CHARACTERIZATION OF TRYPANOSOMA BRUCEI GP63 PROTEIN

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Trypanosoma brucei, the causative agent of African trypanosomiasis, multiplies extracellularly in the bloodstream of its mammalian hosts but is resistant to complement mediated lysis (CML). The objective of this proposed research is to determine the role of T. brucei GP63 (Tb-GP63) genes in resistance to CML. Tb-GP63 is a homolog of leishmania GP63 (L- GP63), a cell surface zinc metalloprotease that is critical for the resistance of leishmania to CML. Two groups of Tb-GP63 genes have been identified A and B. which are approximately 40 % identical and 60 % similar to L- GP63 and to each other. All disulfide bonded cysteines in L-GP63 and nine prolines are conserved in Tb-GP63-A and -B, suggesting that the Tb-GP63 proteins will be structurally similar to L-GP63. To study the role of Tb- GP63 in the resistance of T. brucei to CML Tb-GP63 will expressed in a baculovirus expression system and purified antibodies will be generated against Th-GP63 and cell lines that over-express or that lack Tb-GP63 will be created. With these reagents. the following questions will be addressed: (i) is Tb-Gp63-A is a functional cell surface protease? (ii) is Tb-GP63-B also a functional GP63 homolog? (iii) does Tb-GP63 increase the resistance cells to CML? (iv) is Tb-GP63 is required for growth of T. brucei in mice? and (v) do the Tb-GP63 gene 3' UTRs regulate stage specific expression of Tb-GP63? These studies may identify a new target for anti-trypanosome drugs and will provide insight into the molecular basis for the success of T. brucei as a parasite.