Novel gene, named TILIr ( TIL Ixodes ricinus), that encoded yet unknown protein with TIL domain characteristic to proteins that belong to trypsin inhibitors family was discovered and isolated from hard tick Ixodes ricinus. Two isoforms of novel gene were identified and studied in this project. As the result of this study recombinant TILIr protein was produced in bacterial expression system and analysis of its functional characteristics was conducted. The protein belonging to family of trypsin inhibitors represent a potential candidate for anti-tick vaccine component.

Cistatinas são um grupo de inibidores de cisteíno proteases que atuam na regulação da proteólise e estão presentes em uma ampla variedade de organismos. Estudos sobre essa classe de inibidores em parasitas têm contribuído para elucidar suas funções na regulação de processos fisiológicos como a digestão do sangue e a supressão da resposta imune do hospedeiro durante a alimentação. Por isso, cistatinas são um alvo interessante na pesquisa para o desenvolvimento de novos métodos de controle de parasitas. Além disso, a caracterização de proteínas compartilhadas por diferentes espécies de parasitas representa uma estratégia viável para encontrar potenciais alvos para uma vacina multi-espécie. Entretanto, as funções das cistatinas nos carrapatos permanecem obscuras, especialmente na espécie Ixodes ovatus. Neste trabalho, foi caracterizado o perfil inibitório de rJpIocys2a, uma cistatina de I. ovatus, para as catepsinas B, C e L. O perfil de inibição enzimática de rJpIocys2a se mostrou variável entre catepsinas envolvidas na digestão do sangue pelo carrapato e na evasão da resposta imune do hospedeiro. Além disso, um peptídeo baseado na sequência de aminoácidos do inibidor JpIocsy2a (STQ-pep) foi sintetizado e utilizado para imunização de coelhos. O soro anti-STQ-pep foi usado para analisar a antigenicidade cruzada entre cistatinas dos carrapatos Rhipicephalus microplus e I. ovatus. A análise da antigenicidade cruzada revelou que anticorpos gerados contra o peptídeo são capazes de reconhecer cistatinas nativas e recombinante de R. microplus. A habilidade de rJpIocys2a de inibir catepsinas relacionadas a diferentes funções sugere múltiplas funções para esse inibidor. Os resultados obtidos neste trabalho forneceram bases para outros experimentos que utilizem esta proteína como antígeno vacinal. / Cystatins are a group of cysteine protease inhibitors that act on the regulation of physiological proteolysis and are present in a wide range of organisms. Researches on this class of inhibitors in parasites have contributed to clarify their roles in the regulation of important physiological processes, such as blood digestion and suppression of the host immune response during blood feeding. Thus, cystatins are a topic of research for the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to identify potential targets for a multi-species vaccine. However, cystatin functions in ticks remain undetermined, especially in Ixodes ovatus. This work characterized the inhibitory profile of rJpIocys2a, an I. ovatus cystatin, to cathepsins B, C, and L. The enzymatic inhibition profile of JpIocys2a shows a distinct modulation of cathepsins related to tick blood digestion and evasion of host immune response. In addition, a peptide from JpIocys2a amino acid sequence (STQ-pep) was synthesized and used for rabbit immunization. Anti-STQ-pep serum was used to analyze the cross-antigenicity between Rhipicephalus microplus and I. ovatus cystatins. Crossantigenicity assays revealed that antibodies against the JpIocys2a peptide are able to recognize native and recombinant R. microplus ticks cystatins. The ability of rJpIocys2a to inhibit cathepsins related to different functions suggests multiple roles for JpIocys2a. The results obtained in this work provided basis for further experiments using this protein as a vaccine antigen.

Antimicrobial proteins (AMPs) are effector molecules and an important part of the innate immune system. AMPs have a broad antimicrobial spectrum and lyse microbial cells by interaction with biomembranes. Besides direct impact in host defence, AMPs are mediators of inflammation with impact on epithelial and inflammatory cells influencing diverse processes such as cytokine release, cell proliferation, angiogenesis, wound healing, chemotaxis, immune induction, and protease-antiprotease balance. AMPs could replace antibiotics which efficiency has decreased due to extensive clinical use. Therefore knowledge of mechanism of action of antimicrobial peptides, their properties and possible usage is essential for their further use as therapeutics. Ticks are blood-feeding ectoparisites that serve as extremely effective vectors of pathogens. Analysis of the ticks molecules that are involved in immune response to the pathogens invasion represent one of the strategies in searching for new compounds that might be used in future as theurapeutic agents. This study represents analysis of newly identified antimicrobial peptide form in the hard tick Ixodes ricinus (IRAMP). IRAMP revealed the high similarity to the recently described antimicrobial peptide isolated from hard tick Ixodes scapularis (protein ISAMP). Analysis and characterization of novel AMP, testing its antimicrobial potential and expression pattern are the main objectives of this study

