African trypanosomes (Trypanosoma brucei ssp.) are parasitic protozoa that cause human African trypanosomiasis (HAT, sleeping sickness) and nagana in livestock. These diseases have devastating impact throughout Africa where the tsetse fly vector is found. ~60 million people in 36 countries are at risk of tsetse bite and transmission, consequently HAT is considered to be a great neglected tropical disease. Few drugs are available, the best of which (eflornithine) is costly and requires a prolonged regimen, and the worst (melarsoprol) kills up to 10% of recipients. Infection is inevitably fatal without treatment, and since vaccination is not an option there is a critical need for new drug development. Thus a better understanding of the basic parasite biology is essential, particularly of aspects amenable to therapeutics. One such area is the lysosome because it impacts the host-pathogen balance in multiple ways. Expression of lysosomal activities is differentially regulated during the life cycle [1], and there are stage specific differences in the trafficking of essential lysosomal components [2]. The lysosome is the final repository of endocytic cargo acquired from the host for nutrition [3], as well as for lytic immune complexes removed from the cell surface [4]. Release of lysosomal proteases is a factor in the signature event of human infection, penetration of the central nervous system [5]. Lysosomal physiology is also critical to the activity of an innate human serum resistance trait, trypanolytic factor, which limits the host range of Trypanosoma species [6]. And finally, lysosomal hydrolytic activities have drawn considerable attention as potential chemotherapeutic targets [7]. However, only three lysosomal components have been characterized in T. brucei, p67 an essential membrane glycoprotein [6, 8], the a cathepsin L orthologue, TbCatL [1, 9], and a cathepsin B orthologue, TbCatB [10]. Mammalian lysosomes contain ~75 proteins that have been validated by biochemical and/or proteomic analyses, and of these ~half are associated with human genetic in-born errors in metabolism (also known as lysosomal storage diseases) [11]. Of the mammalian total only 11 obvious orthologues (~1/7th) can be identified in the T. brucei genome (treating the multi-subunit vacuolar protein pump as a single entity), despite the fact that biochemical analyses indicate many of these activities should be present. One would predict based on the percent of all genes in the genome for which function is known or can predicted by homology (~1/3rd) that this number would be much higher. The overarching goal of this proposal is to rectify this situation by direct proteomic analysis. Given the circumstances this is the only approach to filling the gaps that cannot be filled by standard genomic approaches. The uber-rationale is that these proteins will provide ample opportunities for subsequent drug development, and for further basic studies of lysosomal biogenesis. This rationale is amply supported by the critical functions of the lysosome in trypanosome biology, and the frequency that human disease is associated with lysosomal deficiencies.

Public Health Relevance

African trypanosomes (Trypanosoma brucei ssp.) are parasitic protozoa that cause human African trypanosomiasis (HAT, sleeping sickness), as well as nagana in cattle and other livestock. Current drugs are toxic and vaccination is not an option, consequently new chemotherapeutic targets are needed. We propose a protoeomic analysis of purified lysosomes with the rationale that identification of critical parasite-specific enzymes will provide unique opportunities for subsequent parasite-specific drug development.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Pathogenic Eukaryotes Study Section (PTHE)
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Mcgugan, Glen C
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University of Wisconsin Madison
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