Caracterização funcional do sistema complemento e análise da microbiota de Anopheles darlingi em resposta à infecção com plasmodium vivax
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2019-02-28
Autores
Voges, Kamila
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Universidade Estadual Paulista (Unesp)
Resumo
A malária é uma doença ocasionada por protozoários do gênero Plasmodium e transmitida ao homem por meio da picada de mosquitos do gênero Anopheles. No Brasil a maior parte dos casos da doença concentra-se na região Amazônica, onde grande parte das infecções é causada por Plasmodium vivax, e o A. darlingi é o principal vetor. Estudos recentes indicam que a microbiota e que o sistema imune dos mosquitos do gênero Anopheles, em particular, componentes do sistema complemento, possuem uma importante função na determinação da competência vetorial, podendo modular o desenvolvimento do parasita no mosquito. Apesar da importância epidemiológica de A. darlingi, pouco se sabe a cerca da composição de sua microbiota intestinal, e sobre as interações moleculares deste vetor com P. vivax. Neste cenário, temos como principais objetivos: 1) Avaliar o papel de LRIM1 (Leucine-rich repeat protein 1), um dos genes do complemento, na interface A. darlingi-P.vivax, e 2) Averiguar determinados aspectos da interação entre microbiota- A.darlingi- P.vivax. Quanto às análises de LRIM1, as topologias das árvores filogenéticas mostram maior relação filogenética entre A. darlingi e A. albimanus, apresentando-os como táxons irmãos. Para a realização dos ensaios funcionais, sintetizamos RNA dupla-fita com base na sequência codificadora de LRIM1 de A. darlingi e microinjetamos no tórax de fêmeas desta espécie, as quais foram posteriormente submetidas a uma infecção por P. vivax. Quanto aos ensaios funcionais, nossos resultados apresentaram níveis de oocistos e prevalência similares entre o grupo teste e controle, não havendo diferenças estatisticamente significativas em todas as réplicas analisadas. Deste modo, com os dados obtidos até o momento, é necessário considerar a hipótese de que LRIM1 não desempenhe um papel na defesa anti P.vivax em A. darlingi. Neste trabalho, nós também analisamos a composição da microbiota intestinal de mosquitos A. darlingi infectados com P.vivax, por meio de sequenciamento em larga escala da região hipervariavel V4 do gene 16S rRNA. As classes bacterianas mais abundantes observadas em nossas amostras foram: Gammaproteobacteria, Alphaproteobacteria, e Flavobacteria. Quanto ao nível taxonômico de família, Enterobacteriaceae foi observada em maior abundância nos mosquitos apresentando baixa infecção com P. vivax, sugerindo que esta família possa antagonizar o parasita. Ao passo que, Flavobacteriaceae foi obtida em maior proporção nos mosquitos que apresentaram alta infeção com P. vivax. Isso sugere que esta família, de alguma maneira, possa favorecer o parasita, e aumentar sua carga nos mosquitos. Diante disso, uma vez que na literatura trabalhos sobre a resposta imune de anophelinos brasileiros (especialmente A. darlingi) são escassos, dados sobre LRIM1, tais quais análises funcionais e filogenia, proveem informações que podem impulsionar outros estudos a respeito desta proteína, bem como de outros genes do sistema imune. Ademais, nossos resultados corroboram estudos anteriores em anophelinos, indicando que as bactérias associadas ao intestino de A.darligi podem desempenhar papel importante na infecção e resposta imune ao parasito.
