Origins and consequences of kinetoplast loss in trypanosomes

Abstract

The kinetoplast is the large mitochondrial genome present in the eponymous Kinetoplastida. Trypanosoma brucei is an African trypanosome that can lose kinetoplast DNA (kDNA), however, when the nuclear-encoded gamma subunit of the mitochondrial F1FO-ATP synthase ({gamma}ATPase) is mutated. These mutations, analogous to a broken camshaft at the core of the ATP synthase rotary motor, are associated with multidrug resistance, and correlated with tsetse-fly independent mechanical transmission, and geographical spread of these parasites beyond Africa. Here we engineer kinetoplast-independent T. brucei to explore origins and consequences of kDNA loss. We used oligo targeting to edit the native{gamma} ATPase gene, and selection with the ATP synthase targeting drug oligomycin to enrich the desired mutants. Using this approach, we identified novel M282F, M282W, and M282Y mutants, and subsequently generated precision-edited strains expressing the previously described L262P or A273P mutants, or the novel M282F mutant. Heterozygous M282F mutants retained sensitivity to the kDNA-targeting drug acriflavine, while homozygous M282F mutants were acriflavine resistant and readily tolerated acriflavine-induced kDNA loss. Proteomics analysis of the homozygous mutant pre-kDNA-loss revealed highly specific depletion of ATP synthase-associated proteins, but not the F1 subunits. Complete kDNA-loss in these cells was associated with substantial depletion of kDNA-binding proteins and mitochondrial RNA-processing factors. In contrast, mitochondrial membrane-associated transporters were increased in abundance. We conclude that T. brucei cells with a homozygous{gamma} ATPase M282F mutation assemble a remodelled ATP synthase and readily tolerate kDNA loss, which is accompanied by substantial remodelling of the mitochondrial proteome Author summaryMutations in the gamma subunit of the mitochondrial ATP synthase in parasitic African trypanosomes can have major consequences. Specifically, the entire large and complex mitochondrial genome, the kinetoplast, is rendered dispensable, and the cells become resistant to important kinetoplast-targeting drugs. Veterinary parasites with these mutations have also spread outside Africa through simple mechanical transmission, either sexually or by biting flies or vampire bats. We precision-edited the gamma subunit to replicate previously described mutants and identified a novel mutant that readily tolerated kinetoplast loss. Using quantitative proteomics, we demonstrated highly specific depletion of ATP synthase-associated proteins pre-kinetoplast-loss. We then use genome sequencing to show that the kinetoplast could be completely lost by these cells and demonstrated that cells lacking mitochondrial nucleic acids displayed specific depletion of mitochondrial nucleic acid-binding proteins. Notably, several mitochondrial membrane-associated transporter complexes were increased in abundance. Thus, we establish a method to test precise {gamma}ATPase mutations and to identify new mutations associated with kinetoplast loss. We also show that trypanosomes with a dispensable kinetoplast specifically remodel the ATP synthase pre-kinetoplast-loss and substantially remodel the mitochondrial proteome post-kinetoplast-loss.