Two cysteine and two serine protease inhibitors from a tick Ixodes ricinus saliva were overexpressed using a prokaryotic overexpression system and refolded to their native state. Both cysteine protease inhibitors were tested as potential antigens for an anti-tick vaccine showing no effect on tick feeding or reproduction. Various immunological methods were employed to test the potential immunomodulatory function of these proteins without success.

The function of chitin binding protein (CBP) and two isoforms of cathepsin B (cathB1, cathB2) were tested by using RNA interference in the tick I. ricinus. Two different methods have been used to deliver dsRNA for RNAi in ticks {--} injection and capillary feeding. The synthesized dsRNA was used to find out the impact of RNAi in the tick tissues, which were tested by RT-PCR and Western blot. The expression of CBP was successfully silenced by RNAi in the salivary glands. The silencing of cathB1 and cathB2 in the gut was less effective, but still limited tick`s ability to feed.

Cistatinas são um grupo de inibidores de cisteíno proteases que atuam na regulação da proteólise e estão presentes em uma ampla variedade de organismos. Estudos sobre essa classe de inibidores em parasitas têm contribuído para elucidar suas funções na regulação de processos fisiológicos como a digestão do sangue e a supressão da resposta imune do hospedeiro durante a alimentação. Por isso, cistatinas são um alvo interessante na pesquisa para o desenvolvimento de novos métodos de controle de parasitas. Além disso, a caracterização de proteínas compartilhadas por diferentes espécies de parasitas representa uma estratégia viável para encontrar potenciais alvos para uma vacina multi-espécie. Entretanto, as funções das cistatinas nos carrapatos permanecem obscuras, especialmente na espécie Ixodes ovatus. Neste trabalho, foi caracterizado o perfil inibitório de rJpIocys2a, uma cistatina de I. ovatus, para as catepsinas B, C e L. O perfil de inibição enzimática de rJpIocys2a se mostrou variável entre catepsinas envolvidas na digestão do sangue pelo carrapato e na evasão da resposta imune do hospedeiro. Além disso, um peptídeo baseado na sequência de aminoácidos do inibidor JpIocsy2a (STQ-pep) foi sintetizado e utilizado para imunização de coelhos. O soro anti-STQ-pep foi usado para analisar a antigenicidade cruzada entre cistatinas dos carrapatos Rhipicephalus microplus e I. ovatus. A análise da antigenicidade cruzada revelou que anticorpos gerados contra o peptídeo são capazes de reconhecer cistatinas nativas e recombinante de R. microplus. A habilidade de rJpIocys2a de inibir catepsinas relacionadas a diferentes funções sugere múltiplas funções para esse inibidor. Os resultados obtidos neste trabalho forneceram bases para outros experimentos que utilizem esta proteína como antígeno vacinal. / Cystatins are a group of cysteine protease inhibitors that act on the regulation of physiological proteolysis and are present in a wide range of organisms. Researches on this class of inhibitors in parasites have contributed to clarify their roles in the regulation of important physiological processes, such as blood digestion and suppression of the host immune response during blood feeding. Thus, cystatins are a topic of research for the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to identify potential targets for a multi-species vaccine. However, cystatin functions in ticks remain undetermined, especially in Ixodes ovatus. This work characterized the inhibitory profile of rJpIocys2a, an I. ovatus cystatin, to cathepsins B, C, and L. The enzymatic inhibition profile of JpIocys2a shows a distinct modulation of cathepsins related to tick blood digestion and evasion of host immune response. In addition, a peptide from JpIocys2a amino acid sequence (STQ-pep) was synthesized and used for rabbit immunization. Anti-STQ-pep serum was used to analyze the cross-antigenicity between Rhipicephalus microplus and I. ovatus cystatins. Crossantigenicity assays revealed that antibodies against the JpIocys2a peptide are able to recognize native and recombinant R. microplus ticks cystatins. The ability of rJpIocys2a to inhibit cathepsins related to different functions suggests multiple roles for JpIocys2a. The results obtained in this work provided basis for further experiments using this protein as a vaccine antigen.