Malaria is a disease caused by protozoa of the genus Plasmodium and transmitted to humans through the bite of mosquitoes of the genus Anopheles. In Brazil, most cases of the disease are concentrated in the Amazon region, where most of the infections are caused by Plasmodium vivax, and A. darlingi is the main vector. Recent studies indicate that the microbiota and the immune system of Anopheles mosquitoes, in particular components of the complement system, play an important role in the determination of vector competence and can modulate the development of the parasite in the mosquito. Despite the epidemiological importance of A. darlingi, little is known about the composition of its intestinal microbiota, and about the molecular interactions of this vector with P. vivax. In this scenario, we have as main objectives: 1) To evaluate the role of LRIM1 (Leucine-rich repeat protein 1), one of the complement genes, in the interface A. darlingi-P.vivax, And 2) To ascertain some aspects of the interaction between microbiota- A.darlingi- P. vivax. As for the LRIM1 analyzes, the topologies of the phylogenetic trees show a higher phylogenetic relationship between A. darlingi and A. albimanus, presenting them as sister taxa. To perform the functional assays, we synthesized double-stranded RNA based on the coding sequence of A. darlingi LRIM1 and microinjections in the thorax of females of this species, which were subsequently subjected to a P. vivax infection. Regarding the functional tests, our results showed similar levels of oocysts and prevalence between the test and control groups, and there were no statistically significant differences in all the replicates analyzed. Thus, with the data obtained so far, it is necessary to consider the hypothesis that LRIM1 does not play a role in the defense against P.vivax in A. darlingi. In this work, we also analyzed the intestinal microbiota composition of A. darlingi mosquitoes infected with P.vivax by means of large-scale sequencing of the V4 hypervariable region of the 16S rRNA gene. The most abundant bacterial classes observed in our samples were: Gammaproteobacteria, Alphaproteobacteria, and Flavobacteria. As for the taxonomic family level, Enterobacteriaceae was observed in greater abundance in the mosquitoes presenting low infection with P. vivax, suggesting that this family can antagonize the parasite. Whereas, Flavobactériaceae was obtained in greater proportion in mosquitoes that showed high infection with P. vivax. This suggests that this family may somehow favor the parasite, and increase its burden on mosquitoes. Therefore, since the literature on the immune response of Brazilian anophelins (especially A. darlingi) is scarce, data on LRIM1, such as functional analyzes and phylogeny, provide information that may boost other studies on this protein, as well as of other genes of the immune system. In addition, our results corroborate previous studies on anophelinos, indicating that bacteria associated with the A.darligi gut may play an important role in infection and immune response to the parasite.
Malaria is a disease caused by protozoa of the genus Plasmodium and transmitted to humans through the bite of mosquitoes of the genus Anopheles. In Brazil, most cases of the disease are concentrated in the Amazon region, where most of the infections are caused by Plasmodium vivax, and A. darlingi is the main vector. Recent studies indicate that the microbiota and the immune system of Anopheles mosquitoes, in particular components of the complement system, play an important role in the determination of vector competence and can modulate the development of the parasite in the mosquito. Despite the epidemiological importance of A. darlingi, little is known about the composition of its intestinal microbiota, and about the molecular interactions of this vector with P. vivax. In this scenario, we have as main objectives: 1) To evaluate the role of LRIM1 (Leucine-rich repeat protein 1), one of the complement genes, in the interface A. darlingi-P.vivax, And 2) To ascertain some aspects of the interaction between microbiota- A.darlingi- P. vivax. As for the LRIM1 analyzes, the topologies of the phylogenetic trees show a higher phylogenetic relationship between A. darlingi and A. albimanus, presenting them as sister taxa. To perform the functional assays, we synthesized double-stranded RNA based on the coding sequence of A. darlingi LRIM1 and microinjections in the thorax of females of this species, which were subsequently subjected to a P. vivax infection. Regarding the functional tests, our results showed similar levels of oocysts and prevalence between the test and control groups, and there were no statistically significant differences in all the replicates analyzed. Thus, with the data obtained so far, it is necessary to consider the hypothesis that LRIM1 does not play a role in the defense against P.vivax in A. darlingi. In this work, we also analyzed the intestinal microbiota composition of A. darlingi mosquitoes infected with P.vivax by means of large-scale sequencing of the V4 hypervariable region of the 16S rRNA gene. The most abundant bacterial classes observed in our samples were: Gammaproteobacteria, Alphaproteobacteria, and Flavobacteria. As for the taxonomic family level, Enterobacteriaceae was observed in greater abundance in the mosquitoes presenting low infection with P. vivax, suggesting that this family can antagonize the parasite. Whereas, Flavobactériaceae was obtained in greater proportion in mosquitoes that showed high infection with P. vivax. This suggests that this family may somehow favor the parasite, and increase its burden on mosquitoes. Therefore, since the literature on the immune response of Brazilian anophelins (especially A. darlingi) is scarce, data on LRIM1, such as functional analyzes and phylogeny, provide information that may boost other studies on this protein, as well as of other genes of the immune system. In addition, our results corroborate previous studies on anophelinos, indicating that bacteria associated with the A.darligi gut may play an important role in infection and immune response to the parasite.
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Palavras-chave
Anopheles darlingi, Plasmodium vivax, Sistema complemento