Cysteine Protease Function in Trypanosoma brucei
Treatment of Trypanosoma brucei with cysteine protease inhibitors demonstrated that clan CA cysteine protease activity was essential for parasite viability in culture and infection in mice. However, the identity and biological function of the essential protease or proteases targeted by these inhibitors was not known. Biochemical, molecular biology, and proteomics techniques were used to identify potential targets of inhibition and to investigate the biological function of cysteine proteases in T. brucei.
A search of the T. brucei genome revealed two cysteine proteases: the previously characterized cathepsin L-like cysteine protease, rhodesain, and a newly-identified cathepsin B-like cysteine protease, tbcatB. Rhodesain is expressed at high levels in procyclic and bloodstream form parasites. TbcatB was found to be expressed at low levels relative to rhodesain, but was more abundant in bloodstream form parasites. RNA interference (RNAi) of rhodesain in cultured parasites did not produce a phenotype. In contrast, a modest decrease in tbcatB mRNA and protein caused by RNAi produced a marked phenotype, with swollen endosomes, accumulation of fluorescein isothiocyanate-transferrin, and a block in cytokinesis, resulting in death. Induction of RNAi against tbcatB in a mouse model of infection confirmed that tbcatB is essential for parasite survival in vivo. The in vivo induction of RNAi against rhodesain did not cure infection, although it extended the survival of five out of ten mice beyond 60 days post-infection. TbcatB-deficiency produced by either TBCATB heterozygosity or RNAi caused morphological abnormalities similar to those seen with cysteine protease inhibitors. TbcatB-deficient parasites exhibited swollen flagellar pockets and accumulated unprocessed or undegraded host and parasite proteins, including transferrin, p67, and rhodesain. Expression and biochemical purification of active, recombinant tbcatB from Pichia pastoris allowed for the determination of the P1-P4 substrate specificity profile. Both results of transferrin cleavage assays and predictions of host substrates based on substrate specificity support the hypothesis that a key function for tbcatB is the degradation of host transferrin. Transferrin receptor expression is specifically upregulated in tbcatB-deficient parasites, consistent with iron starvation.
TbcatB is therefore critical to the survival of T. brucei and a logical drug target for the development of new anti-trypanosomal chemotherapy.