A carregar...
Publicação
Studies on the mosquito immune response effect of antimalarial drugs and Plasmodium sporozoites
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 20.75 MB | Adobe PDF |
Autores
Orientador(es)
Resumo(s)
Este trabalho pretende contribuir para o conhecimento geral da resposta imunológica do mosquito ao parasita da malária, uma vez que a elucidação das interacções entre vector e parasita poderão facilitar o desenvolvimento de medidas eficientes para bloquear a transmissão. As experiências realizadas neste trabalho incluíram o uso de Drosophila melanogaster como modelo de estudo das respostas imunológicas do
mosquito e a avaliação do impacto da presença de esporozoítos de Plasmodium na hemolinfa do mosquito através da determinação de alterações no número de hemócitos, activação da reacção de melanização e do padrão de expressão de proteínas na hemolinfa do mosquito.
O fármaco antimalárico cloroquina promove a transmissão no mosquito e tem sido relacionado com a expressão diferencial de péptidos antimicrobianos no mosquito.
Para avaliar o efeito da cloroquina na sua produção usámos o modelo Drosophila, uma vez que a expressão e síntese de péptidos antimicrobianos na mosca está bem caracterizada, assim como as vias de sinalização da resposta imunológica. Os resultados deste trabalho não conseguiram provar algum efeito do fármaco na expressão e/ou síntese de péptidos antimicrobianos de Drosophila. O tratamento com cloroquina in vivo não afectou as vias de sinalização Toll e Imd, avaliado pela expressão de drosomicina e diptericina em moscas infectadas. Experiências in vitro em que se utilizaram linhas celulares derivadas de hemócitos de moscas produziram os mesmos resultados para a síntese de Drosomicina e Atacina. Experiências de sobrevivência de moscas infectadas e tratadas com cloroquina também não evidenciaram qualquer
efeito do fármaco na resposta imunitária de Drosophila. Como este fármaco antimalárico tem um efeito conhecido na resposta imune do mosquito, propomos que a cloroquina tenha uma acção sobre moléculas específicas dos mosquito ou sobre diferentes vias de activação da sinalização que possam estar presentes apenas no mosquito. Por outro lado, a informação conhecida acerca do efeito da cloroquina na
imunidade foi obtida após tratamento de humanos, ratinhos ou linhas celulares de mamíferos, implicando a metabolização do fármaco. Como tal, não é claro se o efeito observado resulta da acção do própria fármaco ou de um metabolito específico.
Neste trabalho pretendeu-se também determinar as respostas do mosquito aos esporozoítos de Plasmodium na hemolinfa, uma vez que nesta fase da infecção no mosquito o parasita sofre uma grande redução no seu número. No hemocélio domosquito podem ser activadas respostas celulares e humorais. Durante o seu desenvolvimento no oocysto, os esporozoítos são cobertos por uma camada de proteína circumsporozoítica, que constitui o seu maior antigénio de superfície. Quando o oocisto rompe e os esporozoítos são libertados, esta proteína pode ser reconhecida pelas moléculas de reconhecimento presentes na hemolinfa levando à activação de respostas imunes. A activação de respostas imunitárias celulares contra os esporozoítos foi testada com base na determinação de variação do número de hemócitos quando estimulados com a proteína circumsporozoítica de P. falciparum.
Apenas uma das doses (5ng) de proteína utilizadas para estimular linhas celulares de hemóctios causou uma redução significativa no números de hemócitos. Isto pode ser um reflexo de uma cinética de divisão celular mais lenta ou de destruição celular, apoptose, que poderia ser despoletada pela fagocitose de parasitas, por exemplo. Não foi possível obter uma resposta correlacionada com a dose usada para estimulação. No entanto, os hemócitos do mosquito parecem reconhcer a proteína do parasita e responder à sua presença.
