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Estudos de biologia da multiresistência a antimaláricos em Plasmodium falciparum: transportadores ABC e genes de resposta ao stress oxidativo
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Resumo(s)
A malária causada por Plasmodium falciparum é, em conjunto com a tuberculose e o HIV/SIDA, a maior causa de mortalidade mundial entre as doenças infecciosas. Nas últimas décadas, o controlo e tratamento da malária têm sido bastante dificultados pelo surgimento e disseminação da resistência parasitária aos antimaláricos mais utilizados, nomeadamente às quinoleínas.
O parasita encontra-se em fase de elevada actividade metabólica, nos estadios sensíveis às quinoleínas, correspondente à degradação da hemoglobina no vacúolo digestivo, a qual gera ferriprotoporfirina IX (FP-IX) e espécies reactivas de Oxigénio (ROS). A destoxificação destes grupos é efectuada por enzimas de resposta ao stress oxidativo, como a superoxidodismutase e as enzimas dos sistemas dependentes da tiorredoxina e do glutatião (GSH). Os compostos resultantes são demasiado hidrofílicos para difundir através da membrana citoplasmática, necessitando de transportadores específicos da super-família de proteínas ABC. O facto de P. falciparum apresentar resistência a diferentes antimaláricos e a relação existente entre as proteínas ABC (nomeadamente das sub-famílias MDR/TAP e MRP/CFTR) e os fenótipos de multi-resistência, justificaram esta investigação.
No genoma de P. falciparum foram identificadas 18 novas ABCs e posicionadas em 8 sub-famílias.
A susceptibilidade in vitro de isolados de P. falciparum (recorrendo a testes O.M.S.), provenientes de São Tomé e Príncipe, Tailândia e Angola demonstrou resistência simultânea a mais de um fármaco e níveis distintos de susceptibilidade entre aquelas regiões. Aa efectuar um estudo de associação das alterações de sequência nos genes pfmrp1 e pfmrp2 (identificadas no decorrer deste trabalho), os resultados demonstraram
uma correlação positiva apenas na Tailândia, no respeitante à mefloquina, para os polimorfismos de pfmrp1 (191Y e 347A) e para a presença de inserções/delecções nos codões 779 e 3591em de pfmrp2. Usando PCR em tempo real e para os mesmos isolados, foi determinado o número de cópias dos transportadores ABC pfmdr1, pfmrp1 e pfmrp2. Para os dois últimos apenas foi detectada uma cópia, mas para pfmdr1 foi identificado aumento do número de cópias (mais de 2) associado a menor susceptibilidade ao mesmo fármaco, no mesmo País.
Em estudos de expressão dos genes codificantes de enzimas de resposta ao stress oxidativo (pfFe-sod, pfγ-gcs, pfgr, pfgst, pfgpx, pftrxR e pf2-CysPx) e de transportadores VI ABC pfmrp1 e pfmrp2, verificou-se que são regulados no ciclo de desenvolvimento e que o gene pfg6pd apresentou um padrão de regulação consistente com um gene housekeeping. Duma forma geral a amplitude de variação da expressão (no sentido da diferença entre máximo e mínimo) dos genes ao longo do ciclo intra-eritrocitário em 3D7 (clone sensível) é maior do que a observada em Dd2 (clone resistente).
O aumento da expressão dos genes codificantes de enzimas do sistema de destoxificação dependente do glutatião, parece ocorrer na fase inicial do desenvolvimento do parasita (estadio de anel e trofozoíto), antes dos genes codificantes de enzimas do sistema de destoxificação dependente da tioredoxina visível em fase posterior (esquizonte). O aumento da expressão do gene pfmrp1, coincidiu com a função biológica (por analogia) de transporte de xenobióticos atribuída às proteínas da
sub-família ABCC. Pelo contrário, o padrão de expressão do gene pfmrp2, foi compatível com uma função de importação (de ex. glutatião). Este resultado é discutível.
Os perfis de expressão dos referidos 10 genes, indicam que a presença de CQ (dose IC50), altera significativamente a expressão no sentido do aumento de expressão (indução). Mais, a amplitude de variação de expressão dos genes é significativamente mais elevada no clone 3D7 (sensível) do que no Dd2 (resistente). Em geral não foi verificada diminuição (repressão) significativa da expressão dos genes em presença de CQ para as doses utilizadas (IC50). Os resultados dos estudos de estabilidade do mRNA, sugerem que o efeito de regulação da CQ se processa a nível transcripcional, e que a CQ não modifica a velocidade de degradação do mRNA destes genes.
