvimento de Medicamentos , Humanos , Doença de Lyme/tratamento farmacológico , Doença de Lyme/microbiologia , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
In addition to being vectors of pathogenic bacteria, ticks also harbor intracellular bacteria that associate with ticks over generations, aka symbionts. The biological significance of such bacterial symbiosis has been described in several tick species but its function in Ixodes ricinus is not understood. We have previously shown that I. ricinus ticks are primarily inhabited by a single species of symbiont, Midichloria mitochondrii, an intracellular bacterium that resides and reproduces mainly in the mitochondria of ovaries of fully engorged I. ricinus females. To study the functional integration of M. mitochondrii into the biology of I. ricinus, an M. mitochondrii-depleted model of I. ricinus ticks was sought. Various techniques have been described in the literature to achieve dysbiosed or apo-symbiotic ticks with various degrees of success. To address the lack of a standardized experimental procedure for the production of apo-symbiotic ticks, we present here an approach utilizing the ex vivo membrane blood feeding system. In order to deplete M. mitochondrii from ovaries, we supplemented dietary blood with tetracycline. We noted, however, that the use of tetracycline caused immediate toxicity in ticks, caused by impairment of mitochondrial proteosynthesis. To overcome the tetracycline-mediated off-target effect, we established a protocol that leads to the production of an apo-symbiotic strain of I. ricinus, which can be sustained in subsequent generations. In two generations following tetracycline administration and tetracycline-mediated symbiont reduction, M. mitochondrii was gradually eliminated from the lineage. Larvae hatched from eggs laid by such M. mitochondrii-free females repeatedly performed poorly during blood-feeding, while the nymphs and adults performed similarly to controls. These data indicate that M. mitochondrii represents an integral component of tick ovarian tissue, and when absent, results in the formation of substandard larvae with reduced capacity to blood-feed.
Assuntos
Ixodes , Animais , Feminino , Ixodes/microbiologia , Tetraciclina , Antibacterianos , Mitocôndrias , SimbioseRESUMO
Entomopathogenic fungi (EPF) have been widely explored for their potential in the biological control of insect pests and as an environmentally friendly alternative to acaricides for limiting tick infestation in the field. The arthropod cuticle is the main barrier against fungal infection, however, an understanding of internal defense mechanisms after EPF intrusion into the invertebrate hemocoel is still rather limited. Using an infection model of the European Lyme borreliosis vector Ixodes ricinus with the EPF Metarhizium robertsii, we demonstrated that ticks are capable of protecting themselves to a certain extent against mild fungal infections. However, tick mortality dramatically increases when the capability of tick hemocytes to phagocytose fungal conidia is impaired. Using RNAi-mediated silencing of tick thioester-containing proteins (TEPs), followed by in vitro and/or in vivo phagocytic assays, we found that C3-like complement components and α2-macroglobulin pan-protease inhibitors secreted to the hemolymph play pivotal roles in M. robertsii phagocytosis.
Assuntos
Ixodes , Doença de Lyme , Metarhizium , Animais , HemócitosRESUMO
An anti-tick mRNA cocktail vaccine promotes tick detachment and prevents transmission of tick-borne infection in guinea pigs (Sajid et al.).
Assuntos
Carrapatos , Animais , Cobaias , RNA Mensageiro/genética , Carrapatos/imunologiaRESUMO
Ticks are blood-feeding arachnids transmitting a variety of pathogens to humans and animals. A unique trait in tick physiology is their ability to engorge and digest large amounts of host blood, ensuring their high reproductive potential. Activation of the blood digestive machinery in the tick gut, as well as processes controlling maturation of ovaries, are triggered upon blood meal uptake by still largely unknown mechanisms. Sensing of the nutritional status in metazoan organisms is facilitated by the evolutionarily conserved Insulin Signaling Pathway (ISP) and the interlinked Target of Rapamycin (TOR) pathway. Recently, we have identified three components of these pathways in the hard tick Ixodes ricinus midgut transcriptome, namely a putative insulin receptor (InR), and the downstream intracellular serine/threonine kinases AKT and TOR. In this study, we primarily focus on the molecular and functional characterization of the I. ricinus insulin receptor (IrInR), the first InR characterized in Chelicerates. A phylogenetic analysis across the major Arthropod lineages demonstrated that ticks possess only one gene encoding an InR-related molecule. Tissue expression profiling by quantitative PCR in semi-engorged I. ricinus females revealed that the IrInR, as well as AKT (IrAKT) and TOR (IrTOR) are expressed in various organs, with the highest expression being detected in ovaries. We have further evaluated the impact of RNAi-mediated knock-down (KD) of IrInR, IrAKT, and IrTOR on tick blood-feeding and reproductive capacity. Weights of engorged IrInR KD females and laid egg clutches were reduced compared to the control group, and these quantitative parameters clearly correlated with the efficiency of RNAi-KD achieved in individual ticks. The most striking phenotype was observed for IrAKT KD that impaired tick feeding and completely aborted egg production. A recombinant extracellular fragment of the IrInR α-subunit was used to produce antibodies in experimental rabbits to assess its potential as a protective antigen against tick feeding and reproduction. Our data clearly indicate the functionality of the ISP in ticks and demonstrate the need for further investigation of specific roles played by the endogenous insulin-like peptides in tick physiological processes.