A activação da reacção de melanização durante a invasão da hemolinfa por esporozoítos foi testada, através da determinação da activação da enzima profenoloxidase e da actividade da fenoloxidase. Verificou-se que a actividade enzimática da fenoloxidase varia com o tempo em mosquitos submetidos a uma refeição sanguínea não infectante. A infecção por P. berhgei não pareceu impor variações na actividade da fenoloxidase. Diferenças subtis foram observadas aos dias 9, 12 e 15 pós-infecção, sendo a actividade enzimática mais elevada em mosquitos infectados. Os esporozoítos foram detectados na hemolinfa de mosquitos a partir do
dia 9 pós-infecção, indicando que o parasita pode induzir um aumento subtil na activação da melanização. A actividade da fenoloxidase parece ser mantida constitutivamente num nível baixo, mesmo em mosquitos não infectados, o que pode explicar que apenas pequenas diferenças sejam observadas em mosquitos infectados.
Injecções da proteína circumsporozoítica de P. falciparum em mosquitos não revelaram indução da actividade da enzima fenoloxidase. Apesar de não ter sido possível demonstrar conclusivamente a melanização de esporozoítos na hemolinfa, experiências de inibição da fenoloxidase mostraram que a actividade desta enzima é necessária para controlar o número de esporozoítos na hemolinfa e nas glândulas salivares.
A hemolinfa é extremamente rica em proteínas, e conhecida por albergar a maior parte das moléculas do sistema imunológico necessárias ao reconhecimento, sinalização e à resposta efectora. Como tal, de modo a caracterizar o proteoma da hemolinfa durante a infecção por P. berhgei ao dia 13 pós-infecção, usámos umaabordagem que incluiu electroforese bidimensional e espectrometria de massa MALDITOF, visando identificar proteínas diferencialmente reguladas em mosquitos infectados. As proteínas com níveis alterados na hemolinfa de mosquitos infectados
poderão estar envolvidas em processos fisiológicos como metabolismo de ácidos gordos, glicólise e transporte de iões. Estes resultados indicam que o parasita impõe alterações no metabolismo do mosquito, quer directamente quer levando o mosquito a alterar o seu próprio metabolismo como forma de conter a infecção. De facto, não há evidência se as alterações observadas são danosas ou necessárias para o desenvolvimento do parasita. No entanto, os nossos resultados sugerem que mecanismos fisiológicos do mosquito podem ter um papel na resposta imune. Um dado interessante obtido neste trabalho foi a inexistência de correlação entre a regulação a nível proteico e a nível do RNA na emolinfa. Isto pode derivar de uma janela de tempo diferente entre expressão génica e síntese proteica, uma vez que as amostras foram recolhidas ao mesmo tempo, ou pode reflectir um fonte diferente de
RNA e proteína. O RNA amplificado para avaliar a expressão génica era originário dos hemócitos presentes na hemolinfa, enquanto que as proteínas podem ter sido produzidas quer pelos hemócitos quer pelo corpo gordo, que sintetiza a maior parte das moléculas imunes que são secretadas para a hemolinfa. No entanto, é importante tem em atenção que a informação resultante da análise de expressão génica ter de ser
avaliada cuidadosamente, pois pode não ter uma regulação correspondente ao nível da proteína. A biosíntese de eicosanóides teve dois impactos distintos e opostos no desenvolvimento do parasita, promovendo e bloqueando a transmissão. Os eicosanóides parecem ser importantes para o desenvolvimento do parasita numa fase da infecção em que os esporozoítos se desenvolvem nos oocistos, enquanto que numa fasemais tardia, estas moléculas parecem ser importantes para controlar o número de parasitas na hemolinfa. Os nossos resultados sugerem que o parasita possa imunosuprimir o mosquito.
A resposta do mosquito ao Plasmodium parece ser muito complexa, envolvendo acções de ambos os organismos. Para responderà invasão da hemolinfa pelos esporozoítos, o mosquito parece depender de diferentes mecanismos, como a fagocitose e a melanização. Para além destes, moléculas envolvidas em processos fisiológicos do mosquito são também afectadas pela infecção. Os nossos resultados sugerem que a resposta imunológica do mosquito possa envolver mecanismos para além daqueles que são tradicionalmente relacionados com a imunidade, como a
biosíntese de eicosanóides. Verificámos também que pode não existir correlação entre a expressão génica e a síntese de proteínas, e como tal, a resposta imune deveria ser analisada em primeiro lugar por uma abordagem proteómica.
