Malaria remains one of the major global health concerns despite a decrease in both the number of cases and deaths over the past 15 years. The recent description of increase resistance to artemisinin in Southeast Asia heightens the urgency for the discovery of new chemotherapeutic targets in the parasite. An increased understanding of the basic biology of critical pathways and processes will facilitate this discovery process. One of the critical biological processes of the parasite is the pathway of invasion into the host cell. In the case of merozoite invasion of the red blood cell, it is clear that invasion proceeds through a series of coordinated events in a step-wise manner. This process consists of a reversible initial contact between the merozoite and the red blood cell, reorientation of the merozoite such that the apical end of the parasite binds irreversibly to the host cell surface, formation of a tight junction and subsequent internalization of the parasite powered by the parasite's own actin-myosin motor into the newly formed parasitophorus vacuole. Central to this process is the ordered discharge of the contents of the parasite's specialized secretory organelles, the rhoptries, micronemes and dense granules. Numerous merozoite antigens have been localized to these organelles but overall little is known in regards to the structure, biogenesis and functioning of these critical organelles. We will utilize a newly described approach, proximity-dependent biotinylation, coupled to a gene expression control system to discover and identify the molecular components of mature secretory organelles. We will target the investigation to the study of micronemes and dense granules, the latter being the least understood of these organelles. In each case, two proteins with known localization to the specific organelle (PfAMA1 and PfMTRAP for micronemes and PfRESA and PfSUB1 for dense granules) will be fused to a promiscuous biotin ligase (BirA*) that has a decreased affinity for the activated biotin intermediate. Upon addition of exogenous biotin, biotinylation of interacting or proximally located proteins will occur and these proteins will be affinity purified and identified by mass spectrometry (LC-MS-MS). Proteins discovered through this approach, with special attention to proteins classified in the database as ?conserved protein of unknown function, will be epitope-tagged and expressed in the parasite to confirm localization to the specific organelle. These experiments will identify new molecular constituents of these critical secretory organelles and increase our basic knowledge regarding the structure and function of these critical parasite organelles.
The process of malaria parasite invasion into host cells is critical to the growth and development of the parasite and must occur quickly and efficiently to avoid the host immune response. This process involves the stepwise release of the contents of several special secretory organelles located at the apical end of the parasite. The proposed studies will significantly increase our knowledge of the structure and function of these organelles by defining and understanding some of the molecular components comprising these unique organelles.
