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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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Survival analysis for mice infected with T. b. brucei.(A) Kaplan-Meier survival analysis for mice infected with T. b. brucei not transfected with the RNAi construct but given an inducing dose of doxycycline (red) versus standard food and water (blue) (n = 5 in each group). (B) Infection with trypanosomes containing the RNAi plasmid for brucipain plus or minus induction by doxycycline. Brucipain RNAi resulted in prolonged survival of three out of the five mice (p = 0.004) (experiment was conducted twice with the same result). (C) Infection with parasite containing RNAi plasmid for TbCatB plus or minus induction by doxycycline. Note that all five mice infected with parasites in which cathepsin B RNAi was induced survived until the experiment was terminated. (D) Spleen weights in mice infected with cathepsin B RNAi parasites and induced with doxycyline were within normal range compared to uninfected controls.
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pntd-0000298-g001: Survival analysis for mice infected with T. b. brucei.(A) Kaplan-Meier survival analysis for mice infected with T. b. brucei not transfected with the RNAi construct but given an inducing dose of doxycycline (red) versus standard food and water (blue) (n = 5 in each group). (B) Infection with trypanosomes containing the RNAi plasmid for brucipain plus or minus induction by doxycycline. Brucipain RNAi resulted in prolonged survival of three out of the five mice (p = 0.004) (experiment was conducted twice with the same result). (C) Infection with parasite containing RNAi plasmid for TbCatB plus or minus induction by doxycycline. Note that all five mice infected with parasites in which cathepsin B RNAi was induced survived until the experiment was terminated. (D) Spleen weights in mice infected with cathepsin B RNAi parasites and induced with doxycyline were within normal range compared to uninfected controls.

Mentions: 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 [16]. 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 [2],[14]. 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).


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)

Survival analysis for mice infected with T. b. brucei.(A) Kaplan-Meier survival analysis for mice infected with T. b. brucei not transfected with the RNAi construct but given an inducing dose of doxycycline (red) versus standard food and water (blue) (n = 5 in each group). (B) Infection with trypanosomes containing the RNAi plasmid for brucipain plus or minus induction by doxycycline. Brucipain RNAi resulted in prolonged survival of three out of the five mice (p = 0.004) (experiment was conducted twice with the same result). (C) Infection with parasite containing RNAi plasmid for TbCatB plus or minus induction by doxycycline. Note that all five mice infected with parasites in which cathepsin B RNAi was induced survived until the experiment was terminated. (D) Spleen weights in mice infected with cathepsin B RNAi parasites and induced with doxycyline were within normal range compared to uninfected controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553486&req=5

pntd-0000298-g001: Survival analysis for mice infected with T. b. brucei.(A) Kaplan-Meier survival analysis for mice infected with T. b. brucei not transfected with the RNAi construct but given an inducing dose of doxycycline (red) versus standard food and water (blue) (n = 5 in each group). (B) Infection with trypanosomes containing the RNAi plasmid for brucipain plus or minus induction by doxycycline. Brucipain RNAi resulted in prolonged survival of three out of the five mice (p = 0.004) (experiment was conducted twice with the same result). (C) Infection with parasite containing RNAi plasmid for TbCatB plus or minus induction by doxycycline. Note that all five mice infected with parasites in which cathepsin B RNAi was induced survived until the experiment was terminated. (D) Spleen weights in mice infected with cathepsin B RNAi parasites and induced with doxycyline were within normal range compared to uninfected controls.
Mentions: 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 [16]. 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 [2],[14]. 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).

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