This work aimed at contributing to the general knowledge of the mosquito immune responses to the malaria parasite, in hope that the elucidation of vector/parasite interactions will facilitate the development of effective transmission blocking measures. Experiments performed here include the use of Drosophila melanogaster as a model for immunity studies in the mosquito and the evaluation of Plasmodium sporozoites presence in mosquito hemolymph impact on hemocyte numbers, melanization reaction responses and protein expression pattern. Chloroquine promotes malaria transmission in mosquito and it has been linked to differential AMP gene expression in mosquitoes. As Drosophila AMP expression and synthesis is well understood and as we have a good knowledge about immune signaling pathways, we chose this model to evaluate chloroquine effect on AMP production upon infection. Our results failed to show any drug effect on Drosophila AMP expression and/or synthesis. Drug treatment in vivo did not affect either the Toll or Imd immune signaling pathways, as shown when accessing drosomycin and diptericin expression in infected flies, and in vitro experiments using hemocyte-like cell lines produced the same results for Drosomycin and Attacin synthesis. Survival experiments were also performed in drug treated flies and failed to indicate any effect. For all the mechanisms tested, chloroquine did not seem to have any effect on Drosophila immunity. As this antimalarial drug has a known effect on mosquito immunity we propose that chloroquine may act on particular mosquito immune molecules or on different routes for pathway activation operating in the mosquito. Also, data obtained for chloroquine action on immunity were collected from treatment of humans, mice or mammalian cell-lines, implying that the drug is metabolized. Thus it is not clear if the observed effect results from an action of the drug itself, or from a specific metabolite. This would explain why direct drug feeding to flies would fail to produce an effect on Drosophila immunity. Another purpose of this work was to determine the mosquito responses to Plasmodium sporozoites in the hemolymph, as parasite development in the mosquito suffers a major bottleneck at this stage of infection. Both cellular and humoral responses may be triggered in the mosquito hemocel. Upon development inside the oocysts, sporozoites are covered with a layer of circumsporozoite protein, its majorsurface antigen. Recognition molecules present in the hemolymph may recognize sporozoites upon oocyst burst, possibly through its surface protein and activate immune responses towards it. Cellular responses towards sporozoites were tested, based on the evaluation of hemocyte number variation upon stimulation with the circumsporozoite protein of P. falciparum. Only one dose (5ng) stimulated hemocytelike cell lines and led to a significant reduction in cell numbers. This may reflect a slower cell-division kinetics or cell destruction, by apoptosis, following phagocytosis. We failed to show any dose-dependent response. Nevertheless, it seems that the CS protein is recognized by mosquito hemocytes that respond to its presence only in specific conditions. We also tested for activation of melanization reaction upon sporozoite invasion of the hemolymph by accessing PPO activation and PO activity. PO activity was found to vary over time in blood fed mosquitoes. P. berghei infection did not seem to impose variations in PO activity. Subtle differences were observed at D9, 12 and 15pi, when PO activity was higher in infected mosquitoes. Sporozoites were first detected in the hemolymph at D9pi indicating that parasite recognition may induce subtle increases in melanization activation. PO activity seems to be maintained at a low level even in noninfected mosquitoes. This may explain the fact that no great variations were observed upon infection. Pf-CS protein injections in mosquitoes failed to show PO activity induction. Although we could not conclusively determine sporozoite melanization, PO inhibition experiments showed that its activity is necessary for control of sporozoite load in the hemolymph and salivary glands. Hemolymph is an extremely protein rich environment, and known to harbor most of the immune molecules necessary for recognition, signaling and effector mechanisms. As such, we used a two dimensional electrophoresis approach coupled with MALDITOF mass spectrometry to compare the hemolymph proteome of P. berghei infected and non-infected An. gambiae mosquitoes at D13pi, aiming at the identification of differentially regulated protein in infected mosquitoes. Proteins found to have altered levels in the hemolymph of infected mosquitoes are predicted to be involved in physiological processes such