RESUMO
Toxoplasma gondii is an obligate intracellular protozoan parasite whose rapid lytic replication cycles define its pathogenicity. We identified a temperature-sensitive growth mutant, FV-P6, which irreversibly arrests before the middle of the G1 stage of the tachyzoite cell cycle. This arrest is caused by a point mutation in a gene conserved across eukaryotes, Cactin, whose product localizes to the nucleus. To elucidate the role of TgCactin we performed genome-wide expression profiling. Besides the expected G1 expression profile, many genes associated with the extracellular state as well as with the bradyzoite cyst stage were identified. Consistent with these profiles were the expression of AP2 transcription factors typically associated with extracellular and bradyzoite stage parasites. This suggests a role for TgCactin in control of gene expression. As TgCactin does not contain any functionally defined domains we reasoned TgCactin exerts its function through interactions with other proteins. In support of this model we demonstrated that TgCactin is present in a protein complex and can oligomerize. Taken together, these results suggest that TgCactin acts as a pivotal protein potentially regulating gene expression at several transition points in parasite development.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Fase G1 , Proteínas de Protozoários/metabolismo , Toxoplasma/citologia , Toxoplasma/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Ciclo Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Dados de Sequência Molecular , Mutação Puntual , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de Sequência , Toxoplasma/química , Toxoplasma/genética , Toxoplasmose/parasitologiaRESUMO
The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member subfamily of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatiotemporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.
Assuntos
Citoesqueleto/metabolismo , Filamentos Intermediários/metabolismo , Multimerização Proteica , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Fusão Gênica Artificial , Citoesqueleto/ultraestrutura , Genes Reporter , Filamentos Intermediários/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Proteínas de Protozoários/genética , Proteínas de Protozoários/ultraestrutura , Toxoplasma/genéticaRESUMO
Exocytosis is essential to the lytic cycle of apicomplexan parasites and required for the pathogenesis of toxoplasmosis and malaria. DOC2 proteins recruit the membrane fusion machinery required for exocytosis in a Ca(2+)-dependent fashion. Here, the phenotype of a Toxoplasma gondii conditional mutant impaired in host cell invasion and egress was pinpointed to a defect in secretion of the micronemes, an apicomplexan-specific organelle that contains adhesion proteins. Whole-genome sequencing identified the etiological point mutation in TgDOC2.1. A conditional allele of the orthologous gene engineered into Plasmodium falciparum was also defective in microneme secretion. However, the major effect was on invasion, suggesting that microneme secretion is dispensable for Plasmodium egress.