Assuntos
Insulina/genética , Ixodes/genética , Transdução de Sinais , Animais , Proteínas de Artrópodes/análise , Feminino , Insulina/metabolismo , Ixodes/metabolismo , Receptor de Insulina/análiseRESUMO
Ticks are ectoparasitic arthropods that necessarily feed on the blood of their vertebrate hosts. The success of blood acquisition depends on the pharmacological properties of tick saliva, which is injected into the host during tick feeding. Saliva is also used as a vehicle by several types of pathogens to be transmitted to the host, making ticks versatile vectors of several diseases for humans and other animals. When a tick feeds on an infected host, the pathogen reaches the gut of the tick and must migrate to its salivary glands via hemolymph to be successfully transmitted to a subsequent host during the next stage of feeding. In addition, some pathogens can colonize the ovaries of the tick and be transovarially transmitted to progeny. The tick immune system, as well as the immune system of other invertebrates, is more rudimentary than the immune system of vertebrates, presenting only innate immune responses. Although simpler, the large number of tick species evidences the efficiency of their immune system. The factors of their immune system act in each tick organ that interacts with pathogens; therefore, these factors are potential targets for the development of new strategies for the control of ticks and tick-borne diseases. The objective of this review is to present the prevailing knowledge on the tick immune system and to discuss the challenges of studying tick immunity, especially regarding the gaps and interconnections. To this end, we use a comparative approach of the tick immune system with the immune system of other invertebrates, focusing on various components of humoral and cellular immunity, such as signaling pathways, antimicrobial peptides, redox metabolism, complement-like molecules and regulated cell death. In addition, the role of tick microbiota in vector competence is also discussed.
Assuntos
Imunidade Celular , Imunidade Humoral , Saliva/imunologia , Glândulas Salivares/imunologia , Doenças Transmitidas por Carrapatos/imunologia , Carrapatos/imunologia , Animais , Interações Hospedeiro-Parasita , Humanos , Saliva/metabolismo , Glândulas Salivares/metabolismo , Doenças Transmitidas por Carrapatos/metabolismo , Doenças Transmitidas por Carrapatos/transmissão , Carrapatos/metabolismoRESUMO
During feeding on vertebrate hosts, ticks secrete saliva composed of a rich cocktail of bioactive molecules modulating host immune responses. Although most of the proteinaceous fraction of tick saliva is of little immunogenicity, repeated feeding of ticks on mammalian hosts may lead to impairment of tick feeding, preventing full engorgement. Here, we challenged rabbits with repeated feeding of both Ixodes ricinus nymphs and adults and observed the formation of specific antibodies against several tick salivary proteins. Repeated feeding of both I. ricinus stages led to a gradual decrease in engorged weights. To identify the salivary antigens, isolated immunoglobulins from repeatedly infested rabbits were utilized for a protein pull-down from the saliva of pilocarpine-treated ticks. Eluted antigens were first identified by peptide mass fingerprinting with the aid of available I. ricinus salivary gland transcriptomes originating from early phases of tick feeding. To increase the authenticity of immunogens identified, we also performed, for the first time, de novo assembly of the sialome from I. ricinus females fed for six days, a timepoint used for pilocarpine-salivation. The most dominant I. ricinus salivary immunogens identified in our study were zinc-dependent metalloproteases of three different families. To corroborate the role of metalloproteases at the tick/host interface, we fed ticks micro-injected with a zinc metalloprotease inhibitor, phosphoramidon, on a rabbit. These ticks clearly failed to initiate feeding and to engorge. However, neither feeding to ticks immune blood of repeatedly infested rabbits, nor phosphoramidon injection into ticks, prevented their engorgement when fed in vitro on an artificial membrane system. These data show that Zn metalloproteases play a decisive role in the success of tick feeding, mediated by complex molecular interactions between the host immune, inflammatory, and hemostatic processes, which are absent in in vitro feeding. This basic concept warrants further investigation and reconsideration of the current strategies towards the development of an effective "anti-tick" vaccine.
