ABSTRACT. African trypanosome species are causative agents for sleeping sickness in humans and nagana disease in cattle. Trypanosoma brucei can generate ATP via a reverse reaction with glycerol kinase (GK) when alternative oxidase (AOX) is inhibited; thus, GK is considered to be a crucial target for chemotherapy combined with AOX. However, the energy metabolism systems of African trypanosome species other than T. brucei are poorly understood. Thus, GK genes were surveyed from genome databases and cloned by PCR from T. vivax and T. congolense. Then, recombinant GK proteins (rGK) of T. vivax, T. congolense and T. brucei were expressed and purified. Kinetic analysis of these rGK proteins revealed that the K m values of T. congolense rGK for ADP and G-3-P substrates were lower than those of T. vivax and T. brucei. The expression level of GK molecules was highest in T. congolense cells and lowest in T. vivax cells. Based on these results, effective combination dosages of ascofuranone, a specific inhibitor of AOX, and glycerol, an inhibitor of the GK reverse reaction, were determined by using in vitro-cultured trypanosome cells.

Flagellum-mediated motility of Trypanosoma brucei is considered to be essential for the parasite to complete stage development in the tsetse fly vector, while the mechanism by which flagellum-mediated motility is controlled are not fully understood. We thus compared T. brucei whole gene products (amino acid sequence) with Caenorhabditis elegans UNC (uncoordinated) proteins, in order to find uncharacterized motility-related T. brucei genes. Through in silico analysis, we found 88 gene products which were highly similar to C. elegans UNC proteins and categorized them as TbCEUN (T. brucei gene products which have high similarity to C. elegans UNC proteins). Approximately two thirds of the 88 TbCEUN gene products were kinesin-related molecules. A gene product highly similar to C. elegans UNC119 protein was designated as TbUNC119. RNAi-mediated depletion of TbUNC119 showed no apparent phenotype. However, knock-down analysis of both TbUNC119 and its binding protein (TbUNC119BP) which was found by yeast two-hybrid analysis showed characteristic phenotypes, including reduced motility, morphological change (extended cell shape), and cellular apoptosis. Based on the observed phenotypes, possible function of the TbUNC119 and TbUNC119BP is discussed.