Pathogenic African trypanosomes, the causative agent of African sleeping sickness, possess a unique subcellar organelle, the glycosome. This organelle, found only in members of the order Kinetoplastida (including the causative agents of Chagas disease and leishmaniasis) houses many of the enzymes of the Embden- Myerhof pathway of glycolysis. There is no vaccine against any of these diseases and chemotherapy is unfortunately toxic. A further understanding of the glycosomal organelle might lead to the development of targets for new chemotherapies. Glycosomal targeting signals and the regulation of glycosomal gene expression in Trypanosoma brucei will be studied. We have demonstrated translational control of expression of glycosomal phosphoglycerate kinase. The molecular mechanisms of this control will be analyzed. The work on gene regulation will be expanded to include a procyclic-specific glycosomal protein, malate dehydrogenase. The corresponding gene will be cloned, and the regulation of its expression investigated. Using an in vitro glycosomal protein import assay, we will characterize the signals on glycosomal proteins which are responsible for their proper localization in the cell. The requirements for signal function will be delineated both at the amino acid level and at the topographic level. By studying both phosphoglycerate kinase and malate dehydrogenase, hypotheses on the general nature of glycosomal targeting sequences may be formed and ultimately tested. The experiments proposed are designed to elucidate the molecular controls underlying the stage-specific phenotype and to provide an understanding of the properties of glycosomal targeting sequences. Insights into these crucial parameters of glycosomal biogenesis may identify unique aspects which may be selectively disrupted. This information would not only be useful in the specific case of Trypanosoma brucei, but may also provide clues for chemotherapy of diseases caused by Leishmania spp. and Trypanosoma cruzi.
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