Composition Vendor Newgen KnowledgeWorks (P) Ltd. PLoS Pathog plos plospath PLOS Pathogens 1553-7366 1553-7374 Public Library of Science San Francisco, CA USA 10.1371/journal.ppat.1013846 PPATHOGENS-D-25-03269 Research Article Biology and life sciencesCell biologyCellular structures and organellesKinetoplasts Biology and life sciencesBiochemistryEnzymologyEnzymesPhosphatasesAdenosine triphosphatase Biology and life sciencesBiochemistryProteinsEnzymesPhosphatasesAdenosine triphosphatase Biology and life sciencesGeneticsHeredityHomozygosity Biology and life sciencesOrganismsEukaryotaProtozoansParasitic protozoansTrypanosoma Biology and life sciencesBiochemistryBioenergeticsEnergy-producing organellesMitochondria Biology and life sciencesCell biologyCellular structures and organellesEnergy-producing organellesMitochondria Biology and life sciencesGeneticsHeredityHeterozygosity Biology and life sciencesMolecular biologyMolecular biology techniquesCloning Research and analysis methodsMolecular biology techniquesCloning Biology and life sciencesBiochemistryProteomics Genetic origins and proteomic consequences of kinetoplast loss in trypanosomes Kinetoplast DNA loss in trypanosomes Ridgway Melanie Conceptualization Formal analysis Investigation Supervision Writing – original draft Writing – review & editing 1 ‡ ¤ Escrivani Douglas O. Formal analysis Investigation Writing – original draft Writing – review & editing 1 ‡ Novotná Markéta Investigation Writing – review & editing 1 Wood Amy Investigation Writing – review & editing 1 Tinti Michele Data curation Visualization Writing – review & editing 1 Schnaufer Achim Formal analysis Writing – original draft Writing – review & editing 2 https://orcid.org/0000-0001-5173-9284 Horn David Conceptualization Funding acquisition Project administration Supervision Writing – original draft Writing – review & editing 1 * Faculty of Life Sciences, University of Dundee, Dow Street, Dundee, United Kingdom Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, United Kingdom He Cynthia Y. Editor National University of Singapore, SINGAPORE

The authors have declared that no competing interests exist.

‡ These authors are Joint first authors.

¤ Current address: Molecular and Biomedical Science, School of Biological Sciences, Adelaide University, Adelaide, South Australia, Australia

 * E-mail: d.horn@dundee.ac.uk 2532026 32026 22 3 e1013846 23122025 1432026 2026 Ridgway et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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.

 Author summary

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.

 http://dx.doi.org/10.13039/100010269 Wellcome Trust 223608/Z/21/Z https://orcid.org/0000-0001-5173-9284 HornDavid http://dx.doi.org/10.13039/100010269 Wellcome Trust 217105/Z/19/Z https://orcid.org/0000-0001-5173-9284 HornDavid This work was supported by a Wellcome Centre Award (223608/Z/21/Z), DH was co-applicant, and a Wellcome Investigator Award to D.H. (217105/Z/19/Z). The funder played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS Publication Stage vor-update-to-uncorrected-proof Publication Update 2026-03-30 Data Availability The high-throughput sequencing data generated for this study (amplicon sequencing and whole genome sequencing) have been deposited at the Sequence Read Archive under accession code PRJNA1380964 (https://www.ncbi.nlm.nih.gov/bioproject/1380964). The mass spectrometry proteomics data generated for this study have been deposited at the PRIDE repository under accession code PXD071938 (https://www.ebi.ac.uk/pride/archive/projects/PXD071938). Introduction

Trypanosoma brucei brucei is an African trypanosome that is transmitted by tsetse flies, causing nagana disease in cattle and other livestock. Closely related and similarly transmitted African trypanosomes cause sleeping sickness in humans. These parasites are kinetoplastids, flagellated protozoa that contain their mitochondrial genome (mtDNA) in a kinetoplast, hence called kinetoplast DNA, or kDNA. The kDNA is a cytologically prominent feature and comprises a huge network of approximately twenty-five maxicircles and thousands of minicircles, encoding eighteen protein subunits of the mitochondrial respiratory chain, the F1FO-ATP synthase and the mitoribosome, as well as ribosomal RNA and RNA-editing associated guide RNAs [1,2]. Insect stage T. brucei depend on kDNA-encoded proteins for oxidative phosphorylation, while the bloodstream stage requires F1FO-ATPase activity to generate the mitochondrial membrane potential; whereby ATP hydrolysis by F1 is coupled to proton transfer by FO [3]. Key to this coupling is the central F1 γ subunit (γATPase), which acts like a camshaft. γATPase is mechanically coupled to the membrane embedded ring composed of 10 c subunits that, together with the A6 subunit (also known as the a subunit), forms the proton translocating part of FO [4,5]. A6 is the only F1FO-ATPase subunit encoded in kDNA. Because of its essentiality to African trypanosomes and its unique properties, kDNA has proven to be an excellent drug target, albeit with challenges associated with resistance [6,7].