Cistatinas são um grupo de inibidores de cisteíno proteases que atuam na regulação da proteólise e estão presentes em uma ampla variedade de organismos. Estudos sobre essa classe de inibidores em parasitas têm contribuído para elucidar suas funções na regulação de processos fisiológicos como a digestão do sangue e a supressão da resposta imune do hospedeiro durante a alimentação. Por isso, cistatinas são um alvo interessante na pesquisa para o desenvolvimento de novos métodos de controle de parasitas. Além disso, a caracterização de proteínas compartilhadas por diferentes espécies de parasitas representa uma estratégia viável para encontrar potenciais alvos para uma vacina multi-espécie. Entretanto, as funções das cistatinas nos carrapatos permanecem obscuras, especialmente na espécie Ixodes ovatus. Neste trabalho, foi caracterizado o perfil inibitório de rJpIocys2a, uma cistatina de I. ovatus, para as catepsinas B, C e L. O perfil de inibição enzimática de rJpIocys2a se mostrou variável entre catepsinas envolvidas na digestão do sangue pelo carrapato e na evasão da resposta imune do hospedeiro. Além disso, um peptídeo baseado na sequência de aminoácidos do inibidor JpIocsy2a (STQ-pep) foi sintetizado e utilizado para imunização de coelhos. O soro anti-STQ-pep foi usado para analisar a antigenicidade cruzada entre cistatinas dos carrapatos Rhipicephalus microplus e I. ovatus. A análise da antigenicidade cruzada revelou que anticorpos gerados contra o peptídeo são capazes de reconhecer cistatinas nativas e recombinante de R. microplus. A habilidade de rJpIocys2a de inibir catepsinas relacionadas a diferentes funções sugere múltiplas funções para esse inibidor. Os resultados obtidos neste trabalho forneceram bases para outros experimentos que utilizem esta proteína como antígeno vacinal. / Cystatins are a group of cysteine protease inhibitors that act on the regulation of physiological proteolysis and are present in a wide range of organisms. Researches on this class of inhibitors in parasites have contributed to clarify their roles in the regulation of important physiological processes, such as blood digestion and suppression of the host immune response during blood feeding. Thus, cystatins are a topic of research for the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to identify potential targets for a multi-species vaccine. However, cystatin functions in ticks remain undetermined, especially in Ixodes ovatus. This work characterized the inhibitory profile of rJpIocys2a, an I. ovatus cystatin, to cathepsins B, C, and L. The enzymatic inhibition profile of JpIocys2a shows a distinct modulation of cathepsins related to tick blood digestion and evasion of host immune response. In addition, a peptide from JpIocys2a amino acid sequence (STQ-pep) was synthesized and used for rabbit immunization. Anti-STQ-pep serum was used to analyze the cross-antigenicity between Rhipicephalus microplus and I. ovatus cystatins. Crossantigenicity assays revealed that antibodies against the JpIocys2a peptide are able to recognize native and recombinant R. microplus ticks cystatins. The ability of rJpIocys2a to inhibit cathepsins related to different functions suggests multiple roles for JpIocys2a. The results obtained in this work provided basis for further experiments using this protein as a vaccine antigen.

No description available.

