RESUMEN
Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.
Asunto(s)
Eritrocitos/enzimología , Eritrocitos/parasitología , MAP Quinasa Quinasa 1/metabolismo , Plasmodium falciparum/patogenicidad , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Animales , Antimaláricos/farmacología , Eritrocitos/metabolismo , Humanos , Concentración 50 Inhibidora , Plasmodium berghei/patogenicidad , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
PfPK7 is an orphan protein kinase of Plasmodium falciparum with maximal homology to MEK3/6 and to fungal protein kinase A proteins in its C-terminal and N-terminal regions, respectively. We showed previously that recombinant PfPK7 is active on various substrates but is unable to phosphorylate the Plasmodium falciparum mitogen-activated protein kinase homologues, suggesting that it is not a MEK functional homologue. Using a reverse genetics approach to investigate the function of this enzyme in live parasites, we now show that PfPK7(-) parasite clones display phenotypes at two stages of their life cycle: first, a decrease in the rate of asexual growth in erythrocytes associated with a lower number of daughter merozoites generated per schizont, and second, a dramatic reduction in the ability to produce oocysts in the mosquito vector. A normal asexual growth rate and the ability to produce oocysts are restored if a functional copy of the PfPK7 gene is reintroduced into the PfPK7(-) parasites. Hence, PfPK7 is involved in a pathway that regulates parasite proliferation and development.
Asunto(s)
Eritrocitos/metabolismo , Malaria Falciparum/enzimología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Plasmodium falciparum/enzimología , Proteínas Protozoarias/fisiología , Esporas Fúngicas/crecimiento & desarrollo , Animales , Southern Blotting , Western Blotting , Eritrocitos/parasitología , Humanos , Malaria Falciparum/parasitología , Malaria Falciparum/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genéticaRESUMEN
The kinome of the human malaria parasite Plasmodium falciparum includes two genes encoding mitogen-activated protein kinase (MAPK) homologues, pfmap-1 and pfmap-2, but no clear orthologue of the MAPK kinase (MAPKK) family, raising the question of the mode of activation and function of the plasmodial MAPKs. Functional studies in the rodent malaria model Plasmodium berghei recently showed the map-2 gene to be dispensable for asexual growth and gametocytogenesis, but essential for male gametogenesis in the mosquito vector. Here, we demonstrate by using a reverse genetics approach that the map-2 gene is essential for completion of the asexual cycle of P. falciparum, an unexpected result in view of the non-essentiality of the orthologous gene for P. berghei erythrocytic schizogony. This validates Pfmap-2 as a potential target for chemotherapeutic intervention. In contrast, the other P. falciparum MAPK, Pfmap-1, is required neither for in vitro schizogony and gametocytogenesis in erythrocytes, nor for gametogenesis and sporogony in the mosquito vector. However, Pfmap-2 protein levels are elevated in pfmap-1(-) parasites, suggesting that Pfmap-1 fulfils an important function in asexual parasites that necessitates compensatory adaptation in parasites lacking this enzyme.
Asunto(s)
Isoenzimas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Animales , Anopheles/parasitología , Eritrocitos/parasitología , Femenino , Humanos , Isoenzimas/genética , Malaria Falciparum , Masculino , Proteínas Quinasas Activadas por Mitógenos/genética , Fenotipo , Plasmodium falciparum/fisiología , Proteínas Protozoarias/genéticaRESUMEN
The molecular mechanisms regulating the sexual development of malaria parasites from gametocytes to oocysts in their mosquito vector are still largely unexplored. In other eukaryotes, NIMA-related kinases (Neks) regulate cell cycle progression and have been implicated in the regulation of meiosis. Here, we demonstrate that Nek-4, a new Plasmodium member of the Nek family, is essential for completion of the sexual cycle of the parasite. Recombinant Plasmodium falciparum Nek-4 possesses protein kinase activity and displays substrate preferences similar to those of other Neks. Nek-4 is highly expressed in gametocytes, yet disruption of the nek-4 gene in the rodent malaria parasite P. berghei has no effect on gamete formation and subsequent fertilization. However, further differentiation of zygotes into ookinetes is abolished. Measurements of nuclear DNA content indicate that zygotes lacking Nek-4 fail to undergo the genome replication to the tetraploid level that precedes meiosis. Cell cycle progression in the zygote is identified as a likely precondition for its morphological transition to the ookinete and for the successful establishment of a malaria infection in the mosquito.