Molecular and Biochemical Characterization of a Cathepsin B-Like Protease Family Unique toTrypanosoma congolense

Abstract

Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant proTcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis.

Identification of Trans-Sialidases as a Common Mediator of Endothelial Cell Activation by African Trypanosomes

“…5A). TcoTS-A1, TcoTS-D2 and TvTS2 activated the BAE in a dose dependent manner, while BAE incubated with the recombinant cathepsin CB1 produced and purified according to the same protocol as the TS (control) [34] were not activated. Note that activation of the human and murine cell lines by these recombinant TS showed heterogeneity, similarly to the previous activation results by the African trypanosomes (Table S1).…”

“…The recombinant proteins TcoTS-A1, TcoTS-D2, TvTS2 and CB1 were previously obtained in the laboratory [21], [22], [34] …”

Congopain genes diverged to become specific to Savannah, Forest and Kilifi subgroups of Trypanosoma congolense, and are valuable for diagnosis, genotyping and phylogenetic inferences

“…and T. evansi and moderate diversity in T. vivax . Interestingly, analysis of cathepsin B genes from T. congolense IL3000 revealed 13 gene copies with unusual polymorphisms in contrast to the single-copy gene from other trypanosome species (Mendoza-Palomares et al, 2008).…”

The global cysteine peptidase landscape in parasites

“…The biological consequences of increased numbers of active enzymes have proven difficult to recognize or to explain [20], because gene expansions are not uniform among phylogenetic groups. Among the trypanosomatids, the T. brucei genome includes only two C1 peptidases (a cathepsin-L-like and a cathepsin-B-like peptidase) [23] (Figure S1 in the supplementary material online), one of which is necessary for the parasite to cross the blood–brain barrier of the host [24], whereas T. congolense has 13 cathepsin-B-like peptidases [25]. A third trypanosomatid, L. mexicana , expresses at least 19 cathepsin-L-like peptidases and a single cathepsin B [21]; as mentioned earlier, increased cysteine peptidase activity in comparison with L. major might be the cause of differing disease outcomes [18].…”