as fatty acid metabolism, aminoacid synthesis, glycolysis and ion transport. This indicated that the parasite imposes alterations in the overall mosquito metabolism, either directly, or secondarly to combat infection. Actually we have no evidence if the alterations observed are harmful or necessary for parasite development. Yet, the results suggest that mechanisms operating in mosquito physiology may have a role on immune responses. An interesting fact is that protein regulation in the hemolymph did not correlate at any level with gene transcription. This may reflect a different time frame between transcription and protein synthesis, assamples were collected at the same time, or a different source for the RNAs and the tested proteins. RNA amplified to evaluate transcription was hemolymph, ie, hemocyte-derived, while hemolymph proteins may have been produced not only by hemocytes, but also by fat body cells, that synthesize the majority of immune-related molecules secreted into the hemolymph. Nevertheless, it is important to bear in mind that data resulting from gene expression analysis have to be carefully analyzed as it may not indicate direct protein synthesis. Eicosanoid biosynthesis was found to have two distinctive and opposite impacts in parasite development: both promoting and blocking transmission. Eicosanoids seem to be important for parasite biology and development, at a time when sporozoites are developing inside oocysts. At a later stage, these molecules seem to restrain sporozoite infection in the hemolymph. Evidence also point to mosquito immunosuppression by the parasite. Mosquito responses to Plasmodium seem to be highly complex, involving actions from both organisms. To respond to hemolymph invasion by sporozoites, the mosquito seems to rely on different mechanisms, such as phagocytosis and melanization. Additionally, molecules involved in physiological processes are affected by hemolymph infection. Data obtained by this work suggests that immune responses may include mechanisms other than those traditionally related to immunity, as in the case of eicosanoid biosynthesis. Also, our results indicate that correlation between transcription and protein synthesis is not sure to exist and thus, immune responses should be analyzed by proteomics in a first approach.
This work aimed at contributing to the general knowledge of the mosquito immune responses to the malaria parasite, in hope that the elucidation of vector/parasite interactions will facilitate the development of effective transmission blocking measures. Experiments performed here include the use of Drosophila melanogaster as a model for immunity studies in the mosquito and the evaluation of Plasmodium sporozoites presence in mosquito hemolymph impact on hemocyte numbers, melanization reaction responses and protein expression pattern. Chloroquine promotes malaria transmission in mosquito and it has been linked to differential AMP gene expression in mosquitoes. As Drosophila AMP expression and synthesis is well understood and as we have a good knowledge about immune signaling pathways, we chose this model to evaluate chloroquine effect on AMP production upon infection. Our results failed to show any drug effect on Drosophila AMP expression and/or synthesis. Drug treatment in vivo did not affect either the Toll or Imd immune signaling pathways, as shown when accessing drosomycin and diptericin expression in infected flies, and in vitro experiments using hemocyte-like cell lines produced the same results for Drosomycin and Attacin synthesis. Survival experiments were also performed in drug treated flies and failed to indicate any effect. For all the mechanisms tested, chloroquine did not seem to have any effect on Drosophila immunity. As this antimalarial drug has a known effect on mosquito immunity we propose that chloroquine may act on particular mosquito immune molecules or on different routes for pathway activation operating in the mosquito. Also, data obtained for chloroquine action on immunity were collected from treatment of humans, mice or mammalian cell-lines, implying that the drug is metabolized. Thus it is not clear if the observed effect results from an action of the drug itself, or from a specific metabolite. This would explain why direct drug feeding to flies would fail to produce an effect on Drosophila immunity. Another purpose of this work was to determine the mosquito responses to Plasmodium sporozoites in the hemolymph, as parasite development in the mosquito suffers a major bottleneck at this stage of infection. Both cellular and humoral responses may be triggered in the mosquito hemocel. Upon development inside the oocysts, sporozoites are covered with a layer of circumsporozoite protein, its majorsurface