Ticks are blood-feeding arthropods that use the components of their salivary glands to counter the host's hemostatic, inflammatory, and immune responses. The tick midgut also plays a crucial role in hematophagy. It is responsible for managing blood meals (storage and digestion) and protecting against host immunity and pathogen infections. Previous transcriptomic studies revealed the complexity of tick sialomes (salivary gland transcriptomes) and mialomes (midgut transcriptomes) which encode for protease inhibitors, lipocalins (histamine-binding proteins), disintegrins, enzymes, and several other tick-specific proteins. Several studies have demonstrated that mammalian hosts acquire tick resistance against repeated tick bites. Consequently, there is an urgent need to uncover how tick sialomes and mialomes respond to resistant hosts, as they may serve to develop novel tick control strategies and applications. Here, we mimicked natural repeated tick bites in a laboratory setting and analyzed gene expression dynamics in the salivary glands and midguts of adult female ticks. Rabbits were subjected to a primary (feeding on a naive host) and a secondary infestation of the same host (we re-exposed the hosts but to other ticks). We used single salivary glands and midguts dissected from individual siblings adult pathogen-free female Ixodes ricinus to reduce genetic variability between individual ticks. The comprehensive analysis of 88 obtained RNA-seq data sets allows us to provide high-quality annotated sialomes and mialomes from individual ticks. Comparisons between fed/unfed, timepoints, and exposures yielded as many as 3000 putative differentially expressed genes (DEG). Interestingly, when classifying the exposure DEGs by means of a clustering approach we observed that the majority of these genes show increased expression at early feeding time-points in the mid-gut of re-exposed ticks. The existence of clearly defined groups of genes with highly similar responses to re-exposure suggests the existence of molecular swiches. In silico functional analysis shows that these early feeding reexposure response genes form a dense interaction network at protein level being related to virtually all aspects of gene expression regulation and glycosylation. The processed data is available through an easy-to-use database-associated webpage (https://arn.ugr.es/IxoriDB/) that can serve as a valuable resource for tick research.
- MeSH
- klíště * genetika MeSH
- kousnutí klíštětem * MeSH
- králíci MeSH
- obratlovci MeSH
- proteiny členovců genetika metabolismus MeSH
- savci genetika MeSH
- slinné žlázy metabolismus MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA methylation at the fifth position of cytosine (5mC) and at the sixth position of adenine (6 mA) plays an important role in the regulation of the gene expression and, in eukaryotes, is essential for normal development. For Ixodes ricinus, the most common European arthropod vector of human and animal pathogens, the DNA methylation profile and the role of DNA methylation in tick development are still under discussion. Our goal was to analyze the status of I. ricinus DNA methylation at different life stages and identify enzymes that produce this type of DNA modification. We found that 5mC and 6mA are present in I. ricinus genomic DNA at all life stages. In the transcriptome of I. ricinus, we identified the sequences of the putative IrDNMT1, IrDNMT3, and IrDAMT enzymes, and bioinformatic analysis and three-dimensional modeling predicted their DNA methylation activity. This confirms that I. ricinus possesses a complete DNA methylation toolkit. Our results suggest that DNA methylation is important for the physiology and transstadial development of ticks.