Remarkably, African trypanosomes of the T. brucei group can lose their kDNA, and T. b. equiperdum and T. b. evansi, first identified in the 1800’s, present two examples [8,9]. These parasites still infect equids and various mammals, respectively, and grow as bloodstream forms [7] but cannot differentiate to tsetse insect stages [10]. Although unable to complete the usual life cycle in tsetse, they are transmitted mechanically, either sexually in equids (equiperdum), or by biting flies or vampire bats (evansi). Consequently, T. b. equiperdum and T. b. evansi cause diseases known as dourine and surra, respectively, that have spread beyond tsetse endemic regions in Africa, extending to Asia, South America and parts of Europe [11].

kDNA dispensability is caused by specific mutations in γATPase, by allowing for generation of a mitochondrial membrane potential, albeit potentially reduced, in the absence of the kDNA-encoded FO subunit A6 [12–14]. This is thought to involve mechanical uncoupling of the F1 and FO components, enhanced ATP hydrolysis by F1 and electrogenic exchange of mitochondrial ADP3- for cytosolic ATP4- by the mitochondrial ADP/ATP carrier [15]. Remarkably, beyond the A6 subunit, and the mitoribosome subunits required for its translation, no other kDNA-encoded protein appears to be specifically required to maintain the viability of wild-type bloodstream-form T. b. brucei. Trypanosomes with γATPase mutations have emerged several times independently, being equivalent to mutations that enable mtDNA loss in petite-negative yeast [8,14,15]. These parasites are either dyskinetoplastic or akinetoplastic, lacking some or all of their kDNA, respectively. Other subsequent changes appear to have facilitated adaptation to a tsetse fly independent life-cycle [9].

The kDNA has proven to be an excellent drug target, and several veterinary anti-trypanosomal drugs target kDNA, including ethidium bromide and isometamidium. kDNA loss or dispensability in T. b. evansi, T. b. equiperdum, and other γATPase mutants renders these cells multidrug resistant, however [7]. Despite connections to the parasite life cycle, geographical disease distribution, and drug resistance, the mechanisms linking γATPase mutations to kDNA dispensability are not fully understood. To develop our understanding of the origins and consequences of kDNA loss in trypanosomes, we used oligo-targeting [16] and engineered kDNA-independent kinetoplastids. We introduced novel and known mutations in the native γATPase gene and found that a novel homozygous M282F edit rendered the kDNA dispensable. We then used proteomics analysis to assess the complement of proteins impacted by γATPase mutation pre and post kDNA loss, revealing specific impacts on the mitochondrial ATP synthase, mitochondrial nucleic acid binding proteins and mitochondrial membrane-associated transporters.

 Results γATPase editing yielded known and novel oligomycin-resistant mutants

Three distinct non-synonymous substitutions have been identified in the γATPase subunit in trypanosomes that display kDNA dispensability; L262P, A273P, and M282L [12–14]. A273P was identified as a homozygous single-nucleotide mutation in T. b. equiperdum, M282L was identified as a heterozygous single-nucleotide mutation in some isolates of T. b. evansi [14], and L262P was later identified as a homozygous single-nucleotide mutation in T. b. brucei following acriflavine-selection in the laboratory [12]. The link to kDNA dispensability was validated for both the L262P and A273P mutations using ectopic γATPase expression in transgenic T. b. brucei, but a similar assay failed to validate the M282L mutation [12].

To assess the impact of specific mutations at the native γATPase gene locus (Tb927.10.180) in T. b. brucei, we used oligo targeting for precision editing [16], followed by oligomycin selection to enrich those mutants that become independent of the FO component of the ATPase (Fig 1A); oligomycin targets the proton-binding FO subunit c [17]. Given uncertainty regarding the impact of the M282L mutation, we began by assessing edits at this site. For oligo targeting, we typically deliver approximately 50 base ‘reverse-strand’ single-stranded oligodeoxynucleotides (ssODNs) by electroporation, and in this case, we designed a 53-b ssODN to target the M282 site with a centrally located and degenerate ‘NNN’ (N = A, C, T, G) codon (Sheet 1 in S1 Data). Wild type T. b. brucei cells were transfected in duplicate and grown with oligomycin at 200 nM; approximately three times the EC50 (Effective Concentration of drug to inhibit growth by 50%). We then extracted genomic DNA from surviving cells after six days, PCR-amplified the edited region in the γATPase gene, deep-sequenced the γATPase amplicons (Fig 1A), and quantified variant codons. The heatmap in Fig 1B shows relative representation of alternative codons at the targeted site and at flanking sites following oligomycin selection. The analysis revealed highly specific editing at the targeted site, and multiple M282 γATPase edits enriched in the oligomycin-resistant population, all of which encode aromatic residues, M282F, M282W, and M282Y (Fig 1B). Notably, all of the enriched mutants required double or triple nucleotide edits while the naturally occurring M282L mutation of uncertain significance, accessible via two distinct single nucleotide edits, or four distinct double nucleotide edits, was not enriched.

