Date Published: September 24, 2008
Publisher: Public Library of Science
Author(s): Maha-Hamadien Abdulla, Theresa O’Brien, Zachary B. Mackey, Mohamed Sajid, Dennis J. Grab, James H. McKerrow, Serap Aksoy
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.
Partial Text: Subspecies of Trypanosoma brucei are the causative agents of human African trypanosomiasis. In vitro studies utilizing both small molecule cysteine protease inhibitors and RNA interference (RNAi) have implicated the Clan CA (papain) family of cysteine proteases as critical to the successful lifecycle of Trypanosoma brucei brucei (T. b. brucei) ,. In vivo studies have demonstrated that cysteine protease inhibitors prolong the lives of mice infected with lethal inocula of trypanosomes ,. There are two distinct Clan CA cysteine proteases identified in the T. brucei genome. Brucipain (aka trypanopain-Tb, rhodesain) is a cathepsin L-like protease responsible for the bulk of protease activity in the organism . Trypanosoma brucei cathepsin B (TbCatB) is a more recently characterized protease that is upregulated in the bloodstream stage of the parasite . In in vitro studies, RNAi of TbCatB produced swelling of the endosome compartment analogous to that seen with class-specific cysteine protease inhibitors , and led to arrest of trypanosome replication and death. In contrast, knockdown of brucipain by RNAi in vitro produced no detectable phenotypic changes. However, it was hypothesized that this enzyme might play a role in the degradation of mistargeted glycosylphosphatidylinisotol (GPI) anchored proteins, VSG turnover, disruption of the blood–brain barrier, or degradation of host immunoglobulin , While RNAi with cultured parasites can provide important insights into the role of a specific gene product in parasite replication and viability, a role in pathogenesis, as proposed for brucipain, can only be validated in vivo. We show that introduction of RNAi from a tetracycline-inducible promoter can be achieved in vivo in a mouse model of T. b. brucei infection, and show that transcriptional silencing of either of these two proteases alters the course of T. b. brucei infection .
The goal of these experiments was to validate the in vitro effects of RNAi on TbcatB in an in vivo disease model of African trypanosomiasis, and to explore a potential role of brucipain as a virulence factor. For safety reasons we conducted the knockdown experiment in the human non-infective strain T. b. brucei which has been traditionally grown and studied in mice. Doxycycline by itself produced no significant alteration (+/−1 day) in the course of T. b. brucei 90-13 infections (Fig. 1A). Equivalent levels of parasitemia and splenomegaly were observed in mice whether or not they were maintained on a doxycycline-containing diet (not shown). The in vivo induction of RNAi against brucipain in T. b. brucei did not cure infection, but extended the survival of three out of five mice beyond 60 days (Fig. 1B) the experiment was repeated twice with the same result. All mice infected with trypanosomes having the brucipain transcript knockdown had parasitemia and splenomegaly equivalent to that seen in control mice at the time of their sacrifice (not shown). Splenomegaly (quantified by spleen weight) is a convenient gross pathological marker of disease burden . Analysis of mRNA levels in trypanosomes isolated from infected mice confirmed 60% reduction in the level of brucipain mRNA (Fig. 2A). The level of cathepsin B mRNA was not affected by RNAi induction against brucipain in pZJMTbRho induced parasites (Fig. 2B). Active site labeling of brucipain in trypanosomes purified from mouse blood confirmed 60% reduction in brucipain protease activity (Fig. 3C). Endogenous activity levels of brucipain and cathepsin B, quantified by DCG-04 labeling of purified parasites from mice infected with 90-13 strain, confirmed that brucipain was more abundant than cathepsin B (Fig. 3D), consistent with previously published data ,. A control cell line with an insert of GFP was generated to investigate the role of RNAi plasmid construct itself on the parasites in vivo. No difference was seen in mouse pathology or in brucipain or cathepsin B levels with GFP-induced parasites (data not shown).