- MeSH
- epigeneze genetická * MeSH
- genetická transkripce MeSH
- klíště enzymologie genetika růst a vývoj MeSH
- larva genetika růst a vývoj MeSH
- methyltransferasy chemie genetika MeSH
- molekulární konformace MeSH
- nymfa genetika růst a vývoj MeSH
- ovum růst a vývoj MeSH
- proteiny členovců chemie genetika MeSH
- regulace genové exprese * MeSH
- transkriptom * MeSH
- věkové faktory MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- arachnida jako vektory mikrobiologie MeSH
- bakteriální vakcíny aplikace a dávkování MeSH
- Borrelia burgdorferi komplex účinky léků MeSH
- infestace klíšťaty genetika mikrobiologie prevence a kontrola přenos MeSH
- klíště účinky léků MeSH
- lymeská nemoc genetika mikrobiologie prevence a kontrola přenos MeSH
- myši MeSH
- proteiny členovců genetika MeSH
- slinné žlázy mikrobiologie MeSH
- transkriptom * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The allergen repertoire of the house dust mite, Dermatophagoides farinae, is incomplete despite most mite allergens having been described in this species. Using proteogenomics, we aimed to compare proteins and allergens between sexes and provide a foundation for the identification of novel allergens. Overall, 6297 protein hits were identified, and 2899 and 886 were male- and female-specific, respectively. Removal of trace results narrowed the dataset to 3478 hits, including 275 and 157 male- and female-specific hits, respectively. All 34 WHO/IUIS-approved D. farinae allergens (omitting Der f 17) were identified, and we also identified homologs of the yet undescribed Der f 9 and 38. Der f 27/serpin exhibited the largest sex-dependent difference and was dominant in females. Using official protein sequences, Der f 11, 14, 23, 28 and 30 were identified with low success. However, identification success of Der f 11 and 14 was greatly increased by using longer/complete sequences. Der f 30 is characterized by the same tryptic digests as the more abundant Der f 30 (isoform) identified here. Der f 23 appears to be of low abundance in mite bodies. Der f 28.0101 and Der f 28.0201 were detected at low abundance and in trace amounts, respectively. SIGNIFICANCE: In this work, we performed a proteogenomic annotation of the house dust mite, Dermatophagoides farinae, which is the most important source of house dust allergens. The proteogenomic analysis performed here provides a foundation for not only understanding the biology of the mite but also the identification of novel allergens. This study generated a robust proteomic dataset for D. farinae and reviewed existing and candidate allergens in this species. We stress some pitfalls of high-throughput analyses, especially that improper headers of allergen protein records provided in databases can lead to confusion. Using partial sequences in proteomic identification and quantification can lead to low identification success (low signal intensity or MS/MS counts). Thus, we individually curated the protein sequences for proper identification and quantification. The discovered sex differences can be one factor affecting allergen/immunogen variations in mite extracts. Overall, this work provides a benchmark for accurate identification of mite immunogenic proteins using proteomics.
- MeSH
- alergeny genetika imunologie metabolismus MeSH
- Dermatophagoides farinae genetika imunologie metabolismus MeSH
- proteiny členovců genetika imunologie metabolismus MeSH
- proteogenomika metody MeSH
- proteom metabolismus MeSH
- Pyroglyphidae genetika imunologie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- sexuální faktory MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
BACKGROUND: The hard tick Hyalomma dromedarii is one of the most injurious ectoparasites affecting camels and apparently best adapted to deserts. As long-term blood feeders, ticks are threatened by host defense system compounds that can cause them to be rejected and, ultimately, to die. However, their saliva contains a cocktail of bioactive molecules that enables them to succeed in taking their blood meal. A recent sialotranscriptomic study uncovered the complexity of the salivary composition of the tick H. dromedarii and provided a database for a proteomic analysis. We carried out a proteomic-informed by transcriptomic (PIT) to identify proteins in salivary glands of both genders of this tick species. RESULTS: We reported the array of 1111 proteins identified in the salivary glands of H. dromedarii ticks. Only 24% of the proteins were shared by both genders, and concur with the previously described sialotranscriptome complexity. The comparative analysis of the salivary glands of both genders did not reveal any great differences in the number or class of proteins expressed their enzymatic composition or functional classification. Indeed, few proteins in the entire proteome matched those predicted from the transcriptome while others corresponded to other proteins of other tick species. CONCLUSION: This investigation represents the first proteomic study of H. dromedarii salivary glands. Our results shed light on the differences between the composition of H. dromedarii male and female salivary glands, thus enabling us to better understand the gender-specific strategy to feed successfully.