10.1371/journal.ppat.1013846.g001γATPase editing yielded known and novel oligomycin-resistantmutants.

(A) The schematic illustrates oligo targeting for saturation mutagenesis of the T. b. brucei γATPase M282 residue. A sixty-four fold degenerate ssODN was transfected into wild-type T. b. brucei cells, followed by oligomycin selection and γATPase amplicon-sequencing. (B) The heat map shows relative representation of each possible amino acid variant at the targeted M282 site and at adjacent sites; averages for two independent oligomycin-resistant cultures relative to an unedited control. More than 8 M reads were mapped per site on average. Unedited codons are indicated (black) as is the previously reported M282L mutation (red outline). (C) The Sanger sequencing traces show single allele edits encoding the L262P, A273P and M282F mutations, each involving a double nucleotide edit; edited nucleotides are indicated by asterisks. (D) Simplified schematic of the trypanosome F1FO-ATP synthase and key components discussed here. Names of F1 subunits are in white letters (F1 subunits p18, delta and epsilon were omitted for simplicity). The α/β hexamer (cyan) is held in place by attachment to the peripheral ‘stator’ stalk (green) via the OSCP subunit (orange). The proton-translocating part consists of the c10-ring (grey) and the kDNA-encoded A6 subunit (red). PMS, peripheral membrane subcomplex (blue); IMM, inner mitochondrial membrane. The AlphaFold models for wild-type (WT) and mutant γATPase were generated using the AlphaFold server, showing mutant residues in green.

We next designed specific ssODNs to introduce one of the novel edits identified above, M282FTTT, or the other non-synonymous substitutions previously linked to kDNA dispensability, L262PCCA and A273PCCC (Fig 1C, Sheet 1 in S1 Data); a double nucleotide edit in each case allowed us to distinguish between true edits and spontaneous mutations, the vast majority of which are limited to single nucleotides. We transfected wild type T. b. brucei cells with each ssODN, selected the cells with oligomycin at 200 nM, and sub-cloned the resistant cells that emerged. We extracted genomic DNA from the sub-clones, PCR-amplified the edited region in the γATPase gene, and Sanger sequenced the amplicons. Sequencing analysis revealed that all three heterozygous edits were effectively introduced (Fig 1C). We then used AlphaFold [18] to visualise how these edits may impact γ-subunit function. The F1 component of the ATP synthase comprises a rotor made up of three α-subunits and three β-subunits with a central γ-subunit, which is analogous to a camshaft (Fig 1D). The models predict conformational defects associated with each γATPase mutation, in the extended α-helical camshaft-like segment, and more clearly apparent in the A273P mutant. These mutations may interfere with interaction with the α/β hexamer, perhaps uncoupling F1 from FO as described for mitochondrial genome integrity mutations in yeast [19], thereby also reducing sensitivity to the FO inhibitor oligomycin. Thus, γATPase precision editing yielded known and novel heterozygous oligomycin-resistant mutants.

 Only bi-allelic M<sup>282</sup>F <italic>γATPase</italic> editing yielded acriflavine-resistant mutants

Prior analyses suggested that γATPase mutant dosage may be important. Specifically, a heterozygous A281Δ mutant γATPase allele was reported to be preferentially expressed in T. b. evansi [14], while both L262P and A273P γATPase substitutions with a validated link to kDNA dispensability are present as homozygous mutations in T. b. brucei [12] and T. b. equiperdum [14], respectively. Since differential expression of mutant alleles could impact the behaviour of heterozygous mutants, we favoured the analysis of homozygous mutants. Although we had not previously observed homozygous editing using oligo targeting, we identified a homozygous γATPase M282FTTT edited clone following oligomycin selection as detailed above. Indeed, a synonymous polymorphism present seven codons downstream of the targeted codon allowed us to show that both heterozygous and homozygous M282FTTT strains remained diploid at this locus, having retained both γATPase alleles (Fig 2A). Since oligomycin, used above to enrich for edited cells, targets the FO subunit of the ATPase rather than kDNA, we determined whether the edited mutants retained kDNA. Indeed, more than 99% of heterozygous L262P and A273P mutant cells and homozygous M282F mutant cells remained kDNA positive as assessed by DNA-staining and microscopy.

