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Origins and consequences of kinetoplast loss in trypanosomes

ABSTRACT: The kinetoplast is the large mitochondrial genome present in the eponymous Kinetoplastida. African trypanosomes can lose their kinetoplast DNA (kDNA), however, when the nuclear-encoded gamma subunit of the mitochondrial ATP-synthase (g-ATPase) is mutated. These mutations are also associated with multidrug resistance, tsetse-fly independent mechanical transmission, and geographical spread of these parasites beyond Africa. Here we engineer kinetoplast-independent kinetoplastids and explore origins and consequences of kDNA loss in Trypanosoma brucei. We used oligo targeting to edit the native g-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 M282F mutant or the previously described L262P or A273P mutants. These heterozygous mutants retained sensitivity to the kDNA-targeting drug acriflavine, however, and failed to yield kDNA negative cells following acriflavine selection. In contrast, T. brucei with a homozygous M282F edit were acriflavine resistant and readily tolerated acriflavine-induced kDNA loss. Proteomics analysis of the homozygous mutants revealed highly specific depletion of ATP synthase-associated proteins. 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. These results reveal g-ATPase defects that are analogous to a broken camshaft at the core of the ATP synthase rotary motor. In summary, T. brucei cells with a bi-allelic g-ATPase defect assemble a remodelled ATP synthase, and readily tolerate kDNA-loss, accompanied by substantial remodelling of the mitochondrial proteome.

INSTRUMENT(S):

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Michele Tinti

LAB HEAD: David Horn

PROVIDER: PXD071938 | Pride | 2025-12-13

REPOSITORIES: Pride

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			Action	DRS
	056_2025_DUN_DH-MR-2-2-A.raw	Raw
	056_2025_DUN_DH-MR-2-2-B.raw	Raw
	056_2025_DUN_DH-MR-2-2-C.raw	Raw
	056_2025_DUN_DH-MR-2-4-A.raw	Raw
	056_2025_DUN_DH-MR-2-4-B.raw	Raw

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Project description:The gene TbFUT1 encodes an essential fucosyltransferase which, unexpectedly, localises to the mitochondrion of the protist parasite Trypanosoma brucei. The expression of TbFUT1 is required for the maintenance of mitochondrial membrane potential (ΨΔm) in the bloodstream form (BSF) of the parasite, but the precise functions of TbFUT1 are unknown. Here, we demonstrate that depletion of TbFUT1 causes the accumulation of dyskinetoplastid cells; i.e., cells lacking concatenated complexes of mini- and maxicircle kinetoplast DNA (kDNA), the mitochondrial DNA of these organisms. Morphological analysis by serial face block-scanning electron microscopy showed that the dyskinetoplastid mitochondria were otherwise unperturbed with respect to structure and volume. Proteomics analyses showed that TbFUT1 depletion caused a decrease in the steady-state levels of several subunits of the Fo-subcomplex and peripheral stalk components of the mitochondrial FoF1-ATP synthase, as well as a pronounced reduction in mitochondrial ribosomal large subunit (LSU) proteins and more minor reduction in small subunit (SSU) proteins. TbFUT1 was rendered redundant with respect to cell survival and ΨΔm generation upon F1-γWT/L262P mutation; a mutation that allows the generation of ΨΔm in the absence of mitochondrial translation. Additionally, depletion of TbFUT1 no longer perturbs kDNA replication in these cells, indicating that dyskinetoplasty is a downstream consequence of impaired ΨΔm. Depletion of TbFUT1 in wild type cells leads to the collapse of ΨΔm via a functional FoF1-ATP synthase complex. We therefore conclude these mutants are inhibited in the synthesis of Fo-subcomplex components and, thus, impairing the assembly of functional FoF1-ATP synthase complexes. Curiously, mitochondrial transcript levels exhibit similar changes in abundance after FUT1 ablation in the parental and F1-γWT/L262P mutants. Further, the ∼5-fold overexpression of TbFUT1 in the TbFUT1 conditional knockout mutant under permissive conditions selectively inhibits the formation of the fully RNA-edited A6 transcript by an unknown mechanism, partially suppressing FoF1-ATP synthase assembly in these mutants. Together, these data suggest that mitochondrial fucosylation is essential for the assembly of protein complexes containing kDNA encoded subunits.

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Origins and consequences of kinetoplast loss in trypanosomes

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