What do kinetoplastids need a kinetoplast for? Life cycle progression of Trypanosoma brucei in the presence and absence of mitochondrial DNA
Item statusRestricted Access
Embargo end date17//2/28/0
Dewar, Caroline E.
The parasitic protist Trypanosoma brucei is the causative agent of human African trypanosomiasis. The parasite undergoes a complex life cycle involving stages within the mammalian bloodstream and its tsetse fly vector. The fundamental differences between energy metabolism in the procyclic insect form (PCF) and long slender bloodstream form (BSF) T. brucei involve a switch in the directionality of the F1Fo- ATPase. In PCF, the need for oxidative phosphorylation in low glucose conditions requires the enzyme to generate ATP. In the slender BSF, the enzyme uses ATP from glycolysis to drive proton pumping to maintain the essential mitochondrial membrane potential. Fo-ATPase subunit 6 (A6) is critical for proton translocation in either direction and is encoded in the mitochondrial DNA (kDNA). The parasite’s kDNA is therefore essential in the slender BSF, and also in PCF where it encodes multiple subunits of the respiratory chain complexes that constitute the oxidative phosphorylation pathway. Specific point mutations in the nuclearly encoded γ subunit of the mitochondrial F1Fo-ATPase allow survival in the absence of kDNA in the slender BSF T. brucei (Dean et al., 2013). These mutations, even in the heterozygous genotype, cause an increase in resistance to multiple drugs in vitro (Gould and Schnaufer, 2014). This thesis investigates two questions: (1) What is the molecular mechanism of compensation for kDNA loss? (2) Are kDNA and a functional FoF1-ATPase required for life cycle progression? Slender BSF T. brucei were generated expressing ATPase L262Pγ. The effects of this γ mutation and kDNA loss, respectively, on structure/function of the F1Fo- ATPase were probed. Cells expressing L262Pγ show decreased sensitivity to Fo inhibitor oligomycin compared to WT cells, suggesting that the L262Pγ mutation functionally uncouples the enzyme. The impact of the L262Pγ mutation on the structure of the enzyme was probed by high resolution clear native electrophoresis. This shows there are dramatic consequences to F1Fo structure in the presence of the L262Pγ mutation. The apparent selection for cells that no longer express intact F1Fo suggests that L262Pγ uncouples the enzyme, resulting in a lethal proton leak. Pleomorphic T. brucei with and without kDNA were also generated by expressing mutant γ in strain AnTat1.1 90:13. Differentiation studies demonstrate kDNA0 cells can differentiate to insect-transmissible stumpy forms. These cells show a decreased lifespan, suggesting a critical role for a kDNA-encoded product in the stumpy form. Tsetse fly infections show kDNA is indispensable for progression to the PCF. Unexpectedly, parasites homozygous for L262Pγ can establish a midgut infection, while they do not infect the salivary glands. Heterozygous parasites, on the other hand, can form animal-transmissible metacyclics in the salivary glands, providing a potential mechanism for spreading decreased sensitivity to multiple drugs.