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.
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- akaricidy farmakologie MeSH
- aminoacyl-tRNA-synthetasy antagonisté a inhibitory genetika MeSH
- Borrelia burgdorferi komplex MeSH
- klíšťata účinky léků mikrobiologie MeSH
- lidé MeSH
- lymeská nemoc farmakoterapie mikrobiologie přenos MeSH
- proteosyntéza účinky léků MeSH
- vyvíjení léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- Ixodidae * MeSH
- klíště * MeSH
- lidé MeSH
- mikrobiota * MeSH
- ovarium MeSH
- Rhipicephalus * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To identify the gut-associated tick aspartic hemoglobinase, this work focuses on the functional diversity of multiple Ixodes ricinus cathepsin D forms (IrCDs). Out of three encoding genes representing Ixodes scapularis genome paralogs, IrCD1 is the most distinct enzyme with a shortened propeptide region and a unique pattern of predicted post-translational modifications. IrCD1 gene transcription is induced by tick feeding and is restricted to the gut tissue. The hemoglobinolytic role of IrCD1 was further supported by immunolocalization of IrCD1 in the vesicles of tick gut cells. Properties of recombinantly expressed rIrCD1 are consistent with the endo-lysosomal environment because the zymogen is autoactivated and remains optimally active in acidic conditions. Hemoglobin cleavage pattern of rIrCD1 is identical to that produced by the native enzyme. The preference for hydrophobic residues at the P1 and P1' position was confirmed by screening a novel synthetic tetradecapeptidyl substrate library. Outside the S1-S1' regions, rIrCD1 tolerates most amino acids but displays a preference for tyrosine at P3 and alanine at P2'. Further analysis of the cleavage site location within the peptide substrate indicated that IrCD1 is a true endopeptidase. The role in hemoglobinolysis was verified with RNAi knockdown of IrCD1 that decreased gut extract cathepsin D activity by >90%. IrCD1 was newly characterized as a unique hemoglobinolytic cathepsin D contributing to the complex intestinal proteolytic network of mainly cysteine peptidases in ticks.
- MeSH
- genetická transkripce fyziologie MeSH
- genom fyziologie MeSH
- hemoglobiny genetika metabolismus MeSH
- kathepsin D genetika metabolismus MeSH
- klíště enzymologie genetika MeSH
- posttranslační úpravy proteinů fyziologie MeSH
- proteiny členovců genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- střeva enzymologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Intracellular proteolysis of ingested blood proteins is a crucial physiological process in ticks. In our model tick, Ixodes ricinus, cathepsin L (IrCL1) is part of a gut-associated multi-peptidase complex; its endopeptidase activity is important in the initial phase of haemoglobinolysis. We present the functional and biochemical characterisation of this enzyme. We show, by RNA interference (RNAi), that cathepsin L-like activity that peaks during the slow feeding period of females is associated with IrCL1. Recombinant IrCL1 was expressed in bacteria and yeast. Activity profiling with both peptidyl and physiological protein substrates (haemoglobin and albumin) revealed that IrCL1 is an acidic peptidase with a very low optimum pH (3-4) being unstable above pH 5. This suggests an endo/lysosomal localisation that was confirmed by indirect fluorescence microscopy that immunolocalised IrCL1 inside the vesicles of digestive gut cells. Cleavage specificity determined by a positional scanning synthetic combinatorial library and inhibition profile indicated that IrCL1 has the ligand-binding characteristics of the cathepsin L subfamily of cysteine peptidases. A non-redundant proteolytic function was demonstrated when IrCL1-silenced ticks had a decreased ability to feed compared with controls. The data suggest that IrCL1 may be a promising target against ticks and tick-borne pathogens.
- MeSH
- albuminy metabolismus MeSH
- endozomy enzymologie MeSH
- exprese genu MeSH
- fluorescenční mikroskopie MeSH
- hemoglobiny metabolismus MeSH
- kathepsin L chemie genetika metabolismus MeSH
- klíště enzymologie MeSH
- koncentrace vodíkových iontů MeSH
- proteolýza MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- RNA interference MeSH
- stabilita enzymů MeSH
- umlčování genů MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
