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A malária é um dos problemas mais graves de saúde pública em todo o mundo, afectando anualmente a vida de milhões de pessoas. O uso extensivo de antimaláricos, tais como a cloroquina e mefloquina, levou ao aparecimento de parasitas Plasmodium falciparum resistentes, impactando negativamente os esforços globais para o controlo da doença. Por esta razão, o controlo da malária depende de terapias de combinação baseadas em artemisinina (ACTs) que incluem mefloquina-artesunato, amodiaquina-artesunato e lumefantrina-artemeter. Torna-se assim necessário e urgente adquirir conhecimento sobre os mecanismos envolvidos na resistência aos antimaláricos, de modo a abrandar ou evitar a evolução da resistência, no intuito de prolongar a eficácia dos antimaláricos actualmente usados e desenvolver novas terapias. Neste contexto, foram utilizadas neste trabalho ferramentas de genética e genómica aplicadas ao modelo de malária de roedores Plasmodium chabaudi, no intuito de identificar os determinantes genéticos de resistência aos diferentes componentes de ACTS.
Inicialmente, a progenia não clonada obtida de um cruzamento genético previamente efectuado entre o clone resistente à mefloquina, AS-15MF e um clone sensível geneticamente distinto, AJ, foi seleccionada com uma dose de mefloquina aqui optimizada. A progenia resultante foi cruzada novamente com o AJ e o producto obtido foi analisado por Linkage Group Selection (LGS) de modo a investigar as assinaturas de selecção após tratamento com a cloroquina (CQ), mefloquina (MF), lumefantrina (LM) ou artemisinina (ART). Adicionalmente, as alterações genéticas acumuladas no clone AS-15MF foram identificadas por re-sequenciação do genoma inteiro, através da tecnologia Solexa.
Os resultados obtidos mostraram que a MF, LM e ART seleccionam parasitas contendo um segmento duplicado no cromossoma 12, que translocou para o cromossoma 4. A análise da leitura de sequências por Solexa revelou uma duplicação da cobertura estendendo-se por >392 kb e contendo cerca de 112 genes, includindo o gene de multi-resistência (mdr1) codificante da P-glicoproteína 1. O fragmento translocado foi mapeado com precisão no cromossoma 4. Adicionalmente, os antimaláricos, MF and ART, geraram também assinaturas de selecção no cromossoma 2, onde está contida uma mutação numa desubiquitinase, codificada pelo gene ubp1. Deste modo, estes dados constituem uma demonstração clara e directa que a resistência aos componentes de ACT, quimicamente distintos, pode ser determinada pelo(s) mesmo(s) gene(s), realçando a possível limitação destas terapias.
Adicionalmente, uma única mutação no clone AS-15MF, foi identificada em um gene codificante de uma putativa lisina descarboxilase. Esta mutação não é seleccionada marcadamente pela MF e não segrega com a resposta à MF na progenia clonada do cruzamento genético entre os clones AS-15MF e AJ, indicando que não está directamente associada ao fenótipo de resistência da mefloquina.
