Conditional expression of PfAP2-G for controlled massive sexual conversion in Plasmodium falciparum [array]

Malaria transmission requires the conversion of some asexual parasites into sexual forms termed gametocytes. The initial stages of sexual development, including sexually-committed schizonts and sexual rings, remain poorly characterized, in part because only a subset of parasites undergo sexual development and they are morphologically identical to their asexual counterparts. Here we present a system based on conditional expression of PfAP2-G, the master regulator of sexual conversion, for controlled sexual induction. Induction resulted in ~90% sexual conversion, which enables the characterization of early sexual stages without further purification. Transcriptomic analysis identified the alterations that follow pfap2-g activation, including identification of genes that are down-regulated. We also show that the inducible system enables the characterization of committed stages at the phenotypic level. Altogether, the inducible lines will facilitate the study of the initial stages of sexual development, which is important for the development of new strategies to stop malaria transmission. Time-course transcriptomic analysis was performed using tightly synchronized E5ind cultures (0-5 h) induced with 10 nM rapamycin (Rapa) for 1 h at 25-30 hpi or with DMSO as control (Dmso). Samples for transcriptomic analysis were collected 15 h (T1), 28 h (T2) and 38 h (T3) after induction (corresponding to 40-45 hpi schizonts of the first cycle, and 5-10 or 15-20 hpi rings of the next cycle, respectively). The reference pool consited of RNA from E5 wild-type cultures and E5ind cultures, in non-inducing conditions, throughout the asexual blood cycle. The analysis was performed with the results obtained from two independent biological replicates. A total of 12 individual microarray samples were processed. For the first replicate we used a microarray design AMADID-084561, which was later modified to include a small number of new probes and used for the second replicate (AMADID-085763).