PfAP2-G target gene promoter mutants expression timecourse

In the malaria parasite Plasmodium falciparum, the switch from asexual multiplication to sexual differentiation into gametocytes is essential for transmission to mosquitos. One of the key determinants of sexual commitment is the transcription factor PfAP2-G, which has been proposed to orchestrate this crucial cell fate decision by driving expression of gametocyte genes. We show conclusively that PfAP2-G is a transcriptional activator of gametocyte genes and identify the earliest known markers expressed during commitment. Remarkably, we also find that in sexually committed cells, PfAP2-G is associated with the promoters of genes important for red blood cell invasion and activates them through its interactions with a second transcription factor. We thus demonstrate an intriguing transcriptional link between the apparently opposing processes of red blood cell invasion and gametocytogenesis that is coordinated by the master regulator PfAP2-G. This finding has important implications for the development of new anti-malarial drugs that block the invasion of red blood cells by sexually committed cells, thereby preventing parasite transmission. Two lines were tested: AP2-G-DD and AP2-G-DD-ap2-g-mut. At 24 hpi, highly synchronous parasites were treated with Shld1 to stabilise the AP2-G-DD protein. RNA was collected from each culture in parallel at intervals of six hours for the next 66 hours, to give a total of 22 samples (11 for each line). Cy5-labelled cDNA generated from these samples was hybridized against a Cy3-labeled reference pool of 3D7 mixed staged parasites spiked with cDNA from the samples (to make up 50% of the new reference pool) on a two-color array.