Aujourd'hui, l'émergence ou la réémergence de maladies transmises par les tiques (TBDs) devient un problème majeur. En raison des problèmes générés par l'utilisation des acaricides (pollution, résistance), il est donc urgent d'identifier de nouvelles approches pour contrôler les populations de tiques. Parmi ces stratégies, la vaccination visant des molécules conservées chez les tiques et impliquées dans leur capacité vectorielle, sont devenues particulièrement attractives. En conséquence, l'identification de cibles antigéniques appropriées est un défi majeur pour la mise en œuvre de ces stratégies de contrôle des tiques et des TBDs. Dans le présent travail, l'objectif principal est d'élucider les interactions moléculaires entre I. ricinus et B. henselae, afin d'identifier des molécules qui pourraient représenter des cibles vaccinales contre les tiques et les agents pathogènes qu'elles transmettent. Dans ce but, nous avons identifié, par séquençage à haut débit, des transcrits d'Ixodes ricinus différentiellement exprimés au niveau des glandes salivaires de la tique en réponse à une infection par B. henselae. Dans un second temps, l'implication d'un de ces transcrits surexprimés lors de l'infection dans la transmission de B. henselae, a été évaluée. Enfin, et en premier lieu, nous avons validé l'utilisation de la technique de gorgement artificiel sur membrane pour infecter I. ricinus par B. henselae et évalué l'impact de différents paramètres sur le gorgement des tiques. Les résultats ont montré que la technique de gorgement sur membrane est bien adaptée à l'infection d'I. ricinus par B. henselae en laboratoire, et que la proportion et le poids des tiques gorgées sont diminués lors de l'infection du sang par la bactérie Le séquençage en 454 des glandes salivaires de tiques a généré une banque de référence contenant 24, 539 transcrits, et la comparaison des glandes salivaires d'I. ricinus infectés et non-infectés par B. henselae a montré que 839 et 517 transcrits étaient respectivement significativement surexprimés et sous-exprimés en réponse à l'infection par des bactéries. Parmi les gènes de fonction connue, 161 transcrits correspondent à 9 familles déjà identifiées, quand les autres correspondent à des gènes de fonction inconnue. L'extinction par RNA interférence du gène le plus surexprimé, IrSPI qui appartient à la famille des inhibiteurs de sérine protéase BPTI/Kunitz, a entraîné une réduction de la taille du repas sanguin prit par les tiques (et donc sa descendance) ainsi que du niveau d'infection au niveau des glandes salivaires. En conclusion, cette étude a démontré que la technique de gorgement artificiel des tiques sur membrane est un outil puissant pour étudier les interactions entre les tiques et les agents pathogènes qu'elles transmettent comme B. henselae. Ce travail apporte aussi une nette avancée en termes de données génétiques sur I. ricinus (dont le génome n'est pas séquencé) et sur les interactions moléculaires entre une bactérie et son vecteur. Enfin, ce travail a permis la mise en évidence d'une molécule représentant un candidat vaccinal très prometteur à la fois pour diminuer la population de tiques et lutter contre les agents pathogènes qu'elles transmettent. Dans le futur, et en fonction de la confirmation du rôle des gènes identifiés ici dans la transmission bactérienne, de nombreux candidats vaccins pourront ainsi être évalués, ouvrant alors de nouvelles perspectives dans la lutte contre les tiques et les maladies dues aux agents qu'elles transmettent / Ticks are obligate blood-feeding ectoparasites of many hosts including mammals, birds and reptiles. After mosquitoes, they are the most important vectors worldwide, and are able to transmit the highest variety of pathogens including virus, bacteria and parasites. Ixodes ricinus (Acari: Ixodidae), the most common tick species in Europe, is a three-life stage hard tick. It is frequently associated with bites in humans, and transmits several pathogens, including Tick-Borne Encephalitis, Babesia spp., Borrellia spp., Anaplasma spp., and to a lesser extent Bartonella spp. Bartonella spp. are facultative intracellular bacteria associated with a number of emerging diseases in humans and animals. It has been demonstrated that I. ricinus is a competent vector for B. henselae that causes cat scratch disease as well as being increasingly associated with a number of other syndromes, particularly ocular infections and endocarditis. Recently, emergence or re-emergence of tick-borne diseases (TBDs) is increasingly becoming a problem. Indeed, and because of the limited success and disadvantages of controlling TBDs via acaricides, new approaches are urgently needed. Therefore, vaccine strategies that target conserved components of ticks that play roles in vector infestation and vector capacity have become particularly attractive. Accordingly, the identification of suitable antigenic targets is a major challenge for the implementation of tick and TBDs control strategies. In the present work, the main objective is to elucidate molecular interactions between I. ricinus and B. henselae in order to identify some targets that may be used as vaccines against ticks and tick-borne pathogens. Two principal points are focused on: primarily, to identify I. ricinus salivary gland differentially expressed transcripts in response to B. henselae infection with next generation sequencing techniques (454 pyrosequencing and HiSeq 2000); secondly, to validate the implication of one of these transcripts in the transmission of B. henselae. For that purpose, and at first, we validated artificial membrane feeding technique for ticks infection by B. henselae and evaluated the impact of several parameters on tick feeding. Results showed that membrane feeding technique is a suitable method to infect I. ricinus with B. henselae and that the proportion and weight of engorged ticks are decreased by B. henselae infection of the blood meal. Transcriptional analysis of the tick salivary glands generated a reference databank containing 24,539 transcripts, and the comparison of B. henselae-infected and non-infected I. ricinus female salivary glands showed that 839 and 517 transcripts were significantly up- and down-regulated in response to bacteria infection, respectively. Among them, 161 transcripts corresponded to 9 groups of ticks salivary gland gene families already described, when the other ones corresponded to genes of unknown function. Silencing the most up-regulated gene IrSPI, which belongs to BPTI/Kunitz family of serine protease inhibitor, resulted in reduction of tick feeding and bacteria load in tick salivary gland. In conclusion, this work demonstrated that artificial-membrane feeding technique is a powerful tool for investigating the interactions between tick and tick-borne pathogens as B. henselae. It also increases the available genomic information for I. ricinus and the knowledge to improve our understanding of the molecular interaction between tick and tick-borne pathogens. At last, it provides a potential vaccine candidate to control tick-borne diseases. In the future, and depending of differentially expressed genes' role confirmation, more and more vaccine candidate will be provided by this work, and the strategy of controlling tick and tick-borne disease will come to a new stage