Malaria, tuberculosis and AIDS are presently the most important infectious diseases worldwide. In malaria, this is related to the emergence and spread of multi-drug resistant strains of Plasmodium falciparum, namely to quinolines and has greatly complicated malaria control programs. Quinoline-containing antimalarial drugs interfere with hemoglobin digestion in the blood stages of the parasite cycle. Digestion of hemoglobin in the food vacuole, produces high quantities of Ferriprotoporphyrin IX (FP-IX) and reactive Oxygen species (ROS) as toxic by-products. ROS and FP-IX that reaches parasite cytoplasm are detoxified by stress response enzymes like: superoxide dismutase and thioredoxin and glutathione (GSH) dependent detoxification systems. Multidrug resistance associated proteins (ABC transporters) are fundamental cellular detoxifying factors supposed to transport a wide range of compounds across cell membranes either as GSH conjugates, GSSG or as co-transport accompanying GSH transposition. The observations that some P. falciparum strains exhibit resistance to different antimalarials and taking under consideration the association of ABC proteins (namely MDR/TAP and MRP/CFTR sub-families) to multidrug resistant phenotypes in other biological systems justified this research. We identified 18 new ABCs and positioned them in 8 different sub-families. Drug susceptibility studies with isolates from São Tomé Príncipe, Angola and Thailand (using WHO assays) demonstrated presence of multidrug resistance in isolates as well different levels of susceptibility among the regions. Correlating sequence variation on pfmrp1 e pfmrp2 genes (identified in this work) data demonstrated positive association only in Thailand, concerning mefloquine and pfmrp1 (191Y e 347A) as well as pfmrp2 concerning tandem repeats in condoms 779 e 3591. Gene copy numbers of pfmdr1, pfmrp1 and pfmrp2 were analysed by Real-time PCR, for the referred isolates. For the later two only one copy of the gene was detected, while for pfmdr1 a positive correlation was detected between altered copy number (more than 2) and resistance to the same drug and country. Oxidative stress response genes (pfFe-sod, pfγ-gcs, pfgr, pfgst, pfgpx, pftrxR e pf2-CysPx) and the ABC transporters pfmrp1 e pfmrp2, are cell cycle regulated in P. falciparum. Only pfg6pd exhibit a housekeeping gene consistent behaviour. Generally, gene expression was greater for the sensitive (3D7) clone than for the resistant one (Dd2). Genes coding for enzymes of the GSH dependent detoxification system, increase expression earlier (late ring and trophozoite) than those coding for enzymes of the thioredoxin dependent detoxification system (schizont stage). The transcription of pfmrp1 peaks consistently with what would be expected from their inferred functional interaction as xenobiotics, GSH conjugates and GSSG efflux pumps. However, the second MRP gene pfmrp2 is transcribed much earlier, during the ring stage and peaks again towards the end of the cycle, behaviour more consistent with an importer activity (namely of GSH). This is arguable. Expression profiles of the 10 studied genes under CQ challenge (IC50) indicate that this drug significantly enhances their expression in both clones. However, for the sensitive clone (3D7), upregulation is significantly higher than for the resistant Dd2, for most of the genes. Significant gene downregulation was not observed. CQ did not induce gene expression in the presence of an mRNA transcription inhibitor, suggesting regulation at the transcriptional level.
Malaria, tuberculosis and AIDS are presently the most important infectious diseases worldwide. In malaria, this is related to the emergence and spread of multi-drug resistant strains of Plasmodium falciparum, namely to quinolines and has greatly complicated malaria control programs. Quinoline-containing antimalarial drugs interfere with hemoglobin digestion in the blood stages of the parasite cycle. Digestion of hemoglobin in the food vacuole, produces high quantities of Ferriprotoporphyrin IX (FP-IX) and reactive Oxygen species (ROS) as toxic by-products. ROS and FP-IX that reaches parasite cytoplasm are detoxified by stress response enzymes like: superoxide dismutase and thioredoxin and glutathione (GSH) dependent detoxification systems. Multidrug resistance associated proteins (ABC transporters) are fundamental cellular detoxifying factors supposed to transport a wide range of compounds across cell membranes either as GSH conjugates, GSSG or as co-transport accompanying GSH transposition. The observations that some P. falciparum strains exhibit resistance to different antimalarials and taking under consideration the association of ABC proteins (namely MDR/TAP and MRP/CFTR sub-families) to multidrug resistant phenotypes in other biological systems justified this research. We identified 18 new ABCs and positioned them in 8 different sub-families. Drug susceptibility studies with isolates from São Tomé Príncipe, Angola and Thailand (using WHO assays) demonstrated presence of multidrug resistance in isolates as well different levels of susceptibility among the regions. Correlating sequence variation on pfmrp1 e pfmrp2 genes (identified in this work) data demonstrated positive association only in Thailand, concerning mefloquine and pfmrp1 (191Y e 347A) as well as pfmrp2 concerning tandem repeats in condoms 779 e 3591. Gene copy numbers of pfmdr1, pfmrp1 and pfmrp2 were analysed by Real-time PCR, for the referred isolates. For the later two only one copy of the gene was detected, while for pfmdr1 a positive correlation was detected between altered copy number (more than 2) and resistance to the same drug and country. Oxidative stress response genes (pfFe-sod, pfγ-gcs, pfgr, pfgst, pfgpx, pftrxR e pf2-CysPx) and the ABC transporters pfmrp1 e pfmrp2, are cell cycle regulated in P. falciparum. Only pfg6pd exhibit a housekeeping gene consistent behaviour. Generally, gene expression was greater for the sensitive (3D7) clone than for the resistant one (Dd2). Genes coding for enzymes of the GSH dependent detoxification system, increase expression earlier (late ring and trophozoite) than those coding for enzymes of the thioredoxin dependent detoxification system (schizont stage). The transcription of pfmrp1 peaks consistently with what would be expected from their inferred functional interaction as xenobiotics, GSH conjugates and GSSG efflux pumps. However, the second MRP gene pfmrp2 is transcribed much earlier, during the ring stage and peaks again towards the end of the cycle, behaviour more consistent with an importer activity (namely of GSH). This is arguable. Expression profiles of the 10 studied genes under CQ challenge (IC50) indicate that this drug significantly enhances their expression in both clones. However, for the sensitive clone (3D7), upregulation is significantly higher than for the resistant Dd2, for most of the genes. Significant gene downregulation was not observed. CQ did not induce gene expression in the presence of an mRNA transcription inhibitor, suggesting regulation at the transcriptional level.
Descrição
Palavras-chave
Malária Plasmodium falciparum Antimaláricos Resistência Stress oxidativo Proteínas ABC Parasitologia
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical