Molecular & Biochemical Parasitology 182 (2012) 7–16 Contents lists available at SciVerse ScienceDirect Molecular & Biochemical Parasitology Trypanosoma brucei brucei oligopeptidase B null mutants display increased prolyl oligopeptidase-like activity Richard T. Kangethe a , Alain F.V. Boulangé a,b,1 , Virginie Coustou c , Théo Baltz c , Theresa H.T. Coetzer a,∗ a School of Biochemistry, Genetics and Microbiology, University of KwaZulu-Natal (Pietermaritzburg campus), Private Bag X01, Scottsville 3209, South Africa b CIRAD, UMR Intertryp, F-34398 Montpellier, France c UMR-CNRS 5234 Microbiologie Cellulaire et Moléculaire et Pathogénicité, University Victor Segalen, Bordeaux 2, Bât 3A, 1 er étage, 146, Rue Léo Saignat, F-33076 Bordeaux, France a r t i c l e i n f o Article history: Received 23 June 2011 Received in revised form 7 October 2011 Accepted 14 November 2011 Available online 22 November 2011 Keywords: Trypanosoma brucei brucei Oligopeptidase B opb null mutants Prolyl oligopeptidase a b s t r a c t African trypanosomosis is a parasitic disease in man and animals caused by protozoan parasites of the genus Trypanosoma. Nagana, the cattle form of the disease, is caused by Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei brucei. An option for developing vaccines and chemother- apeutic agents against trypanosomosis is to target pathogenic factors released by the parasite during infection, namely an “anti-disease” approach. One such pathogenic factor is oligopeptidase B (TbOPB), a trypanosome peptidase that hydrolyses Arg/Lys containing peptides smaller than 30 amino acid residues and is suspected to be involved in the hormonal deregulation associated with the disease. To better under- stand the role TbOPB plays in parasite physiology and host pathogenesis, oligopeptidase B null mutant parasites (opb) were generated in the T. b. brucei Lister 427 strain. opb Trypanosoma brucei parasites grew at a significantly faster rate in vitro, and were as virulent as wild type strains during infection in mice. Immunohistopatholgy of infected mouse testes revealed opb parasites in extra vascular regions showing that TbOPB is not involved in assisting T. brucei parasites to cross microvascular endothelial cells. Gelatine gel analysis of opb null mutants showed an increase in discrete cysteine peptidase activ- ities when compared to wild type strains. Enzymatic activity assays were carried out to identify how closely related oligopeptidases are affected by TbOPB gene deletion. A significant increase of T. brucei prolyl oligopeptidase (TbPOP) activity was observed, but no concomitant increase in TbPOP protein lev- els, suggesting that a POP-like enzyme might compensate for a loss in OPB activity in opb null mutants. © 2011 Elsevier B.V. All rights reserved. 1. Introduction African trypanosomosis, also referred to as sleeping sickness in humans and nagana in cattle, is a parasitic disease caused by protozoan haemoparasites of the genus Trypanosoma, with Trypanosoma congolense, Trypanosoma vivax and Trypanosoma bru- cei causing nagana [1]. Nagana puts large numbers of cattle in Abbreviations: AEBSF, 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochlo- ride; AMC, aminomethylcoumarin; E-64, trans-Epoxysucciny-L-leucyl-amido(4- guanidino) butane; IgG, immunoglobulin G; IgY, immunoglobulin Y; MES, 2-(N-morpholino)ethanesulfonic acid; 4MNA, 4-methoxy-beta-naphthylamide; pGLbla, blasticidin resistance plasmid; pGLneo, neomycin resistance plasmid; SBTI, soybean trypsin inhibitor; TbOPB, T. brucei oligopeptidase B; TbPOP, T. brucei prolyl oligopeptidase; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; Z, benzyloxy- carbonyl. ∗ Corresponding author. Tel.: +27 33 260 5463; fax: +27 33 260 6127. E-mail address: coetzer@ukzn.ac.za (T.H.T. Coetzer). 1 Present address: Centro de Biotecnologia, Universidade Eduardo Mondlane, Maputo, Mozambique. sub-Saharan Africa at risk with an estimated revenue loss of up to five billion US $ annually [2–5]. Symptoms include anaemia, prolif- eration of lymphoid cells, increased permeability of blood vessels, disseminated coagulation and immunosuppression [6]. Many of these symptoms appear as the invading parasites are cleared by the host immune system and most are attributed to parasite products released by dead and dying parasites [2]. These products include trypanosome lipids, haemolysins, mitogens, inflammatory factors, hepatotoxins and enzymes such as peptidases, phospholipases and acid phosphatases [7,8]. This observation has led to the idea of developing vaccines and chemotherapeutic agents that target pathogenic factors released by the parasite during infection, a strat- egy referred to as an “anti-disease” approach [9]. Several enzymes have been identified as possible pathogenic factors, with oligopep- tidase B (TbOPB) and prolyl oligopeptidase (TbPOP) implicated in neuroendocrine symptoms during T. brucei infection [10–13]. Both TbPOP and TbOPB belong to the S9 prolyl oligopeptidase family of clan SC serine proteases but cleave peptides differently with TbPOP hydrolysing substrates at the carboxyl end of proline and ala- nine residues [10]. TbOPB on the other hand only hydrolyses low 0166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2011.11.007