The parasitic disease, malaria, is one of the major causes of mortality and morbidity worldwide (Oaks). Control efforts have been complicated by the emergence of drug-resistant parasites and new approaches are needed. The sexual stage of the parasite life-cycle offers unique opportunities for blocking parasite development. Rhofs230 is processed from a 360 kDa protein to a 310 kDa protein as the parasite emerges from the red blood cell to become an extracellular gamete (Williamson et al). This is the first demonstration of stage-specific processing in sexual stage parasites. Proteolytic processing is known to activate a large number of enzymes and hormones; likewise the proteolytic processing of rhofs230 may play a critical functional role in gamete development and its evasion of host defenses. The objective of this project is to determine the mechanism of stage-specific rhofs230-processing and its role in gamete development and host defense evasion. To determine the mechanism of rhofs230 processing, specific aim 1, the rhofs230 cleavage site will be identified and the protease responsible will be localized and characterized. The amino acid sequence of the cleavage site will be used to evaluate protease specificity and to design subtrates that could inhibit the protease. This information will also be used to design recombinant vaccine candidates. The location of the protease and the determination of optimal reaction conditions will contribute to the understanding of the process involved in rhofs230 cleavage and will be sued to develop an assay to screen for agents that specifically inhibit rhofs230 proteolysis. Reagents that block proteolyis of rofs230 will be characterized and tested for their effect on gamete decelopment and its evasion of host defenses. The role of rhofs230 processing in gamete development, specific aim 2, will be assessed by evaluating gamete emergemce from the red blood cell, exflagellation, fertilization, surface antigen expression, and mosquito infectivity. The ability of the parasite to evade host defenses, such as complement and antibodies produced against gametocytes, specific aim 3, will also be evaluated. Reagents that block gamete development will be further evaluated as transmission- blocking reagents. The results will be used to direct the design of a rhofs230 subunit vaccine and identifying new drug targets, such as a critical protease. This work will also increase our understanding of the molecular mechanisms involved in malaria parasite development which may be used in the future to modify or alter malaria control efforts.
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