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RNA interference of Trypanosoma brucei cathepsin B and L affects disease progression in a mouse model.

Abdulla MH, O'Brien T, Mackey ZB, Sajid M, Grab DJ, McKerrow JH - PLoS Negl Trop Dis (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Sandler Center for Basic Research in Parasitic Diseases, California Institute for Quantitative Biomedical Research, University of California San Francisco, San Francisco, California, USA. maha.abdulla@ucsf.edu

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.

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Parasite traversal across human BMEC.Transwell inserts containing human BMEC (initial TEER = 26.3 Ω) were incubated with 3×105 pZJMTbRho RNAi trypanosomes (+/−tetracycline) and the number of parasites that crossed the BMEC monolayers into the bottom wells determined. All values represent the mean±SEM of triplicate determinations.
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pntd-0000298-g004: Parasite traversal across human BMEC.Transwell inserts containing human BMEC (initial TEER = 26.3 Ω) were incubated with 3×105 pZJMTbRho RNAi trypanosomes (+/−tetracycline) and the number of parasites that crossed the BMEC monolayers into the bottom wells determined. All values represent the mean±SEM of triplicate determinations.

Mentions: While the residual brucipain activity seen after RNAi induction might be responsible for disease progression in two of the mice shown in (Fig. 1B), an alternative conclusion is that brucipain plays a specific role in Trypanosoma pathogenesis in vivo, but not in parasite viability per se. Nikolskaia et al. [5] showed that a cysteine protease inhibitor, known to target brucipain, blocked the ability of African trypanosomes to cross a model of the blood–brain barrier (BBB) [5]. Using this in vitro model of the blood–brain barrier, we confirmed that brucipain is required for African trypanosomes to effectively cross the brain endothelial barriers. Without tetracycline 3.54E+04±1.41E+03 (mean±SEM) of the initial brucipain RNAi trypanosome (pZJMTbRho–tet) inoculum crossed the endothelial cell barrier (1–2%) (Fig. 4). This is comparable to those noted for T. b. brucei 427 and TREU 927 in previously published reports [5],[11]. However when brucpain RNAi was induced by tetracycline, the number of parasites migrating across the barrier was reduced by 50% (1.10E+03±6.35E+02), (p = 0.003). The human BMEC transendothelial electrical resistance (TEER) at the end of the experiment was 30.4±1.2 ohms (p = 0.00002), indicating that barrier integrity was maintained for all T. b. brucei treatment conditions. To rule out any effect of tetracycline on the in vitro BBB model other than induces RNAi, trypanosomes (pZJMTbRho) were pretreated with tetracycline, but the antibiotic was then removed and the parasites incubated with human BMEC overnight. The number of parasites crossing the BMEC was the same as control (with tetracycline), demonstrating that tetracycline has no effect on endothelial cells (data not shown). Experiments were repeated twice with the same result. In summary, the data show that knockdown of brucipain transcripts by RNAi led to reduced protease activity but no effect on parasitemia or splenomegaly. However the prolonged survival of some of the infected mice might be due to inability of the parasite to efficiently enter the CNS.


RNA interference of Trypanosoma brucei cathepsin B and L affects disease progression in a mouse model.

Abdulla MH, O'Brien T, Mackey ZB, Sajid M, Grab DJ, McKerrow JH - PLoS Negl Trop Dis (2008)

Parasite traversal across human BMEC.Transwell inserts containing human BMEC (initial TEER = 26.3 Ω) were incubated with 3×105 pZJMTbRho RNAi trypanosomes (+/−tetracycline) and the number of parasites that crossed the BMEC monolayers into the bottom wells determined. All values represent the mean±SEM of triplicate determinations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553486&req=5

pntd-0000298-g004: Parasite traversal across human BMEC.Transwell inserts containing human BMEC (initial TEER = 26.3 Ω) were incubated with 3×105 pZJMTbRho RNAi trypanosomes (+/−tetracycline) and the number of parasites that crossed the BMEC monolayers into the bottom wells determined. All values represent the mean±SEM of triplicate determinations.
Mentions: While the residual brucipain activity seen after RNAi induction might be responsible for disease progression in two of the mice shown in (Fig. 1B), an alternative conclusion is that brucipain plays a specific role in Trypanosoma pathogenesis in vivo, but not in parasite viability per se. Nikolskaia et al. [5] showed that a cysteine protease inhibitor, known to target brucipain, blocked the ability of African trypanosomes to cross a model of the blood–brain barrier (BBB) [5]. Using this in vitro model of the blood–brain barrier, we confirmed that brucipain is required for African trypanosomes to effectively cross the brain endothelial barriers. Without tetracycline 3.54E+04±1.41E+03 (mean±SEM) of the initial brucipain RNAi trypanosome (pZJMTbRho–tet) inoculum crossed the endothelial cell barrier (1–2%) (Fig. 4). This is comparable to those noted for T. b. brucei 427 and TREU 927 in previously published reports [5],[11]. However when brucpain RNAi was induced by tetracycline, the number of parasites migrating across the barrier was reduced by 50% (1.10E+03±6.35E+02), (p = 0.003). The human BMEC transendothelial electrical resistance (TEER) at the end of the experiment was 30.4±1.2 ohms (p = 0.00002), indicating that barrier integrity was maintained for all T. b. brucei treatment conditions. To rule out any effect of tetracycline on the in vitro BBB model other than induces RNAi, trypanosomes (pZJMTbRho) were pretreated with tetracycline, but the antibiotic was then removed and the parasites incubated with human BMEC overnight. The number of parasites crossing the BMEC was the same as control (with tetracycline), demonstrating that tetracycline has no effect on endothelial cells (data not shown). Experiments were repeated twice with the same result. In summary, the data show that knockdown of brucipain transcripts by RNAi led to reduced protease activity but no effect on parasitemia or splenomegaly. However the prolonged survival of some of the infected mice might be due to inability of the parasite to efficiently enter the CNS.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Sandler Center for Basic Research in Parasitic Diseases, California Institute for Quantitative Biomedical Research, University of California San Francisco, San Francisco, California, USA. maha.abdulla@ucsf.edu

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.

Show MeSH
Related in: MedlinePlus