Characterization of Alanine Aminotransferase and Transaldolase in T. brucei

T. brucei is the causative agent of sleeping sickness in humans and nagana in cattle. The parasite is transmitted by the tsetse fly, which is endemic in many areas of Africa. Considerable metabolic changes occur between the two life cycle stages, the procyclic fly form (PCF) and the mammalian bloodstream form (BSF). This work focuses on two enzymes of T. brucei (1) Transaldolase (TbTA) is part of the pentose phosphate pathway. The pathway is complete in the PCFS, but show abnormalities in the BSF. In particular, the enzymes preceding and succeeding the TbTA reaction are not detectable in BSF. This peculiarity could mean that the enzyme has an altered function in BSFs. Preliminary characterization of the recombinant enzyme and RNA silencing was carried out. RNAi suppression led to a residual activity of ~40% in both life cycle forms. No change in phenotype was observed under these conditions. (2) The second enzyme studied was alanine aminotransferase (TbAAT) which links carbohydrate and amino acid metabolism. This cytosolic enzyme is expressed in the BSF with a reported specific activity of ~.5umol/min/mg, whereas in the PCFS it is expressed with a reported specific activity of ~.05umol/min/mg. The function of TbAAT is to convert the glycolyic end product pyruvate into alanine. Characterization of the recombinant protein was performed. RNA interference was carried out on both life forms. Incomplete knockdown of the enzyme led to residual activity of ~20% and no phenotype could be observed under normal growth conditions in both life cycle forms. In media with reduced fetal calf serum, the BSF exhibited a reduced growth rate. To probe these findings further, an inducible gene knockout was carried out in PCF and both alleles were ablated. The ablation of this gene had a sever growth effect, proving the gene was essential for PCF. In BSF, a combination of RNAi and single allele knockout was performed. The activity of TbAAT was lowered t

Identification and Characterization of an Unusual Class I Myosin Involved in Vesicle Traffic in Trypanosoma brucei

Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1) that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of ∼90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites