Recombinant expression and biochemical characterisation of two alanyl aminopeptidases of Trypanosoma congolense.

Trypanosoma congolense is a haemoprotozoan parasite that causes African animal trypanosomosis, a wasting disease of cattle and small ruminants. Current control methods are unsatisfactory and no conventional vaccine exists due to antigenic variation. An anti-disease vaccine approach to control T. congolense has been proposed requiring the identification of parasitic factors that cause disease. Immunoprecipitation of T. congolense antigens using sera from infected trypanotolerant cattle allowed the identification of several immunogenic antigens including two M1 type aminopeptidases (APs). The two APs were cloned and expressed in Escherichia coli. As the APs were expressed as insoluble inclusion bodies it was necessary to develop a method for solubilisation and subsequent refolding to restore conformation and activity. The refolded APs both showed a distinct substrate preference for H-Ala-AMC, an optimum pH of 8.0, puromycin-sensitivity, inhibition by bestatin and amastatin, and cytoplasmic localisation. The two APs are expressed in procyclic metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro.

Trypanosoma brucei brucei oligopeptidase B null mutants display increased prolyl oligopeptidase-like activity.

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 chemotherapeutic 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 understand the role TbOPB plays in parasite physiology and host pathogenesis, oligopeptidase B null mutant parasites (Δopb) were generated in the T. b. brucei Lister 427 strain. Δopb 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 Δopb 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 Δopb null mutants showed an increase in discrete cysteine peptidase activities 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 levels, suggesting that a POP-like enzyme might compensate for a loss in OPB activity in Δopb null mutants.