Genetic origins and proteomic consequences of kinetoplast loss in trypanosomes

TY - JOUR

T1 - Genetic origins and proteomic consequences of kinetoplast loss in trypanosomes

AU - Ridgway, Melanie

AU - Escrivani, Douglas O.

AU - Novotna, Marketa

AU - Wood, Amy

AU - Tinti, Michele

AU - Schnaufer, Achim

AU - Horn, David

N1 - © 2026 Ridgway et al

PY - 2026/3/25

Y1 - 2026/3/25

N2 - The kinetoplast incorporates 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 F1 FO-ATP synthase (γATPase) 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 kDNA-independent T. brucei to explore origins and consequences of kDNA loss. We use oligo targeting to edit the native γATPase gene, and selection with the ATP synthase targeting drug oligomycin to enrich the desired mutants. Using this approach, we identify novel M282F, M282W, and M282Y mutants, and subsequently generate precision-edited strains expressing the previously described L262P or A273P mutants, or the novel M282F mutant. Heterozygous M282F mutants retain sensitivity to the kDNA-targeting drug acriflavine, while homozygous M282F mutants are acriflavine resistant. Proteomic analysis of the kDNA-positive homozygous M282F mutant reveals highly specific depletion of ATP synthase-associated proteins, but not the F1 subunits. Proteomic analysis following acriflavine-induced kDNA loss then reveals depletion of kDNA-binding proteins and mitochondrial RNA-processing factors alongside increased expression of mitochondrial membrane-associated transporters. We conclude that T. brucei cells with a homozygous γATPase M282F mutation remodel ATP synthase subunit expression and readily tolerate kDNA loss, which is accompanied by substantial remodelling of the mitochondrial proteome.

AB - The kinetoplast incorporates 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 F1 FO-ATP synthase (γATPase) 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 kDNA-independent T. brucei to explore origins and consequences of kDNA loss. We use oligo targeting to edit the native γATPase gene, and selection with the ATP synthase targeting drug oligomycin to enrich the desired mutants. Using this approach, we identify novel M282F, M282W, and M282Y mutants, and subsequently generate precision-edited strains expressing the previously described L262P or A273P mutants, or the novel M282F mutant. Heterozygous M282F mutants retain sensitivity to the kDNA-targeting drug acriflavine, while homozygous M282F mutants are acriflavine resistant. Proteomic analysis of the kDNA-positive homozygous M282F mutant reveals highly specific depletion of ATP synthase-associated proteins, but not the F1 subunits. Proteomic analysis following acriflavine-induced kDNA loss then reveals depletion of kDNA-binding proteins and mitochondrial RNA-processing factors alongside increased expression of mitochondrial membrane-associated transporters. We conclude that T. brucei cells with a homozygous γATPase M282F mutation remodel ATP synthase subunit expression and readily tolerate kDNA loss, which is accompanied by substantial remodelling of the mitochondrial proteome.

U2 - 10.1371/journal.ppat.1013846

DO - 10.1371/journal.ppat.1013846

M3 - Article

C2 - 41880374

SN - 2767-3170

VL - 22

JO - PLoS Digital Health

JF - PLoS Digital Health

IS - 3

M1 - e1013846

ER -