Global Transcriptional Repression: Initial and Essential Step for Plasmodium Sexual Development [expression array]

Gametocytes are nonreplicative sexual forms that mediate malaria transmission to a mosquito vector. They are generated from asexual blood stage parasites, which proliferate in the circulation. However, it remains largely unknown as to how this transition is genetically regulated. Here, we report that an Apetala2 (AP2) family transcription factor, AP2-G2, regulates the transition as a transcriptional repressor. Disruption of AP2-G2 in the rodent malaria parasites, Plasmodium berghei, did not prevent commitment to the sexual stage but halted their development before manifesting sex-specific morphologies. ChIP-seq analysis revealed that AP2-G2 targets approximately 1,500 genes and recognizes a five-base motif on their promoters. Most of these target genes are required for asexual proliferation in the blood by the parasites, thereby suggesting that AP2-G2 blocks the program for asexual replication of parasites in the blood. DNA microarray analysis showed that the identified targets constituted approximately 70% of the upregulated genes in AP2-G2-depleted parasites, and a promoter assay using a centromere plasmid demonstrated that the binding motif functions as a cis-acting negative regulatory element. These results suggest that global transcriptional repression, which occurs during the initial phase of gametocytogenesis, is an essential step to promote conversion to the sexual stage. To identify target genes of AP2-G2, microarray analysis was performed between AP2-G2(−) and wild-type parasites. To establish synchronous infection in Wistar rats, whole blood was harvested from rats infected with AP2-G2(-) or wild-type parasites and cultured for 16 h. Mature schizonts were purified from the culture with Nicodenz and inoculated into rats. Whole blood was harvested from these rats at 19 h after the innoculation. Four biologically independent experiments were performed in both AP2-G2(-) and wild-type parasites. To kill asexual stage parasites, eight-week-old female Balb/c mice infected with AP2-G2(-) or wild-type parasites were treated with sulfadiazine in drinking water (10 mg/l) for two days. Five biologically independent experiments were performed in both AP2-G2(−) and wild-type parasite samples.