antigen. Recognition molecules present in the hemolymph may recognize sporozoites upon oocyst burst, possibly through its surface protein and activate immune responses towards it. Cellular responses towards sporozoites were tested, based on the evaluation of hemocyte number variation upon stimulation with the circumsporozoite protein of P. falciparum. Only one dose (5ng) stimulated hemocytelike cell lines and led to a significant reduction in cell numbers. This may reflect a slower cell-division kinetics or cell destruction, by apoptosis, following phagocytosis. We failed to show any dose-dependent response. Nevertheless, it seems that the CS protein is recognized by mosquito hemocytes that respond to its presence only in specific conditions. We also tested for activation of melanization reaction upon sporozoite invasion of the hemolymph by accessing PPO activation and PO activity. PO activity was found to vary over time in blood fed mosquitoes. P. berghei infection did not seem to impose variations in PO activity. Subtle differences were observed at D9, 12 and 15pi, when PO activity was higher in infected mosquitoes. Sporozoites were first detected in the hemolymph at D9pi indicating that parasite recognition may induce subtle increases in melanization activation. PO activity seems to be maintained at a low level even in noninfected mosquitoes. This may explain the fact that no great variations were observed upon infection. Pf-CS protein injections in mosquitoes failed to show PO activity induction. Although we could not conclusively determine sporozoite melanization, PO inhibition experiments showed that its activity is necessary for control of sporozoite load in the hemolymph and salivary glands. Hemolymph is an extremely protein rich environment, and known to harbor most of the immune molecules necessary for recognition, signaling and effector mechanisms. As such, we used a two dimensional electrophoresis approach coupled with MALDITOF mass spectrometry to compare the hemolymph proteome of P. berghei infected and non-infected An. gambiae mosquitoes at D13pi, aiming at the identification of differentially regulated protein in infected mosquitoes. Proteins found to have altered levels in the hemolymph of infected mosquitoes are predicted to be involved in physiological processes such as fatty acid metabolism, aminoacid synthesis, glycolysis and ion transport. This indicated that the parasite imposes alterations in the overall mosquito metabolism, either directly, or secondarly to combat infection. Actually we have no evidence if the alterations observed are harmful or necessary for parasite development. Yet, the results suggest that mechanisms operating in mosquito physiology may have a role on immune responses. An interesting fact is that protein regulation in the hemolymph did not correlate at any level with gene transcription. This may reflect a different time frame between transcription and protein synthesis, assamples were collected at the same time, or a different source for the RNAs and the tested proteins. RNA amplified to evaluate transcription was hemolymph, ie, hemocyte-derived, while hemolymph proteins may have been produced not only by hemocytes, but also by fat body cells, that synthesize the majority of immune-related molecules secreted into the hemolymph. Nevertheless, it is important to bear in mind that data resulting from gene expression analysis have to be carefully analyzed as it may not indicate direct protein synthesis. Eicosanoid biosynthesis was found to have two distinctive and opposite impacts in parasite development: both promoting and blocking transmission. Eicosanoids seem to be important for parasite biology and development, at a time when sporozoites are developing inside oocysts. At a later stage, these molecules seem to restrain sporozoite infection in the hemolymph. Evidence also point to mosquito immunosuppression by the parasite. Mosquito responses to Plasmodium seem to be highly complex, involving actions from both organisms. To respond to hemolymph invasion by sporozoites, the mosquito seems to rely on different mechanisms, such as phagocytosis and melanization. Additionally, molecules involved in physiological processes are affected by hemolymph infection. Data obtained by this work suggests that immune responses may include mechanisms other than those traditionally related to immunity, as in the case of eicosanoid biosynthesis. Also, our results indicate that correlation between transcription and protein synthesis is not sure to exist and thus, immune responses should be analyzed by proteomics in a first approach.
Descrição
Palavras-chave
Parasitologia médica Malária Mosquitos Antimaláricos Plasmodium sporozoites
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical