# Genetic origins and proteomic consequences of kinetoplast loss in trypanosomes

## TL;DR

This study explores how mutations in a key mitochondrial protein in African trypanosomes lead to loss of the large mitochondrial genome and drug resistance.

## Contribution

The paper introduces a method to precisely edit the gamma subunit of ATP synthase and identifies novel mutations associated with kinetoplast DNA loss.

## Key findings

- Homozygous M282F γATPase mutants become resistant to kDNA-targeting drugs.
- kDNA loss is accompanied by specific depletion of mitochondrial RNA-processing factors and kDNA-binding proteins.
- Mitochondrial membrane-associated transporters increase in abundance after kDNA loss.

## Abstract

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 F1FO-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.

Mutations 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 DNA (kDNA), is rendered dispensable, and the cells become resistant to important kDNA 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. Here, we precision-edit the gamma subunit to replicate previously described mutants and identify a novel mutant that readily tolerates kDNA loss. Using quantitative proteomics, we demonstrate highly specific depletion of ATP synthase-associated proteins pre kDNA loss. We then use genome sequencing to show that the kDNA can be completely lost by these cells and demonstrate that cells lacking mitochondrial nucleic acids display specific depletion of mitochondrial nucleic acid-binding proteins. Notably, several mitochondrial membrane-associated transporter complexes are increased in abundance. Thus, we establish a method to test precise γATPase mutations and to identify new mutations associated with kDNA loss. We also show that trypanosomes with dispensable kDNA specifically remodel expression of ATP synthase subunits pre kDNA loss and substantially remodel the mitochondrial proteome post kDNA loss.