Due to climate change, marked developments are tracked in various links of pathogen-host distribution. Markedly increased number of ectoparasites is observed in Central and Eastern Europe. Ticks are blood feeding wide group of arthropods of utmost medical, epidemiological and veterinary significance throughout the world. The ecology of ticks, the outcome of their interactions with their natural environment, is fundamental to the spatial and temporal variation in the risk of infection by tick-borne pathogens. The aim of the study was to investigate: the distribution of Ixodes ricinus ticks and their transmitted parasites prevalence in different biotopes in Lithuania; and to find out factors influencing the activity dynamics of ticks and prevalence of endoparasites. The scientific researches on ectoparasite-endoparasite system (I. ricinus-B.burgdorferi s.l., B.divergens and Ehrlichia sp.) in different biotopes in Lithuania have been carried out for the first time. Also, it was the first investigation of prevalence of ticks (according to development phases and different sexes) in different biotopes and ticks infectious by microparasites. Influence of the main factors (air temperature, precipitations) on seasonal and daily activity changes of ticks have been described. Experiments on ectoparasites and endoparasites have been employed by the procedures on molecular level, including PCR method. As the result of the study the specificity of ticks’ species has been identified... [to full text] / Vykstant klimato atšilimui yra stebimi ryškūs pokyčiai įvairiose parazitas-šeimininkas sistemos grandyse. Pastebimas ektoparazitų skaitlingumo padidėjimas Vidurio ir Rytų Europoje. Erkės yra įvairių zoonozių ir antropozių, kurias sukelia įvairūs virusai, bakterijos ir protistai, pernešėjai. Erkių platinamų ligų sukėlėjų patogeninių bakterijų (Borrelia sp., Ehrlichia sp. ir kt.) ir protistų (Babesia sp. ir kt.) tyrimus turi apimti visus ekologinius lygmenis: patogeninio organizmo, vektoriaus ir rezervuarinio šeimininko. Šio darbo tikslas – nustatyti Ixodes ricinus L. erkių ir jų pernešamų endoparazitų paplitimą skirtinguose biotopuose Lietuvoje bei išaiškinti veiksnius, turinčius įtakos erkių aktyvumui ir endoparazitų paplitimui. Lietuvoje pirmą kartą buvo atlikti ektoparazito - endoparazito sistemos (I. ricinus–B.burgdorferi s.l., B.divergens ir Ehrlichia sp.) tyrimai skirtinguose Lietuvos biotopuose. Buvo įvertintas bendras erkių (pagal vystymosi stadiją ir lytį) paplitimas skirtinguose biotopuose, jų užsikrėtimas mikroparazitais ir išanalizuoti veiksniai, lemiantys erkių aktyvumo sezoninius ir paros kitimus, gausumą ir užsikrėtimą. Ektoparazitų ir endoparazitų tyrimams buvo įdiegtas polimerazės grandininės reakcijos metodas ir identifikuotas erkių rūšinis specifiškumas, nustatyti mikroparazitai bei jų genotipai. Buvo nustatytas mikroparazitų paplitimas skirtinguose Lietuvos biotopuose ir ištirti veiksniai, galintys sąlygoti šį paplitimą. Pirmą kartą Lietuvoje buvo... [toliau žr. visą tekstą]