For Plasmodium falciparum, the most widespread and virulent human malaria parasite, persistence within the human host requires asexual replication within red blood cells, while transmission depends on differentiation to non-replicative sexual stages, gametocytes. At the time of invasion parasites are already committed to one of these fates, with the developmental decision having been made in the previous cell cycle. Sexual commitment requires two proteins: AP2-G and GDV1. Expression of AP2-G, the transcriptional master regulator of sexual development, during this ?commitment cycle? poises gene expression to initiate sexual development upon re- invasion. Derepression of the epigenetically silenced ap2-g locus through a heretofore-unknown mechanism leads to transcription activation of ap2-g and subsequent sexual commitment of newly forming parasites. Unlike AP2-G, GDV1 is expressed in every replication cycle. However, previous work showed that this protein localizes to the nuclear periphery in a subpopulation of committed parasites, an ideal position for regulation of epigenetically silenced genes, including ap2-g. RNA microarray data suggests that the timing of maximal GDV1 expression coincides with initial ap2-g transcription. These data suggest that GDV1 may play a role in activation of ap2-g transcription. This activation may be facilitated by interaction with key upstream regulatory elements. In the proposed study, I will investigate mechanisms of ap2-g activation using two complementary strategies by identifying the key upstream sequence elements of ap2-g that are necessary and sufficient for its silencing and transcriptional activation (AIM 1) and defining the role of GDV1 in ap2-g activation (AIM 2). The first question to be addressed regards the specific roles of upstream regulatory elements in activating or silencing transcription of ap2-g. We address this by first identifying the transcriptional start site (TSS) of ap2-g at different stages of parasite development using 5? RACE. Secondly we will create a system for integrative recombination that will allow for rapid modification of upstream sequence elements in the endogenous ap2-g locus. qRT-PCR, immunofluorescence, and gametocyte formation will be used to monitor affects on ap2-g activation with each TSS and following upstream modification of the locus. The next question to be addressed focuses on the necessity of GDV1 and its nuclear localization for transcriptional activation of ap2-g. To address this, a system for conditional misloclaization of GDV1 will be engineered allowing for precise post-translational regulation of the protein. ap2-g activation will be measured using qRT-PCR, immunofluorescence and gametocyte formation. Furthermore, nuclear localization of GDV1 as well as direct interaction of the protein with the ap2-g locus will be analyzed using combined IFA/FISH and GDV1 ChIP. Successful completion of this work will yield new insights into the mechanism responsible for regulation of gametocyte formation. The ability to disrupt the regulation of sexual commitment in malaria parasites holds significant promise for therapeutic intervention, as blocking activation would interrupt disease transmission.
Malaria is among the world?s most devastating infectious diseases, causing an estimated 429,000 deaths worldwide, for which repeated emergence of drug resistance in both malaria parasites and their mosquito vectors continue to cripple current control strategies. Our research plan tests a mechanistic hypothesis for how AP2-G, the master switch controlling transmission to the mosquito, is activated. Unveiling the molecular mechanism underlying ap2-g activation will facilitate the development of novel drugs targeting parasite development and transmission.
