RNA Interference of Trypanosoma brucei Cathepsin B and L Affects Disease Progression in a Mouse Model

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Abstract
We investigated the roles played by the cysteine proteases cathepsin B and cathepsin L (brucipain) in the pathogenesis of Trypansoma brucei brucei in both an in vivo mouse model and an in vitro model of the blood–brain barrier. Doxycycline induction of RNAi targeting cathepsin B led to parasite clearance from the bloodstream and prevent a lethal infection in the mice. In contrast, all mice infected with T. brucei containing the uninduced Trypanosoma brucei cathepsin B (TbCatB) RNA construct died by day 13. Induction of RNAi against brucipain did not cure mice from infection; however, 50% of these mice survived 60 days longer than uninduced controls. The ability of T. b. brucei to cross an in vitro model of the human blood–brain barrier was also reduced by brucipain RNAi induction. Taken together, the data suggest that while TbCatB is the more likely target for the development of new chemotherapy, a possible role for brucipain is in facilitating parasite entry into the brain. African trypanosomiasis, or sleeping sickness, is caused by the single-cell parasite Trypanosoma brucei (T. brucei). Two parasite-derived enzyme proteins have been hypothesized to play an important role in the viability of the parasite or its ability to produce disease in the human host. Utilizing RNA interference that blocks the production of these proteins in the parasite, we show that elimination of parasite cathepsin B cures infection in mice. RNAi of the second enzyme protein, brucipain, results in the prolongation of life of half the infected mice, but does not cure. Further experiments carried out in a culture system show that brucipain facilitates the migration of parasites across a model of the blood–brain barrier. This suggests that while brucipain is not necessary for the viability of the organisms, it may play a role in infection by allowing parasites to reach the central nervous system and produce the severe second stage of sleeping sickness.