10.1371/journal.ppat.1013846.g002Only bi-allelic <italic>γ</italic><italic>ATPase</italic> M<sup>282</sup> editing yielded acriflavine-resistant mutants.

(A) The Sanger sequencing traces show T. b. brucei γATPase M282F edits, both heterozygous and homozygous. A GCT/G, alanine polymorphism can be seen on the right-hand side of each panel, confirming retention of both alleles. Edited nucleotides are indicated by asterisks. (B) Dose-response curves for oligomycin, measured in duplicate. EC50 values are shown. (C) Dose-response curves for acriflavine, measured in duplicate. EC50 values are shown.

Access to both heterozygous and homozygous M282FTTT strains presented an opportunity to compare impacts on oligomycin sensitivity. We performed dose response assays comparing wild-type, heterozygous and homozygous mutants. An oligomycin dose response assay revealed that heterozygous M282FTTT parasites displayed 3-fold increased EC50 while homozygous M282FTTT mutant parasites displayed 10-fold increased EC50 (Fig 2B); the heterozygous L262P and A273P mutants displayed 8-fold and 22-fold increased EC50, respectively (S1 Fig). Thus, all of the γATPase edits yielded significant increases in oligomycin resistance (P < 1e-4), as expected, but the dosage of mutant γATPase alleles in the M282FTTT edited cells impacted the relative shift in EC50.

We next performed dose response assays using acriflavine, a DNA-intercalating agent that targets kDNA, again comparing wild-type, heterozygous and homozygous mutants (Fig 2C). We observed that homozygous M282FTTT parasites displayed a significant, 3-fold increased EC50 (P < 1e-4), while heterozygous M282FTTT parasites displayed only 1.1-fold (P = 0.4) increase in EC50 (Fig 2C); the heterozygous L262P and A273P mutants both displayed 2-fold and 2.2-fold increased EC50 respectively (S1 Fig). Thus, homozygous M282FTTT edits yielded acriflavine resistant cells, while heterozygous edits failed to do so, indicating that this mutation is recessive with respect to acriflavine-resistance. We concluded that both heterozygous and homozygous γATPase M282FTTT editing conferred oligomycin-resistance, albeit to differing degrees, while only homozygous γATPase M282FTTT editing conferred acriflavine-resistance.

 ATP synthase remodelling and kDNA loss in homozygous <italic>γATPase</italic> mutants

To further elucidate the mechanism underpinning oligomycin and acriflavine cross-resistance, wild-type and homozygous γATPase M282FTTT edited parasites were assessed using high resolution quantitative proteomics on an Orbitrap Astral mass spectrometer with data-independent acquisition (Sheet 2 in S1 Data). The analysis revealed highly specific depletion of all eighteen known nuclear-encoded subunits of the FO component of the T. b. brucei ATP synthase (Fig 3, see Fig 1D); the peripheral stalk proteins, including the oligomycin sensitivity conferring protein OSCP, membrane region proteins and peripheral membrane subcomplex proteins [5]. In striking contrast, subunits of the F1 component of the ATP synthase, including the mutated γ subunit itself, were not depleted (Fig 3A). Thus, proteomic analysis revealed highly specific depletion of subunits of the FO component of the T. b. brucei ATP synthase in homozygous M282FTTT mutants. To determine whether depletion of FO subunits had major impacts on kDNA or on mitochondrial membrane potential, we examined both wild-type cells and homozygous M282FTTT mutants by microscopy following DNA-staining, and by flow cytometry following MitoTracker staining. More than 99% of these cells were kDNA positive by microscopy (Fig 3B), and MitoTracker staining appeared unperturbed when assessed using flow cytometry (Fig 3C).

10.1371/journal.ppat.1013846.g003ATP synthase remodelling in homozygous<italic>γ</italic><italic>ATPase</italic> mutants.

(A) Proteomics analysis of wild-type T. b. brucei and homozygous γATPase M282F mutants. Subunits of the F1 and Fo γATPase components are highlighted. Averages from three replicates; n = 6847 proteins. (B) The microscopy images show nuclei (larger structures) and kDNA (smaller structures) in wild-type T. b. brucei and homozygous γATPase M282F mutant cells; DNA was stained with DAPI. Scale bars, 5 μm. (C) Flow cytometry analysis of MitoTracker-stained cells, wild-type and homozygous γATPase M282F mutants. The data are representative of three technical replicates. Control, unstained cells.

