Mitochondrial metabolism in livestock Trypanosomes: acetate production in Trypanosoma congolense

Abstract

African Animal Trypanosomiasis (AAT) is a debilitating disease affecting livestock in sub-Saharan Africa. AAT is caused by protozoan parasites of the genus Trypanosoma, with T. congolense being the species primarily associated with chronic infections in cattle. Chemotherapy and chemoprophylaxis are the primary means of controlling this disease, however the lack of a detailed understanding of the metabolism of livestock trypanosomes (compared to the more intensively studied T. brucei) remains a significant barrier to the identification and development of adequate drug compounds. A comprehensive understanding of the parasites’ growth requirements in vitro is crucial to successful drug development. Recent data suggests that the various trypanosome species implicated in AAT may exhibit differences in metabolic activity. Identifying such differences is necessary in order to culture parasites in a stable, biologically relevant manner, which will allow for a rational, targeted approach to the development of chemotherapeutic compounds.

This thesis is an exploration of the metabolic characteristics of one livestock trypanosome species in particular, T. congolense. A recent study employing omics technologies has brought to light numerous interesting differences in the metabolism of this trypanosome species compared to T. brucei, particularly with respect to mitochondrial metabolism. For instance, metabolomics and transcriptomics datasets suggest that the generation of acetate (a mitochondrially-produced end-product of glucose degradation) may be much more highly active in T. congolense compared to T. brucei. The production of acetate was therefore chosen as a starting point for the work outlined in this thesis. First, the production and release of this molecule was compared in T. congolense and T. brucei. The carbon sources which contribute to its synthesis were then interrogated using nuclear magnetic resonance spectroscopy (NMR). These data added a degree of functional validation to previously published omics data, but also provided an unexpected finding: pyruvate, as well as glucose and L-threonine is taken up in large amounts and converted to acetate.

Pyruvate has traditionally been considered the primary waste product of bloodstream form trypanosomes, therefore its uptake and degradation was chosen as a focus for further experiments. Based on previously published studies involving the procyclic form of T. brucei, it was hypothesised that in T. congolense the uptake and degradation of pyruvate may be linked to the generation of cellular ATP. A simple assay was developed in order to test this hypothesis. The results showed that supplementation of pyruvate results in ATP production and motility in BSF T. congolense (but not BSF T. brucei) even in the complete absence of glucose – a major difference between the two species. Further iterations of this experiment were then performed, and showed that ATP could also be produced using malic acid as a substrate, and that the utilisation of these two carbon sources in the absence of glucose was entirely dependent on buffer composition.

Using recently developed genetic methods, the enzyme pyruvate dehydrogenase (PDH) was then repressed in T. congolense by generating an RNA interference (RNAi) cell line. These RNAi experiments demonstrated that PDH is an essential gene in T. congolense, and that repressing its expression results in sustained decreases in growth, as well as reductions in cellular ATP. The metabolic consequences of PDHE2 RNAi were further explored using a combination of NMR and liquid chromatography mass spectrometry (LCMS). Results from these experiments showed increases in glycolytic intermediates such as phosphoenolpyruvate, as well as significant increases in a metabolite putatively identified as lipoic acid, implying that the repression of PDH may have broad metabolic implications. In combination, these data strengthen existing observations that BSF T. congolense exhibit metabolic activities that are distinct from T. brucei. The emerging picture is of an approach to carbon source utilisation that is more flexible than originally thought, where glucose intermediaries can be taken up and re-used for energy generation. These findings are therefore of importance to the on-going refinement of relevant culturing methods for livestock trypanosomes.