Human African Trypanosomiasis (HAT, sleeping sickness) is a neglected but fatal vector bone disease caused by Trypanosoma brucei ssp. (T. brucei). Sixty million people are at risk of infection with HAT, and 50,000 new cases and an equal number of deaths are reported annually in sub-Saharan Africa. Currently, available anti-trypanosomals are extremely toxic, and often cause post-treatment encephalopathy in treated patients. The lack of suitable drugs for treating Trypanosomiasis is due to deficiencies in the understanding of molecular and physiological mechanisms utilized by parasites to maintain homeostasis and proliferate. Ideal anti-trypanosomal agents will target vital physiological processes and/or non-variant parasite-derived pathogenic molecules without adversely affecting the human host. Many studies indicate that Ca 2+ pumps (Ca2+ ATPases) are involved in Ca2+ translocation, signal transduction, proliferation, cation homeostasis and apoptosis in pathogenic eukaryotes and are potential targets for cation pump inhibitor therapy. Cytosolic calcium ion concentration [Ca2+]i in blood stages of T. brucei is 4-10 orders of magnitude below that encountered in extracellular millieu. Therefore, parasites require effective Ca2+ pumps to maintain [Ca2+]i homeostasis, survival, and proliferation. We have identified, cloned, partially sequenced, and generated antibodies of two plasma- membrane-like calcium ATPases (TBCA1 and TBCA2) that are significantly up regulated in blood stages of T. brucei but not in culture procyclics (""""""""insect stage""""""""). Interestingly, treatment of procyclic T. brucei with commercially available Ca2+ pump inhibitors such as ortho-Vanadate and Thapsigargin were able to inhibit parasite proliferation in vitro. These findings indicate that these pumps are essential for parasite proliferation and support our hypothesis that TBCA1 and TBCA2 expression are required to promote successful transition from the insect vector (Glossina sp) to mammalian host blood. A corollary to this hypothesis is that inhibition of TBCA1 and TBCA2 by RNA interference strategies and blocking by antibodies, will prevent T. brucei development and proliferation. We plan to use these probes in conjunction with new technologies, including bioinformatics, gene and protein expression analysis, RNA inhibition assays, as well as immunohistology, to characterize and determine the functional role of these important Ca2+ pumps in T. brucei survival and proliferation.
Our aims are to: (a) structurally characterize TBCA1 and TBCA2, (b) evaluate TBCA1 and TBCA2 expression, regulation, and localization, and (c) investigate the functional role of TBCA1 and TBCA2 in T. brucei at different stages of development. Our long-term goal is to develop reagents such as small molecule drugs and/or immunotherapeutics capable of inhibiting the essential Ca2+ pumps and T. brucei development. The success of this effort has major implications for understanding Ca2+ pumps in T. brucei infection and pathogenesis of this important disease.
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