Malaria is by far one of the most severe public health problems worldwide, devastating the lives of millions of people each year. The extensive use of antimalarial drugs such as chloroquine and mefloquine, has led to the acquisition of drug resistance by Plasmodium falciparum, severely curtailing global efforts to control malaria. For this reason, much hope is now laid on new therapeutic approaches based on the use of artemisinin-based combination therapies (ACTs), which include mefloquine-artesunate, amodiaquine-artesunate and lumefantrine-artemether. A better understanding of the underlying mechanisms of drug resistance is therefore imperative to slow or circumvent the evolution of resistance, to prolong the life span of the current drugs and to develop new drugs. In this context, genetic and genomic tools were applied here to the rodent malaria model Plasmodium chabaudi, to exploit the genetic determinants of resistance to different component drugs of ACTs. First, the uncloned progeny of a genetic cross between a mefloquine-resistant mutant (AS-15MF) and a genetically distinct sensitive clone (AJ) was selected with an optimized dose of mefloquine. The progeny obtained was then backcrossed here with AJ and the resulting product analysed by Linkage Group Selection (LGS) to define the signatures of selection arising after treatment with chloroquine (CQ), mefloquine (MF), lumefantrine (LM) or artemisinin (ART). Additionally, the critical genome-wide changes accumulated in AS-15MF were identified by Solexa whole genome re-sequencing. Results showed that MF, LM and ART selected parasites bearing a duplicated segment on chromosome 12 which has translocated onto chromosome 4. Solexa sequence read-coverage analysis showed that this duplicated fragment extends for >392 kb and contains about 112 genes, including mdr1, the gene encoding the multi-drug resistance P-glycoprotein. The translocated fragment was precisely mapped on chromosome 4. MF and ART also generated selection signatures on chromosome 2, containing a mutation in a deubiquitinating enzyme, encoded by the ubp1 gene. Unambiguous evidence is thus provided for the first time to demonstrate that resistance to chemically distinct components of ACTs share the same underlying genes, highlighting a possible limitation of these therapies. Furthermore, a single mutation, unique to AS-15MF, was identified in a gene encoding a putative lysine decarboxylase. This mutation is not markedly selected by MF and it does not segregate with MF responses in the progeny clones of the genetic cross between AS-15MF and AJ, implying that it is not directly associated with the MF resistance phenotype.
Malaria is by far one of the most severe public health problems worldwide, devastating the lives of millions of people each year. The extensive use of antimalarial drugs such as chloroquine and mefloquine, has led to the acquisition of drug resistance by Plasmodium falciparum, severely curtailing global efforts to control malaria. For this reason, much hope is now laid on new therapeutic approaches based on the use of artemisinin-based combination therapies (ACTs), which include mefloquine-artesunate, amodiaquine-artesunate and lumefantrine-artemether. A better understanding of the underlying mechanisms of drug resistance is therefore imperative to slow or circumvent the evolution of resistance, to prolong the life span of the current drugs and to develop new drugs. In this context, genetic and genomic tools were applied here to the rodent malaria model Plasmodium chabaudi, to exploit the genetic determinants of resistance to different component drugs of ACTs. First, the uncloned progeny of a genetic cross between a mefloquine-resistant mutant (AS-15MF) and a genetically distinct sensitive clone (AJ) was selected with an optimized dose of mefloquine. The progeny obtained was then backcrossed here with AJ and the resulting product analysed by Linkage Group Selection (LGS) to define the signatures of selection arising after treatment with chloroquine (CQ), mefloquine (MF), lumefantrine (LM) or artemisinin (ART). Additionally, the critical genome-wide changes accumulated in AS-15MF were identified by Solexa whole genome re-sequencing. Results showed that MF, LM and ART selected parasites bearing a duplicated segment on chromosome 12 which has translocated onto chromosome 4. Solexa sequence read-coverage analysis showed that this duplicated fragment extends for >392 kb and contains about 112 genes, including mdr1, the gene encoding the multi-drug resistance P-glycoprotein. The translocated fragment was precisely mapped on chromosome 4. MF and ART also generated selection signatures on chromosome 2, containing a mutation in a deubiquitinating enzyme, encoded by the ubp1 gene. Unambiguous evidence is thus provided for the first time to demonstrate that resistance to chemically distinct components of ACTs share the same underlying genes, highlighting a possible limitation of these therapies. Furthermore, a single mutation, unique to AS-15MF, was identified in a gene encoding a putative lysine decarboxylase. This mutation is not markedly selected by MF and it does not segregate with MF responses in the progeny clones of the genetic cross between AS-15MF and AJ, implying that it is not directly associated with the MF resistance phenotype.
Descrição
Palavras-chave
Parasitologia médica Malária Terapêutica Mefloquina Resistência
Contexto Educativo
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Editora
Instituto de Higiene e Medicina Tropical