A carregar...
Publicação
TickProVac: An insight into the Rhipicephalus sanguineus tick salivary gland proteome during Ehrlichia canis infection towards vaccine development
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 5.75 MB | Adobe PDF |
Orientador(es)
Resumo(s)
As carraças são reconhecidas como vectores de diversos agentes patogénicos responsáveis por doenças relevantes na medicina humana e animal. Erliquiose monocítica canina (EMC), causada pela bactéria Ehrlichia canis, é transmitida pela carraça Rhipicephalus sanguineus. O potencial zoonótico da EMC está bem documentado com relatos de infecções humanas em aumento permanente. A infestação por carraças e as doenças por elas transmitidas (DTCs) continuam a representar um sério problema devido à falta de profilaxias eficazes. É, portanto, imperativo adotar novas abordagens, nomeadamente a utilização de vacinas, para a redução de infestações por carraças e DTCs. Actualmente não existe vacina anti-R. sanguineus comercialmente disponível. De forma a solucionar esta lacuna, o objetivo principal do presente trabalho foi a caracterização de genes e proteínas que atuam na interface vector-agente patogénico na presença de infeção para compreender os mecanismos associados à transmissão de DTCs. Neste trabalho estão reportados genes e proteínas diferencialmente expressos obtidos a partir da análise transcriptómica e proteómica das glândulas salivares (GSs) de R. sanguineus durante a infeção com E. canis. Os níveis de expressão dos genes que codificam as proteínas serina carboxipeptidase (psc), proteína de choque térmico (phsrp20) e proibitina (prohib) foram investigados in vitro e in vivo, em amostras infectadas e não infectadas. Posteriormente, o silenciamento de genes foi realizado através de RNA de interferência (RNAi) para avaliar o efeito da sub-expressão de ferritina 1 na alimentação, desenvolvimento ovárico, oogénese e aquisição E. canis pela carraça. O mesmo foi efetuado para determinar o efeito da sub-expressão de psc, prohib e phsrp20 na invasão e multiplicação desta bactéria na linha celular IDE8 e em R. sanguineus. O péptido pPHSRP20, relacionado com o choque térmico, foi sintetizado para avaliar a sua imunogenicidade em ratos CD1. Os resultados mostraram que o silenciamento de ferritina 1 compromete a competência da carraça em se alimentar e provoca alterações morfológicas e histoquímicas nos ovários e oócitos. Os dados transcriptómicos e proteómicos demonstraram que as alterações da expressão génica e proteica estão principalmente relacionadas com processos proteicos celulares e metabólicos e com atividades catalíticas, muito provavelmente um reflexo de alterações de transcrição e tradução em resposta à presença de infecção. Em amostras infectadas verificou-se uma sub-expressão de phsrp20 e prohib em células IDE8 e de phsrp20 e psc nas GSs. Inversamente, os genes psc nas células IDE8 e prohib nas GSs estavam sobre-expressos. Apesar de se terem atingido níveis elevados de silenciamento, o seu efeito na biologia de E. canis não foi determinado devido a limitações moleculares. pPHSRP20 desencadeou uma resposta imunitária detectável e específica nos ratos CD1. Em geral, os resultados demonstraram que a presença de E. canis nas GSs desencadeia respostas celulares para regulação do stress, inflamação e rearranjo do citoesqueleto. A modulação desta maquinaria molecular pela bactéria poderá ser uma estratégia para lidar e escapar à resposta imunitária e para utilizar proteínas do vector necessárias à virulência. Através destes resultados esperou identificar-se potenciais antigénios de carraça que contribuam para o desenvolvimento de uma vacina anti-R. sanguineus e/ou bloqueadora da transmissão de agentes patogénicos.
Ticks are recognised as potent vectors of disease-causing pathogens of medical and veterinary importance. Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis (CME), is transmitted by the brown dog tick Rhipicephalus sanguineus and is acknowledged as a highly infectious disease. The zoonotic potential of CME is widely recognised with reports of human infections steadily increasing. Tick infestation and tick-borne diseases (TBDs) remain a serious and persistent veterinary health problem, due to the lack of efficient control measures. It is therefore vital that novel approaches to tackle TBDs are pursued. Although vaccination to reduce tick infestation is recognised, no anti- R. sanguineus vaccine exists. To address this, we aimed to characterise the crucial gene and protein interactions at the vector-pathogen interface to gain a fundamental understanding of the interactions underpinning disease transmission. Here we report differentially expressed genes and proteins found either in the literature or found in our transcriptomic and proteomic data from R. sanguineus salivary glands (SGs) during E. canis infection. The mRNA expression levels of the putative serine carboxypeptidase (psc), heat-shock related protein (phsrp20) and prohibitin-like protein genes were investigated in vitro and in vivo, in infected and uninfected samples. RNA interference (RNAi) was carried out to determine the effect of ferritin 1 in tick feeding, ovary (OV) development, oogenesis, and pathogen acquisition. We also determined the effect of a downregulation of three selected genes or proteins from our omics data on E. canis invasion and multiplication in the IDE8 tick cell line and R. sanguineus ticks. We synthesised a heat-shock related protein (pPHSRP20) peptide to evaluate its immunogenicity in CD1 mice. Our results have shown that silencing ferritin 1 alters tick competence to normally engorge and causes morphological and histochemical changes in the OV and oocytes. Our transcriptomic and proteomic data has revealed alterations in gene and protein expression, mostly concerning protein cellular and metabolic processes and catalytic activities, perhaps related with transcriptional and translational responses to E. canis infection. When analysing the transcription levels in E. canis-infected samples, a downregulation was verified for phsrp20 and prohib in IDE8 cells and phsrp20 and psc in the SGs, whereas an upregulation was observed for psc in IDE8 cells and prohib for the SGs. Even with high levels of gene silencing, the effect of gene silencing in E. canis biology was not determined due to molecular limitations. pPHSRP20 triggered a detectable and specific immune response in mice. Overall, results show that the presence of E. canis in the SGs leads to regulation of stress response, inflammation and cytoskeletal rearrangement molecules. Modulation of tick molecular machinery is a coping strategy to evade the host immune response and to utilise its proteins for infectivity. From this, we expected to identify tick antigens that will direct the development of an anti-R. sanguineus and/or transmission-blocking vaccine.
Ticks are recognised as potent vectors of disease-causing pathogens of medical and veterinary importance. Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis (CME), is transmitted by the brown dog tick Rhipicephalus sanguineus and is acknowledged as a highly infectious disease. The zoonotic potential of CME is widely recognised with reports of human infections steadily increasing. Tick infestation and tick-borne diseases (TBDs) remain a serious and persistent veterinary health problem, due to the lack of efficient control measures. It is therefore vital that novel approaches to tackle TBDs are pursued. Although vaccination to reduce tick infestation is recognised, no anti- R. sanguineus vaccine exists. To address this, we aimed to characterise the crucial gene and protein interactions at the vector-pathogen interface to gain a fundamental understanding of the interactions underpinning disease transmission. Here we report differentially expressed genes and proteins found either in the literature or found in our transcriptomic and proteomic data from R. sanguineus salivary glands (SGs) during E. canis infection. The mRNA expression levels of the putative serine carboxypeptidase (psc), heat-shock related protein (phsrp20) and prohibitin-like protein genes were investigated in vitro and in vivo, in infected and uninfected samples. RNA interference (RNAi) was carried out to determine the effect of ferritin 1 in tick feeding, ovary (OV) development, oogenesis, and pathogen acquisition. We also determined the effect of a downregulation of three selected genes or proteins from our omics data on E. canis invasion and multiplication in the IDE8 tick cell line and R. sanguineus ticks. We synthesised a heat-shock related protein (pPHSRP20) peptide to evaluate its immunogenicity in CD1 mice. Our results have shown that silencing ferritin 1 alters tick competence to normally engorge and causes morphological and histochemical changes in the OV and oocytes. Our transcriptomic and proteomic data has revealed alterations in gene and protein expression, mostly concerning protein cellular and metabolic processes and catalytic activities, perhaps related with transcriptional and translational responses to E. canis infection. When analysing the transcription levels in E. canis-infected samples, a downregulation was verified for phsrp20 and prohib in IDE8 cells and phsrp20 and psc in the SGs, whereas an upregulation was observed for psc in IDE8 cells and prohib for the SGs. Even with high levels of gene silencing, the effect of gene silencing in E. canis biology was not determined due to molecular limitations. pPHSRP20 triggered a detectable and specific immune response in mice. Overall, results show that the presence of E. canis in the SGs leads to regulation of stress response, inflammation and cytoskeletal rearrangement molecules. Modulation of tick molecular machinery is a coping strategy to evade the host immune response and to utilise its proteins for infectivity. From this, we expected to identify tick antigens that will direct the development of an anti-R. sanguineus and/or transmission-blocking vaccine.
Descrição
Palavras-chave
Parasitologia