Since acriflavine inhibits kDNA replication and segregation, acriflavine resistance displayed by homozygous γATPase M282FTTT edited parasites suggested that kDNA would be dispensable in these cells. To induce kDNA loss, we grew two parallel cultures of homozygous M282FTTT edited cells in the presence of a sub-EC50 dose of acriflavine for 7 days (1.25 nM; the EC50 is 2 nM, see Fig 2C), which yielded populations containing approximately 50% kDNA negative cells, as determined by DNA-staining and microscopy. Each population was then cloned by limiting dilution in the absence of acriflavine and, when sufficient cells were available after 7–8 days, clones were assessed by DNA-staining and microscopy. Clones that appeared to lack kDNA, two from each independent culture, were selected for further analysis (see Fig 4A). Notably, kDNA negative M282FTTT cells displayed a growth defect relative to the kDNA positive parent, with doubling time increased by approximately 1.5-fold to 9.4 h + /-1.5 h (n = 4); parent doubling time was 6.4 h.

10.1371/journal.ppat.1013846.g004kDNA loss in homozygous <italic>γATPase</italic>mutants.

(A) The microscopy images show homozygous γATPase M282F mutant cells with or without kDNA, the smaller blue DNA-stained structures. DNA was stained with DAPI. Scale bars, 5 μm. (B-C) Whole genome sequencing data for wild-type (WT) T. b. brucei, the homozygous γATPase M282F mutant with kDNA (P for parent) and independently generated clones lacking kDNA. (B) The upper circular plot shows genome sequencing data mapped to the T. b. brucei nuclear chromosomes 1–11 (grey). The lower circular plot shows genome sequencing data mapped to the T. b. brucei kDNA (magenta), maxicircle sequence and the most abundant minicircle sequences; the numbers indicate minicircle ID. Mapping is for 150-bp bins and for two independently generated kDNA negative clones. (C) The heatmap shows data for maxicircle sequence, additional minicircle sequences (n = 90), and for all four kDNA negative clones. kDNA negative clone numbers are indicated in each panel.

We next considered a more sensitive approach to determine whether kDNA had been completely lost, and subjected wild-type cells, kDNA positive M282FTTT edited cells, and all four kDNA negative clones, to whole genome sequencing. We used a recent T. b. brucei maxicircle and minicircle DNA assembly for the same strain used in our study [2] as a template for this analysis and observed highly specific loss of both classes of kDNA in independently generated kDNA negative clones without apparent changes in the nuclear genome (Fig 4B). Closer inspection of minicircle abundance indicated that some were already depleted in M282FTTT edited cells prior to acriflavine exposure, with some of the lower abundance minicircles apparently lost entirely (Fig 4C) Thus, genome sequencing revealed some minicircle loss in homozygous M282FTTT mutants pre acriflavine-exposure, and complete elimination of kDNA induced by acriflavine in these cells.

 Mitochondrial proteome remodelling following kDNA loss

To explore the consequences of kDNA loss, we again used high resolution quantitative proteomics to compare homozygous γATPase M282FTTT mutants with or without kDNA. Analysis of these proteomes (Sheets 3–6 in S1 Data) revealed substantial changes, including further specific depletion of subunits of the FO component of the T. b. brucei ATP synthase, except for subunit c, which displayed increased abundance. As above (Fig 3A), subunits of the F1 component of the ATP synthase, including the mutated γ subunit itself, were not depleted (Fig 5A, S2 Fig). DNA and MitoTracker staining followed by super resolution microscopy confirmed complete loss of kDNA in the M282FTTT mutants following acriflavine treatment and revealed broadly maintained mitochondrial structure (Fig 5B, S3 Fig). We also assessed these MitoTracker stained cells by flow cytometry, which suggested that mitochondrial membrane potential was maintained following kDNA loss (Fig 5C).

10.1371/journal.ppat.1013846.g005Proteomic and mitochondrial impacts of kDNA loss.

(A) Proteomics analysis of homozygous γATPase M282F mutants with and without kDNA with subunits of the F1 and Fo γATPase components highlighted. Averages from three replicates; n = 6768 proteins; clone 1.2 is shown here and three other clones are shown in Supplementary Fig 2. (B) The representative super resolution microscopy images show wild-type T. b. brucei and homozygous γATPase M282F mutant cells lacking kDNA, following growth in acriflavine. DNA was stained with DAPI (cyan) and mitochondria were stained with MitoTracker (yellow). Nuclear DNA (N) and kDNA (K) are indicated. Scale bars, 2 μm. A gallery of additional images is shown in Supplementary Fig 3. (C) Flow cytometry analysis of MitoTracker-stained cells, homozygous γATPase M282F mutants with or without kDNA. Data are shown for two independent biological replicates without kDNA and are representative of three technical replicates in each case.

We next examined changes in the abundance of nuclear and mitochondrial proteins following kDNA loss and found that mitochondrial proteins were selectively and significantly reduced in abundance in all four kDNA negative clones (Fig 6A). More proteins reported a highly significant (-log10 False Discovery Rate [FDR] >4) reduction in abundance relative to proteins that reported increased abundance following kDNA loss (168 v 51), and a closer inspection of >2-fold depleted proteins revealed kDNA-binding proteins and mitochondrial RNA-processing factors (Fig 6B), consistent with destabilisation of these proteins after loss of all mitochondrial DNA and RNA. These included the kDNA-associated proteins involved in DNA compaction [20], and kDNA anchoring to the tripartite attachment complex [21], primases [22], polymerases [23], topoisomerase involved in DNA replication, and mRNA polyadenylation factors [24]. Other depleted proteins were the calcium uniporter, known to interact with subunit c of the ATP-synthase [25], and PUF9, an RNA-binding protein involved in nuclear cell cycle regulation [26]. Notably, IF1, an inhibitor of F1-mediated ATP hydrolysis, was also depleted. Expression of this protein was thought to be restricted to the insect stage, where ATP synthesis, but not hydrolysis, is essential [27]. This observation might indicate increased ATP hydrolysis following kDNA loss. In contrast, PUF9 target 1 (PNT1), a kDNA replication-associated peptidase [28], is notably increased in abundance in kDNA negative cells (Fig 6B).

10.1371/journal.ppat.1013846.g006Mitochondrial proteome remodelling following kDNA loss.

(A) Proteomics analysis of homozygous γATPase M282F T. b. brucei mutants and all four M282F clones lacking kDNA. The boxplot shows nuclear proteins (n = 1185, GO:0005634) and mitochondrial proteins (n = 1431, GO:0005739). Boxes indicate the interquartile range (IQR) and the whiskers show the range of values within 1.5 × IQR. (B) Proteomics analysis showing all proteins with log2 average expression >16 and with some notable proteins highlighted. (C) Gene Ontology profiles for proteins with log2 average expression >16 that are significantly (>2 –log10 FDR) decreased or increased in abundance in kDNA negative cells. Mito’, mitochondrial. (D) Selected cohorts of proteins that are significantly decreased or increased in abundance in kDNA negative cells. Boxes indicate the interquartile range (IQR) and the whiskers show the range of values within 1.5 × IQR. MCP, mitochondrial carrier proteins; TIM, Translocases of the Inner Membrane; ATOM, Archaic Translocase of the Outer Membrane; MICOS, Mitochondrial contact site and Cristae Organizing System; POMP, Present in the Outer mitochondrial Membrane Proteome; EMC, ER-Membrane Complex; ER, Endoplasmic Reticulum. Cohorts were derived using GO-terms except for kinetoplast-associated proteins from [31] and MCP, TIM and POMP, derived using wild-card searches, MCP*, TIM* and POMP* at https://tritrypdb.org [43]. Data are shown for kDNA negative clones 1.2 (blue) and 2.1 (red).

Finally, we profiled proteins that were significantly (FDR < 0.01) reduced (n = 507) or increased (n = 761) in abundance following kDNA loss (Sheets 3–6 in S1 Data), using Gene Ontology (GO) terms. The top GO-term hits for depleted proteins were exclusively associated with the mitochondrion, again including DNA and RNA binding proteins, and ATP synthase (Fig 6C, upper panel). Kinetoplast (P = 1.4e-89) and mitochondrial matrix (P = 4.1e-148) registered the highest significance, and components of the NADH dehydrogenase, complex I of the electron transport chain, were also significantly depleted; perhaps unsurprisingly since several components of this complex are encoded in kDNA [1,2]. In contrast, mitochondrial membrane-associated transporters, and endoplasmic reticulum (ER) associated proteins, were significantly increased in abundance (Fig 6C, lower panel). The telomere-telomerase complex and nuclear pore proteins were also increased, perhaps reflecting connections between nuclear and kDNA replication [29]. Changes in abundance for several of these cohorts of proteins are shown in Fig 6D, revealing substantial depletion of the mitochondrial ribosome, RNA editing complex [30] and kinetoplast-associated proteins [31]. The tripartite attachment complex itself [21] is notably not substantially depleted, and only the p166 component (Tb927.11.3290) achieved an FDR < 0.01 following kDNA loss (log2 fold-change = -0.52 + /-0.12); indeed, kDNA is not required for assembly of this complex [32]. The mitochondrial carrier proteins [33], translocase of the inner mitochondrial membrane (TIM) [34], archaic translocase of the outer mitochondrial membrane (ATOM), and mitochondrial contact site and cristae organization system [35], were all increased in abundance (Fig 6D), perhaps compensating for defects in mitochondrial import and supporting the maintenance of mitochondrial membrane potential [13,36]. Proteins present in the outer mitochondrial membrane proteome (POMP), many of which remain otherwise uncharacterised [37], were also increased in abundance. Finally, the ER-membrane complex (EMC), previously connected to kDNA dependency [6], and now known to localise to the mitochondrial – ER interface [38], was increased in abundance (Fig 6D). Taken together, our results reveal ATP synthase complex remodelling associated with bi-allelic γATPase mutation and resistance to both ATPase and kDNA-targeting drugs; oligomycin and acriflavine, respectively. These cells readily tolerate kDNA loss, which is associated with substantial mitochondrial proteome remodelling.

 Discussion

γATPase mutations in T. brucei are associated with kDNA loss and multidrug resistance and are also correlated with tsetse-fly independent mechanical transmission and geographical spread of these parasites beyond Africa. Here, we explore γATPase mutations and connections to kDNA loss in T. b. brucei. We precision-edited the native γATPase gene, confirming that L262P and A273P mutants are resistant to the ATP synthase targeting drug oligomycin and to the kDNA-targeting drug, acriflavine. We also identified novel oligomycin-resistant aromatic amino-acid mutants replacing M282. Quantitative proteomics analysis of homozygous M282 mutants revealed specific depletion of ATP synthase FO components prior to kDNA loss. Following acriflavine-induced kDNA loss, confirmed to be complete by genome sequencing, we observed substantial mitochondrial proteome remodelling; the abundance of mitochondrial DNA and mRNA binding proteins was reduced while the abundance of proteins involved in mitochondrial import was increased. While we observed further depletion of ATP synthase FO components following kDNA loss, c subunit abundance was increased, perhaps reflecting accumulation of F1-ATP synthase associated with only the c-ring of the FO moiety (see Fig 1D).

In terms of the origins of kDNA loss, we found that precision-editing could be used to generate both heterozygous and homozygous γATPase mutants in otherwise wild-type trypanosomes. While a heterozygous M282F edit failed to confer resistance to acriflavine, a homozygous M282F mutant was acriflavine-resistant and readily tolerated kDNA loss. We used quantitative proteomics to explore the impact of the homozygous M282F edit prior to kDNA loss and observed highly specific depletion of ATP synthase-associated proteins; possibly due to increased turnover when unassembled. Importantly, we selected for edited cells using oligomycin rather than a DNA-damaging agent, avoiding direct selective pressure on the kDNA and likely reducing the potential for off-target mutations at this stage of the process. It is also worth noting in this regard that the widespread use of veterinary anti-trypanosomal drugs that target the kDNA, such as the DNA-damaging agents, ethidium bromide and isometamidium, could induce γATPase mutations and/or other mutations, and promote kDNA loss in the field [39].

A homozygous M282F γATPase edit generated here in T. b. brucei was sufficient to confer kDNA dispensability, while a heterozygous edit was insufficient. This recessive effect with respect to kDNA dispensability suggested a dosage effect and prompted further consideration of naturally occurring non-synonymous γATPase mutations implicated in conferring kDNA dispensability; a heterozygous M282L mutation in some isolates of T. b. evansi [14], a homozygous A273P mutation in T. b. equiperdum [14], and a homozygous L262P mutation in T. b. brucei [12]. Among these non-synonymous edits, we only failed to recover M282L using oligomycin selection. Indeed, ectopic mutant γATPase expression assays in T. b. brucei also indicated that the M282L mutation is insufficient to confer kDNA dispensability [12]. On the other hand, ectopic expression assays suggested that a single A273P or L262P allele may be sufficient to confer kDNA dispensability, and our demonstration that heterozygous A273P or L262P edits are sufficient to confer acriflavine resistance is consistent with this view. Homozygous editing described here is the first example of dual-allele oligo targeting, suggesting that this approach may be exploited to generate and assess further homozygous γATPase mutants.

We also used quantitative proteomics to elucidate the consequences of kDNA loss in homozygous M282F γATPase mutants and observed extensive proteome remodelling in this case. Perhaps unsurprisingly, kDNA-binding proteins and mitochondrial RNA-processing factors were significantly depleted in the absence of mitochondrial DNA and RNA; residual editing complexes in kDNA-negative cells have been reported to retain function, however [40]. Mitochondrial membrane-associated transporters on the other hand were significantly increased in abundance, suggesting a boost in mitochondrial protein import capacity and inter-organellar trafficking capacity. Indeed, we found increased abundance of components of the ER membrane complex particularly intriguing, since we previously linked expression of this complex to kDNA dispensability in the absence of γATPase mutation [6]; this complex is now known to localise to the mitochondrial – ER interface [38]. These adaptations may compensate for mitochondrial import defects associated with changes in F1FO-ATP synthase assembly and maintenance of mitochondrial membrane potential. Similarly, these adaptations may reflect a response to depletion of other multiprotein complexes that contain kDNA-encoded proteins, such as respiratory complex I or the mitoribosome [13,36]. Proteome remodelling was not sufficient to recover a wild-type growth rate here, however, consistent with further adaptation thought to have occurred in T. b. evansi andT. b. equiperdum [9].

