: Malaria caused by Plasmodium falciparum affects 300-500million people globally and causes 1-2 million deaths annually. The asexual cycle is responsible for the inmune pathology and the clinical manifestations of malaria. Despite intense efforts, no vaccine has been devel.0ped against malaria and disease control is mainly through drugs and insecticide use against the mosquito vectors. Drug resistance by the parasite is on the rise and an immunological approach in the form of vaccines, focusing on specific parasite antigens remains the best approach for controlling the disease. We isolated rhoptries from segmented schizonts of P. yoelii and performed preliminary rhoptry proteome analysis using multidimensional protein identification technology (MudPIT). We identified 26 proteins enriched and unique to the merozoite rhoptries. Seven of the proteins are hypothetical proteins with no known homologies to proteins in the data bases, suggesting they are Plasmodium specific. The seven proteins possess C-terminal tyrosine rhoptry targeting motifs, signal peptides and transmembrane domains. The rationale for proteomic analysis of the rhoptries is that low abundance proteins, proteins with unusual modification, membrane proteins and regulatory proteins not usually identified by conventional screening methods can be identified. The goal of this study is to characterize the 7 novel rhoptry genes and to investigate their patterns of gene expression during the asexual stage. We will express the rhoptry gene products,"""""""" generate recombinant specific antibodies and verify the rhoptry origin of the proteins. Rhoptry gene expression will be evaluated using a custom-made high-density oligonucleotide microarray. The proposed study is part of a long-term research goal to understand the biological role of the rhoptries in asexual stage parasite development and in merozoite invasion. The novel rhoptry proteins might serve as candidates for a malaria vaccine or as drug targets. The following specific aims will serve as guidelines to the research: 1. We will express the novel rhoptry proteins from cDNAs, generate recombinant specific antibodies and characterize the antibodies. We will evaluate stage specific rhoptry gene expression using a custom-made high-density oligonucleotide microarray. 2) We will analyze rhoptries during schizont development by MudPIT analysis and evaluate the distribution of rhoptry proteins during rhoptry biogenesis. ? ?
