RESUMEN
Malaria parasites undergo a complex life cycle in the human host and the mosquito vector. The ApiAP2 family of DNA-binding proteins plays a dominant role in parasite development and life cycle progression. Most ApiAP2 factors studied to date act as transcription factors regulating stage-specific gene expression. Here, we characterized an ApiAP2 factor in Plasmodium falciparum that we termed PfAP2-HC. We demonstrate that PfAP2-HC specifically binds to heterochromatin throughout the genome. Intriguingly, PfAP2-HC does not bind DNA in vivo and recruitment of PfAP2-HC to heterochromatin is independent of its DNA-binding domain but strictly dependent on heterochromatin protein 1. Furthermore, our results suggest that PfAP2-HC functions neither in the regulation of gene expression nor in heterochromatin formation or maintenance. In summary, our findings reveal PfAP2-HC as a core component of heterochromatin in malaria parasites and identify unexpected properties and substantial functional divergence among the members of the ApiAP2 family of regulatory proteins.
RESUMEN
The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.
Asunto(s)
Sistemas CRISPR-Cas , Malaria Falciparum/sangre , Plasmodium falciparum/genética , Esporas Protozoarias/genética , Animales , Anopheles/parasitología , Células Cultivadas , Eritrocitos/parasitología , Hepatocitos/citología , Hepatocitos/parasitología , Interacciones Huésped-Parásitos , Humanos , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Microscopía Fluorescente , Mosquitos Vectores/parasitología , Plasmodium falciparum/fisiología , Esporas Protozoarias/fisiología , Esporozoítos/genética , Esporozoítos/fisiologíaRESUMEN
The Plasmodium falciparum variant surface antigen PfEMP1 expressed on the surface of infected erythrocytes is thought to play a major role in the pathology of severe malaria. As the sequence pool of the var genes encoding PfEMP1 expands there are opportunities, despite the high degree of sequence diversity demonstrated by this gene family, to reconstruct full-length var genes from small sequence tags generated from patient isolates. To test whether this is possible we have used a set of recently laboratory adapted ICAM-1-binding parasite isolates to generate sequence tags and, from these, to identify the full-length PfEMP1 being expressed by them. In a subset of the strains available we were able to produce validated, full-length var gene sequences and use these to conduct biophysical analyses of the ICAM-1 binding regions.
Asunto(s)
Molécula 1 de Adhesión Intercelular/genética , Malaria Falciparum/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos/genética , Animales , Antígenos de Superficie/genética , Simulación por Computador , Eritrocitos/química , Eritrocitos/parasitología , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/patogenicidad , Unión Proteica , Proteínas Protozoarias/química , Alineación de SecuenciaRESUMEN
Malaria is caused by Plasmodium parasites that proliferate in the bloodstream. During each replication cycle, some parasites differentiate into gametocytes, the only forms able to infect the mosquito vector and transmit malaria. Sexual commitment is triggered by activation of AP2-G, the master transcriptional regulator of gametocytogenesis. Heterochromatin protein 1 (HP1)-dependent silencing of ap2-g prevents sexual conversion in proliferating parasites. In this study, we identified Plasmodium falciparum gametocyte development 1 (GDV1) as an upstream activator of sexual commitment. We found that GDV1 targeted heterochromatin and triggered HP1 eviction, thus derepressing ap2-g Expression of GDV1 was responsive to environmental triggers of sexual conversion and controlled via a gdv1 antisense RNA. Hence, GDV1 appears to act as an effector protein that induces sexual differentiation by antagonizing HP1-dependent gene silencing.