Project description:ApiAP2 are a family of conserved transcription factors (TF) that play an important role in regulating gene expression in apicomplexan parasites. ApiAP2 proteins that may control the cell cycle dependent expression program are to be discovered. To better understand how these cell-cycle dependent gene expression profiles are established, we identified ApiAP2 proteins showing a cell cycle dependent expression. Using the Auxin-degradation system, we produced several inducible knock-down (iKD) mutant for these ApiAP2 proteins. Among them, a cell-cycle regulated ApiAP2 is expressed during the late S-phase of the tachyzoite cell-cycle. The iKD mutant parasites for this gene are unable to proliferate. In presence of Auxin, the mutant parasites do not produce daughter cells and are arrested in the early phase of budding. This indicates that this TF may specifically regulate the early steps of the daughter cell formation. Using RNA-Seq, we demonstrate that the level of expression of number of transcripts is affected by the knock-down of this potential TF.
Project description:Toxoplasma gondii is an apicomplexan parasite infecting human and animals, causing huge health concerns and economic losses. Calcium ion, a critical second messenger in cells, can regulate related vital activities, particularly in parasite invasion and escape processes. Calmodulin (CaM) is a short, highly conserved Ca2+ binding protein found in all eukaryotic cells, including apicomplexan parasites. After binding to Ca2+, CaM can be activated to interact with a variety of proteins (such as enzymes). Since direct destruction of CaM is impossible, few studies have been conducted on the function of CaM in T. gondii. We generated the CaM indirect knockout strain (iCaM) using a tetracycline-off system with CaM promoter sequence in T. gondii TATI strain, and compared the transcriptomes of tachyzoites with and without Calmodulin.
Project description:Important regulation occurs at the level of transcription in Plasmodium falciparum and growing evidence suggests that these apicomplexan parasites have complex regulatory networks. Recent studies implicate long noncoding RNAs (lncRNAs) as transcriptional regulators in P. falciparum and as part of these regulatory networks. However, due to limited research and the lack of necessary experimental tools, our understanding of the role of lncRNAs in P. falciparum remains largely unelucidated. In this study, we improve the annotation of lncRNAs in order to provide researchers with a tool that will facilitate the study of P. falciparum lncRNAs in vitro.
Project description:Apicomplexan parasites cause numerous diseases in humans and animals, including malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). Despite being a major drug target, the protein composition of the coenzyme Q:cytochrome c oxidoreductase complex (Complex III) in these parasites was previously unknown. Our work highlights the divergence of mitochondrial ETC Complex III composition in apicomplexan parasites and provides a broader understanding of Complex III evolution. Our study also provides important insights into what sets this major drug target apart from the equivalent complex in host species. Future studies can build on our findings to reveal how novel subunits contribute to Complex III function to further investigate this important drug target.
Project description:In this study we have investigated PfAP2-HC (PF3D7_1456000), a protein that was identified by co-immunoprecipitation with PfHP1 coupled with liquid chromatography-tandem mass spectrometry. PfAP2-HC belongs to the ApiAP2 family, the main transcription factor family in Apicomplexan parasites. We have confirmed that AP2-HC colocalises with HP1 with the use of immunofluorescence assays and chromatin immunoprecipitation-sequencing. We show that PfAP2-HC is not required for heterochromatin maintenance and inheritance with the use of PfAP2-HC Kock out and knockdown. We show with transcriptome-wide microarray time course analysis that PfAP2-HC does not act as a transcription factor in blood stage parasites. We demonstrate that the AP2 domain is dispensable for heterochromatin targeting by introducing a premature stop codon before the AP2 domain. We show that PfAP2-HC binding to heterochromatin dependents on PfHP1. and PfAP2-HC is likely not involved in de novo heterochromatin formation
Project description:<p>Apicomplexan parasite growth and replication relies on nutrient acquisition from host cells, in which intracellular multiplication occurs, yet the mechanisms that underlie the nutrient salvage remain elusive. Numerous ultrastructural studies have documented a plasma membrane invagination with a dense neck, termed the micropore, on the surface of intracellular parasites. However, the function of this structure remains unknown. Here we validate the micropore as an essential organelle for endocytosis of nutrients from the host cell cytosol and Golgi in the model apicomplexan <em>Toxoplasma gondii</em>. Detailed analyses demonstrated that Kelch13 is localized at the dense neck of the organelle and functions as a protein hub at the micropore for endocytic uptake. Intriguingly, maximal activity of the micropore requires the ceramide de novo synthesis pathway in the parasite. Thus, this study provides insights into the machinery underlying acquisition of host cell-derived nutrients by apicomplexan parasites that are otherwise sequestered from host cell compartments.</p>
Project description:Obligate intracellular parasites must efficiently invade host cells in order to mature and be transmitted. For the malaria parasite Plasmodium falciparum, invasion of host red blood cells (RBCs) is essential. Here we describe a parasite-specific transcription factor belonging to the Apicomplexan Apetala 2 (ApiAP2) family that is responsible for regulating the expression of a subset of merozoite genes involved in RBC invasion (PfAP2-I). Our genome-wide analysis by ChIP-seq shows that PfAP2-I interacts with a specific DNA motif in the promoters of these genes. msp5 transcription levels decrease when the PfAP2-I DNA-binding motif is mutated in PfAP2-I-GFP parasites, showing that PfAP2-I must bind the DNA motif in order for msp5 to be transcribed.