Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

<p>Abstract</p> <p>Background</p> <p>Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets.</p> <p>Results</p> <p>Using the hard tick, <it>Ixodes ricinus</it>, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain), and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood.</p> <p>Conclusion</p> <p>Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.</p

Blood-feeding adaptations and virome assessment of the poultry red mite Dermanyssus gallinae guided by RNA-seq

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.Instituto de Patología VegetalFil: Ribeiro, José M. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research; Estados UnidosFil: Hartmann, David. Czech Academy of Sciences. Biology Centre. Institute of Parasitology. Biology Centre; República ChecaFil: Bartošová-Sojková, Pavla. Czech Academy of Sciences. Biology Centre. Institute of Parasitology. Biology Centre; República ChecaFil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Debat, Humberto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Moos, Martin. Czech Academy of Sciences. Biology Centre. Institute of Entomology; República ChecaFil: Šimek, Petr. Czech Academy of Sciences. Biology Centre. Institute of Entomology; República ChecaFil: Fara, Jiří.International Poultry Testing Station Ústrašice; República ChecaFil: Palus, Martin. Czech Academy of Sciences. Biology Centre. Institute of Parasitology; República ChecaFil: Kučera, Matěj. Czech Academy of Sciences. Biology Centre. Institute of Parasitology; República ChecaFil: Hajdušek, Ondřej. Czech Academy of Sciences. Biology Centre. Institute of Parasitology; República Chec

Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement.

Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity

anti-tick vaccines to prevent tick-borne diseases in Europe

Ixodes ricinus transmits bacterial, protozoal and viral pathogens, causing disease and forming an increasing health concern in Europe. ANTIDotE is an European Commission funded consortium of seven institutes, which aims to identify and characterize tick proteins involved in feeding and pathogen transmission. The knowledge gained will be used to develop and evaluate anti- tick vaccines that may prevent multiple human tick-borne diseases. Strategies encompassing anti-tick vaccines to prevent transmission of pathogens to humans, animals or wildlife will be developed with relevant stakeholders with the ultimate aim of reducing the incidence of tick-borne diseases in humans

Addendum: Kotál et al. Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement. Int. J. Mol. Sci. 2021, 22, 9480

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Reptile-associated Borrelia species in the goanna tick (Bothriocroton undatum) from Sydney, Australia

Abstract Background Knowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. By exploiting the bilateral anatomical symmetry of ticks, we were able to directly compare two DNA extraction methods for 16S rRNA gene diversity profiling and pathogen detection. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp. Results We collected Australian endemic ticks (Bothriocroton undatum), isolated DNA from equal tick halves using two commercial DNA extraction methods and sequenced samples using V1-V3 and V3-V4 16S rRNA gene diversity profiling assays. Two distinct Borrelia spp. operational taxonomic units (OTUs) were detected using the V1-V3 16S rRNA hypervariable region and matching Borrelia spp. sequences were obtained using a conventional nested-PCR. The tick 16S rRNA gene diversity profile was dominated by Rickettsia spp. (98–99%), while the remaining OTUs belonged to Proteobacteria (51–81%), Actinobacteria (6–30%) and Firmicutes (2–7%). Multiple comparisons tests demonstrated biases in each of the DNA extraction kits towards different bacterial taxa. Conclusions Two distinct Borrelia species belonging to the reptile-associated Borrelia group were identified. Our results show that the method of DNA extraction can promote bias in the final microbiota identified. We determined an optimal DNA extraction method and 16S rRNA gene diversity profile assay that maximises detection of Borrelia species

Characterization of ferritin 2 for the control of tick infestations

Ixodes ricinus is one the most abundant tick species in Europe and these ticks transmit pathogens causing human and animal diseases. The cattle ticks, Rhipicephalus (Boophilus) spp., affect cattle production in tropical and subtropical regions of the world. Development of vaccines directed against tick proteins may reduce tick infestations and the transmission of tick-borne pathogens. However, a limiting step in tick vaccine development has been the identification of tick protective antigens. Herein, the tick iron metabolism pathway was targeted in an effort to identify new tick protective antigens. Recombinant I. ricinus (IrFER2) and Rhipicephalus microplus (RmFER2) ferritin 2 proteins were expressed in Escherichia coli and used to immunize rabbits and cattle, respectively. Vaccination with IrFER2 reduced I. ricinus tick numbers, weight and fertility in rabbits with an overall vaccine efficacy (E) of 98%. Control of cattle tick, R. microplus and Rhipicephalus annulatus infestations was obtained in vaccinated cattle with overall E of 64% and 72%, respectively. Notably, the efficacy of the RmFER2 vaccine was similar to that obtained with Bm86 against R. microplus. These collective results demonstrated the feasibility of using ferritin 2 to develop vaccines for the control of tick infestations.This work was supported by SEP-CONACYT, Mexico (project 25772); FOMIX, Tamaulipas, Mexico (project 73622); INIA, Spain (project FAU2008-00014-00-00) and the Consejería de Educación y Ciencia, JCCM, Spain (project PEII09-0118-8907). The Czech research team was supported by grant No. IAA600960910 to PK from the Grant Agency of the Academy of Sciences of the Czech Republic, Research Centre No. LC06009, and research plans Nos. 6007665801 and Z60220518 MSMT CR. M. Villar and M. Canales were funded by the JAE-DOC program (CSIC-FSE), Spain and the Wellcome Trust under the “Animal Health in the Developing World” initiative (project 0757990), respectively.Peer Reviewe