Assuntos
Ixodes , Infestações por Carrapato , Animais , Proteínas de Artrópodes , Feminino , Metaloproteases , Coelhos , Glândulas Salivares , Proteínas e Peptídeos SalivaresRESUMO
Ixodes ricinus is the vector for Borrelia afzelii, the predominant cause of Lyme borreliosis in Europe, whereas Ixodes scapularis is the vector for Borrelia burgdorferi in the USA. Transcription of several I. scapularis genes changes in the presence of B. burgdorferi and contributes to successful infection. To what extend B. afzelii influences gene expression in I. ricinus salivary glands is largely unknown. Therefore, we measured expression of uninfected vs. infected tick salivary gland genes during tick feeding using Massive Analysis of cDNA Ends (MACE) and RNAseq, quantifying 26.179 unique transcripts. While tick feeding was the main differentiator, B. afzelii infection significantly affected expression of hundreds of transcripts, including 465 transcripts after 24 h of tick feeding. Validation of the top-20 B. afzelii-upregulated transcripts at 24 h of tick feeding in ten biological genetic distinct replicates showed that expression varied extensively. Three transcripts could be validated, a basic tail protein, a lipocalin and an ixodegrin, and might be involved in B. afzelii transmission. However, vaccination with recombinant forms of these proteins only marginally altered B. afzelii infection in I. ricinus-challenged mice for one of the proteins. Collectively, our data show that identification of tick salivary genes upregulated in the presence of pathogens could serve to identify potential pathogen-blocking vaccine candidates.
Assuntos
Vetores Aracnídeos/microbiologia , Proteínas de Artrópodes/genética , Vacinas Bacterianas/administração & dosagem , Doença de Lyme/genética , Glândulas Salivares/microbiologia , Infestações por Carrapato/genética , Transcriptoma , Animais , Grupo Borrelia Burgdorferi/efeitos dos fármacos , Feminino , Ixodes/efeitos dos fármacos , Doença de Lyme/microbiologia , Doença de Lyme/prevenção & controle , Doença de Lyme/transmissão , Camundongos , Infestações por Carrapato/microbiologia , Infestações por Carrapato/prevenção & controle , Infestações por Carrapato/transmissãoRESUMO
Culture-independent metagenomic methodologies have enabled detection and identification of microorganisms in various biological systems and often revealed complex and unknown microbiomes. In many organisms, the microbiome outnumbers the host cells and greatly affects the host biology and fitness. Ticks are hematophagous ectoparasites with a wide host range. They vector a number of human and animal pathogens and also directly cause major economic losses in livestock. Although several reports on a tick midgut microbiota show a diverse bacterial community, in most cases the size of the bacterial population has not been determined. In this study, the microbiome was quantified in the midgut and ovaries of the ticks Ixodes ricinus and Rhipicephalus microplus before, during, and after blood feeding. Although the size of bacterial community in the midgut fluctuated with blood feeding, it was overall extremely low in comparison to that of other hematophagous arthropods. In addition, the tick ovarian microbiome of both tick species exceeded the midgut 16S rDNA copy numbers by several orders of magnitude. This indicates that the ratio of a tick midgut/ovary microbiome represents an exception to the general biology of other metazoans. In addition to the very low abundance, the tick midgut diversity in I. ricinus was variable and that is in contrast to that found in the tick ovary. The ovary of I. ricinus had a very low bacterial diversity and a very high and stable bacterial abundance with the dominant endosymbiont, Midichloria sp. The elucidation of this aspect of tick biology highlights a unique tissue-specific microbial-invertebrate host interaction.