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Expressão de proteínas anti-Trypanosoma sp. em Sodalis glossinidius no modelo Glossina morsitans morsitans (Diptera, Glossinidae)
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Orientador(es)
Resumo(s)
As glossinas, moscas do sono ou moscas tsé-tsés (Glossina spp.) são
responsáveis pela transmissão do protozoário flagelado Trypanosoma sspp., causador da tripanossomose animal africana, ou Nagana, e da tripanossomose humana africana (THA). Esta, é endémica em 30 países da África subsariana e estima-se que possam estar em risco de a contrair cerca de 60 milhões de pessoas.
As alterações climáticas e ambientais podem vir a incrementar a incidência da referida patologia humana, assim como a sua distribuição geográfica. O combate às tripanossomoses africanas terá de adotar uma estratégia multidisciplinar, articulada entre as diferentes componentes que constituem o sistema biológico em causa e ser
dirigido, e aplicado, com as populações. Contudo, terá de envolver também organismos públicos oficiais, como exercício de boa governança.
O desenvolvimento das técnicas de Biologia Molecular abriu novas
possibilidades no que diz respeito ao controlo vetorial. Apesar da viviparidade adenotrófica das glossinas dificultar a sua transgénese direta, a técnica da paratransgénese surgiu como alternativa, apresentando-se presentemente mais vantajosa.
No presente trabalho, e com o objetivo de desenvolver um endossimbionte de Glossina spp capaz de expressar no seu genoma proteínas recombinantes com ação tripanocida (atacina e defensina), recorreu-se a diferentes técnicas para proceder à transformação de Sodalis glossinidius, nomeadamente choque térmico, tratamento
químico e eletroporação. A transformação foi alcançada pela combinação de duas destas técnicas: tratamento químico, seguido de eletroporação. A transformação de S. glossinidius, pela referida combinação, foi alcançada pela primeira vez com sucesso nesta bactéria. O genoma do endossimbionte foi transformado com diferentes
construções de plasmídeos, de modo a expressar as proteínas atacina e defensina, individualmente.
Foram também desenvolvidas construções de plasmídeos para expressar controladamente as proteínas, de forma a poder purifica-las e usá-las, futuramente, em ensaios de função tripanocida.
Por último, foi delineado e otimizado um ensaio de quantificação da expressão da atacina e da defensina, no endossimbionte transformado, por qPCR.
Tsetse flies (Glossina spp.) are responsible for the transmission of the flagellated protozoa Trypanosoma sspp. which causes animal African trypanosomiasis (Nagana) and Human African trypanosomiasis (HAT). The later is endemic in 30 countries in sub-Saharan Africa and it is estimated that 60 million people may be at risk of infection. Climate and environmental changes are likely to increase the incidence of the human pathology as well as its geographical distribution. Strategies undertaken to fight African trypanosomiasis will have to be multidisciplinary and articulated between the different components that comprise its biological system. Actions must be directed, as well as applied, together with the populations. Public governmental entities should be involved in a good governance exercise. The development of molecular biology techniques has opened up new possibilities with respect to vector control. Despite the fact that the direct transgenesis of flies is hampered by tsetse´s adenotrofic viviparity, paratransgenesis technique emerged as an alternative, presenting itself more advantageous, nowadays. In this study and in order to develop an endosymbiont of Glossina spp, able to express recombinant proteins with trypanocidal effect (attacin and defensin), different techniques were applied for the transformation of Sodalis glossinidius. These techniques included thermal shock, chemical treatment and electroporation. Transformation was achieved by the combination of two of these techniques: chemical treatment, followed by electroporation. Transformation of S. glossinidius by this combination was, for the first time, successfully achieved. The endosymbiont was transformed with different plasmid constructs to express attacin and defensin proteins, individually. Other plasmid constructs were also designed to controllably express the refered proteins in order to purify and use them in future trypanocidal function tests. Finally, a qPCR assay was designed and optimized to later quantify the expression of attacin and defensin by the transformed endosymbiont
Tsetse flies (Glossina spp.) are responsible for the transmission of the flagellated protozoa Trypanosoma sspp. which causes animal African trypanosomiasis (Nagana) and Human African trypanosomiasis (HAT). The later is endemic in 30 countries in sub-Saharan Africa and it is estimated that 60 million people may be at risk of infection. Climate and environmental changes are likely to increase the incidence of the human pathology as well as its geographical distribution. Strategies undertaken to fight African trypanosomiasis will have to be multidisciplinary and articulated between the different components that comprise its biological system. Actions must be directed, as well as applied, together with the populations. Public governmental entities should be involved in a good governance exercise. The development of molecular biology techniques has opened up new possibilities with respect to vector control. Despite the fact that the direct transgenesis of flies is hampered by tsetse´s adenotrofic viviparity, paratransgenesis technique emerged as an alternative, presenting itself more advantageous, nowadays. In this study and in order to develop an endosymbiont of Glossina spp, able to express recombinant proteins with trypanocidal effect (attacin and defensin), different techniques were applied for the transformation of Sodalis glossinidius. These techniques included thermal shock, chemical treatment and electroporation. Transformation was achieved by the combination of two of these techniques: chemical treatment, followed by electroporation. Transformation of S. glossinidius by this combination was, for the first time, successfully achieved. The endosymbiont was transformed with different plasmid constructs to express attacin and defensin proteins, individually. Other plasmid constructs were also designed to controllably express the refered proteins in order to purify and use them in future trypanocidal function tests. Finally, a qPCR assay was designed and optimized to later quantify the expression of attacin and defensin by the transformed endosymbiont
Descrição
Palavras-chave
Saúde tropical Doenças tropicais Moscas do sono Trypanosoma Sodalis
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical
Instituto de Higiene e Medicina Tropical
Instituto de Higiene e Medicina Tropical