- MeSH
- klíšťata genetika metabolismus MeSH
- proteiny členovců genetika metabolismus MeSH
- proteom metabolismus MeSH
- proteomika MeSH
- slinné žlázy metabolismus MeSH
- sliny metabolismus MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- velbloudi MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Tick innate immunity involves humoral and cellular responses. Among the humoral effector molecules in ticks are the defensins which are a family of small peptides with a conserved γ-core motif that is crucial for their antimicrobial activity. Defensin families have been identified in several hard and soft tick species. However, little is known about the presence and antimicrobial activity of defensins from the Australian paralysis tick Ixodes holocyclus. In this study the I. holocyclus transcriptome was searched for the presence of defensins. Unique and non-redundant defensin sequences were identified and designated as holosins 1 - 5. The antimicrobial activity of holosins 2 and 3 and of the predicted γ-cores of holosins 1-4 (HoloTickCores 1-4), was assessed using Gram-negative and Gram-positive bacteria as well as the fungus Fusarium graminearum and the yeast Candida albicans. All holosins had molecular features that are conserved in other tick defensins. Furthermore holosins 2 and 3 were very active against the Gram-positive bacteria Staphylococcus aureus and Listeria grayi. Holosins 2 and 3 were also active against F. graminearum and C. albicans and 5 μM of peptide abrogate the growth of these microorganisms. The activity of the synthetic γ-cores was lower than that of the mature defensins apart from HoloTickCore 2 which had activity comparable to mature holosin 2 against the Gram-negative bacterium Escherichia coli. This study reveals the presence of a multigene defensin family in I. holocyclus with wide antimicrobial activity.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- antifungální látky chemie farmakologie MeSH
- Candida albicans účinky léků MeSH
- defensiny chemie genetika imunologie MeSH
- Fusarium účinky léků MeSH
- fylogeneze MeSH
- gramnegativní bakterie účinky léků MeSH
- grampozitivní bakterie účinky léků MeSH
- klíště genetika imunologie MeSH
- proteiny členovců chemie genetika imunologie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Austrálie MeSH
To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.
- MeSH
- cystatiny klasifikace genetika farmakologie MeSH
- cytokiny metabolismus MeSH
- epoxidové sloučeniny metabolismus MeSH
- fylogeneze MeSH
- imunosupresiva chemie metabolismus farmakologie MeSH
- klíště chemie genetika metabolismus MeSH
- krystalografie rentgenová MeSH
- makrofágy účinky léků metabolismus MeSH
- oxid dusnatý metabolismus MeSH
- proteiny členovců chemie genetika farmakologie MeSH
- proteolýza účinky léků MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- slinné cystatiny chemie genetika farmakologie MeSH
- T-lymfocyty účinky léků metabolismus MeSH
- tyrosin analogy a deriváty metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
By searching nucleotide databases for the North American Lyme disease vector, Ixodes scapularis, we have complemented the previously characterized European Ixodes ricinus legumain IrAE1 with a full set of nine analogous genes (isae1-9). Six of these were PCR confirmed as genes present in all tick genomes tested. The absolute mRNA copy number examined by quantitative (q)PCR enabled expression profiling and an absolute comparison of mRNA levels for individual I. scapularis (Is)AEs in tick tissues. Four IsAEs (1, 2, 4, 9) were expressed solely in the gut and thus are proposed to be involved in host blood digestion. Expression qPCR profiling over developmental stages confirmed IsAE1, the direct analogue of previously characterized I. ricinus IrAE1, as the principle legumain transcript in partially engorged females, and demonstrated its strong regulation by on-host feeding in larvae, nymphs and females. In contrast, IsAE2 was the predominant gut legumain in unfed nymphs, unfed females and males. In-silico, IsAE1 and IsAE2 protein three-dimensional structural models displayed minimal differences in overall proenzyme structures, even in comparison with recently resolved crystal structures of mammalian prolegumain. Three functional studies were performed in I. ricinus with IsAE1/IsAE2 analogues: double IrAE1/IrAE2 RNA interference silencing, feeding of ticks on IrAE1+IrAE2 immunized hosts and in vitro membrane tick feeding on blood containing a legumain-specific inhibitor. The latter experiment led to reduced weights of fully engorged ticks and limited oviposition, and indicated the potential of legumain inhibitors for novel anti-tick interventions.
