Eukaryotic Cell doi:10.1128/EC.00405-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Molecular and biochemical characterization of a cathepsin B-like protease family unique to Trypanosoma congolense
Carlos Mendoza Palomares,
Nicolas Biteau,
Christiane Giroud,
Virginie Coustou,
Theresa Coetzer,
Edith Authié,
Alain Boulangé,
and
Théo Baltz*
Université Victor Segalen Bordeaux 2, Laboratoire de Microbiologie Cellulaire et Moléculaire et Pathogénicité, CNRS, UMR-5234, 33076 Bordeaux, France; CIRAD, School of Biochemistry, University of KwaZulu-Natal (Pietermaritzburg campus), Scottsville 3209, South Africa; INRA UR1282, Infectiologie Animale et Santé Publique, Centre de recherche de Tours, Nouzilly, 37380, France
* To whom correspondence should be addressed. Email:
theo.baltz{at}parasitmol.u-bordeaux2.fr.
 |
Abstract |
|---|
Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive anti-disease vaccine candidate in 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 compared to one or two highly homologous genes in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1-5 and TcoCBc6, which possess the classical catalytic triad (Cys,His,Asn) and TcoCBs7-13, which contain an unusual catalytic site (Ser,Xaa,Asn). Expression profiles showed that members the TcoCBc1-5 and the TcoCBs7-13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. Expression of recombinant representatives of each group (TcoCB1,6,12) as pro-enzymes showed that TcoCBc1 and TcoCBc6 are able to auto-catalyze their maturation 21 and 31 residues upstream of the predicted start of the catalytic domain, respectively. Both displayed a carboxydipeptidase function while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not auto-mature in vitro and the pepsin-matured enzyme was inactive when tested 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 bloodstream form survival. 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.
