Malaria is one of the major causes of mortality and morbidity worldwide. To continue to formulate new, more effective control strategies, a detailed understanding of the parasite life cycle on the molecular level is critical. The transition from the asexual cycle to sexual differentiation is required for malaria transmission in the field. Once sexual development is initiated, the parasite no longer undergoes asexual replication and dies several days after reaching maturity if not taken up in a blood meal by a mosquito. Once ingested by a mosquito, gametocytes are triggered to emerge from the RBC and, if fertilized, develop into infectious sporozoites. The molecular mechanisms involved in this complex differentiation pathway are largely unknown, although it has been characterized morphologically and several sexual-stage specific proteins have been identified. Antibodies specific for sexual-stage surface molecules, including Pfs230 and Pfs48/45, have been shown to block the ability of the parasite to infect mosquitoes, thus blocking, malaria transmission. These proteins have been studied for many years as vaccine candidates but their actual functions remain unknown. Both Pfs230 and Pfs48/45 are expressed only during sexual differentiation in the human host and through the transition of the parasite into the mosquito midgut. Our hypothesis is that Pfs230 & Pfs48/45 play a significant role in the development of gametocytes into viable fertilized zygotes and that the regulation of their expression is important to their function. As a first step toward the elucidation of the function of Pfs230 & Pfs48/45, their expression will be inhibited by targeted-gone disruption (Specific aim 1) and the effects this has on gametocyte & gamete differentiation will be evaluated (Specific aim 2). Transformed parasites will be selected by drug- resistance, cloned, and analyzed for disruption of each targeted gene. The morphology and gene expression pattern of transformants and wild-type parasites will be compared throughout sexual differentiation. To confirm that any changes observed are due to disruption of the targeted-gene, expression will be restored by complementation and the phenotype reanalyzed. The biological role of Pfs230 & Pfs48/45 is also affected by the time course and level of their expression. To begin to evaluate this, the elements regulating their transcription will be analyzed (Specific aim 3). The time course of MRNA production will be evaluated and the 5' regulatory elements that are involved in stage- specific regulation will be mapped by testing their ability to drive stage-specific expression of chloramphenicol acetyltransferase. The regulatory regions identified will be used to test for nuclear factor binding, to identify similar regions in other genes, and to construct transformation plasmids containing stage-specific, inducible promoters.