- MeSH
- arachnida jako vektory enzymologie MeSH
- cysteinové endopeptidasy klasifikace genetika metabolismus MeSH
- infestace klíšťaty prevence a kontrola veterinární MeSH
- izoenzymy MeSH
- klíště enzymologie MeSH
- klonování DNA MeSH
- konformace proteinů MeSH
- králíci MeSH
- molekulární modely MeSH
- proteiny členovců klasifikace genetika metabolismus MeSH
- regulace genové exprese enzymů MeSH
- rekombinantní proteiny imunologie MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- vakcíny imunologie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mites of the genus Tyrophagus (Acari: Acaridae) are among the most widespread and common mites, inhabiting diverse natural and anthropogenic habitats. Some species are pests of agricultural products and stored food and/or live in house dust, causing allergies to humans. We sequenced 1.2 kb of the mitochondrial COI gene for 38 individuals belonging to seven species of Tyrophagus, including T. curvipenis, T. putrescentiae, T. fanetzhangorum, T. longior, T. perniciosus, and T. cf. similis. Molecular phylogenetic analyses (1) recovered two major clades corresponding to the presence or absence of eyespots, and (2) separated all included morphological species. Tyrophagus curvipenis and T. putrescentiae had the lowest between-species genetic distances (range, mean ± SD): 14.20-16.30, 15.17 ± 0.40 (K2P). The highest within-species variation was found in T. putrescentiae 0.00-4.33, 1.78 ± 1.44 (K2P). In this species, we recovered two distinct groups; however, no geographical or ecological dissimilarities were observed between them. Based on our analyses, we document important morphological differences between T. curvipenis and T. putrescentiae. For the first time, we record the occurrence of T. curvipenis in the New World and suggest that it may be an emerging pest as it is currently spreading in agricultural produce.
- MeSH
- Acaridae anatomie a histologie klasifikace enzymologie genetika MeSH
- biologická evoluce * MeSH
- fylogeneze * MeSH
- mitochondriální proteiny genetika MeSH
- proteiny členovců genetika MeSH
- respirační komplex IV genetika MeSH
- rozšíření zvířat MeSH
- sekvenční analýza DNA MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Blood-feeding parasites are inadvertently exposed to high doses of potentially cytotoxic haem liberated upon host blood digestion. Detoxification of free haem is a special challenge for ticks, which digest haemoglobin intracellularly. Ticks lack a haem catabolic mechanism, mediated by haem oxygenase, and need to dispose of vast majority of acquired haem via its accumulation in haemosomes. The knowledge of individual molecules involved in the maintenance of haem homeostasis in ticks is still rather limited. RNA-seq analyses of the Ixodes ricinus midguts from blood- and serum-fed females identified an abundant transcript of glutathione S-transferase (gst) to be substantially up-regulated in the presence of red blood cells in the diet. Here, we have determined the full sequence of this encoding gene, ir-gst1, and found that it is homologous to the delta-/epsilon-class of GSTs. Phylogenetic analyses across related chelicerates revealed that only one clear IrGST1 orthologue could be found in each available transcriptome from hard and soft ticks. These orthologues create a well-supported clade clearly separated from other ticks' or mites' delta-/epsilon-class GSTs and most likely evolved as an adaptation to tick blood-feeding life style. We have confirmed that IrGST1 expression is induced by dietary haem(oglobin), and not by iron or other components of host blood. Kinetic properties of recombinant IrGST1 were evaluated by model and natural GST substrates. The enzyme was also shown to bind haemin in vitro as evidenced by inhibition assay, VIS spectrophotometry, gel filtration, and affinity chromatography. In the native state, IrGST1 forms a dimer which further polymerises upon binding of excessive amount of haemin molecules. Due to susceptibility of ticks to haem as a signalling molecule, we speculate that the expression of IrGST1 in tick midgut functions as intracellular buffer of labile haem pool to ameliorate its cytotoxic effects upon haemoglobin intracellular hydrolysis.
- MeSH
- fylogeneze * MeSH
- glutathiontransferasa * biosyntéza chemie genetika MeSH
- hemoglobiny chemie metabolismus MeSH
- klíště * enzymologie genetika MeSH
- proteiny členovců * biosyntéza chemie genetika MeSH
- regulace genové exprese enzymů fyziologie MeSH
- substrátová specifita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