Notably, association of mutant F1 with the inner mitochondrial membrane, perhaps proximal to the ATP/ADP carrier, is thought to be required to sustain mitochondrial membrane potential following kDNA loss [15,41]. Subunit c of the ATP synthase was selectively increased in abundance following kDNA loss, and although it was suggested that subunit c interacts with the calcium uniporter [25], the uniporter was reduced in abundance. We note here that analysis by native gel electrophoresis detected putative ‘F1-c’ complexes as a major ATP synthase assembly state in dyskinetoplastic T. brucei [41]. In the ATP synthase assembly pathway elucidated for other eukaryotes, subunit a (A6 in trypanosomes) is attached to F1-c before assembly with the FO part [42]. We therefore suggest that, in the absence of the kDNA-encoded subunit A6, ATP synthase assembly beyond F1-c is impaired; a hypothesis that could be tested in the future. Other adaptations may reflect disrupted communication between the kDNA and nuclear DNA. For example, PUF9 target 1, a kDNA replication-associated peptidase [28], was increased in abundance while the RNA-binding protein PUF9, involved in nuclear cell cycle regulation [26], was reduced in abundance. The telomere-telomerase complex was also increased in abundance. Taken together, these adaptations reveal a remarkable connectivity between the ATP synthase and other mitochondrial, and even other cellular, complexes and compartments.

In conclusion, T. brucei cells with a bi-allelic γATPase defect assemble a remodelled ATP synthase complex, and tolerate kDNA-loss, accompanied by substantial mitochondrial proteome remodelling. Proline mutations with the potential to disrupt helical structure at L262 or A273 in the γATPase [12,14], or bulky aromatic residue mutations at M282, introduce defects analogous to a broken camshaft at the core of this ATP synthase rotary motor. Our findings yield new insights into the origins and consequences of kDNA loss, with implications for the evolution of trypanosome sub-species that have global veterinary impacts.

 Methods <italic>T. brucei</italic> growth and <italic>γATPase</italic> gene editing

Bloodstream form T. b. brucei Lister 427 cells were grown in HMI-11 (Gibco) supplemented with 10% fetal bovine serum (Sigma) at 37°C and with 5% CO2 in a humidified incubator. For site saturation mutagenesis using oligo-targeting, a degenerate ssODN was transfected in duplicate by electroporation with a Nucleofector (Lonza), and a human T-cell kit (Lonza), with the Nucleofector set to Z-001 (Amaxa). Briefly, we used 40 µg of the ssODNs in 10 µl of 10 mM Tris-Cl, pH 8.5, mixed with 25 million cells in 100 µl transfection buffer. 200 nM oligomycin was applied 6 h after transfection. DNA was isolated 7 d later. The γATPase fragment was amplified by PCR using Q5 high fidelity DNA polymerase (New England Biolabs) as per the manufacturer’s instructions, and primers 1 and 2. Annealing was at 63oC and elongation was for 30 s. PCR products were purified using a Qiagen PCR purification kit. For specific mutagenesis using oligo targeting, a specific ssODN was transfected in duplicate with 10 million cells, and 200 nM oligomycin was applied 6 h after transfection. Oligomycin-resistant cultures were sub-cloned by serial dilution in 96-well plates 5 d later and DNA was extracted from the clones. The γATPase fragment was amplified by PCR as above but using primers 1 and 3 in this case. The products were Sanger sequenced using primer 4 at Azenta Life Sciences. To induce kDNA loss, cells were grown in the presence of 1.25 nM acriflavine for 7 days and then subcloned.

 Dose-response assays

To determine the Effective Concentration of drug to inhibit growth by 50% (EC50), cells were plated in 96-well plates at 1 x 103 cells/ml in a 2-fold serial dilution of selective drug. Plates were incubated at 37°C for 72 h. 20 µl resazurin sodium salt (AlamarBlue, Sigma) at 0.49 mM in PBS was then added to each well, and plates were incubated for a further 6 h. Fluorescence was determined using an Infinite 200 pro plate reader (Tecan) at an excitation wavelength of 540 nm and an emission wavelength of 590 nm. EC50 values were derived using Prism (GraphPad).

 Mass spectrometry

Approx. 5 x 107 PBS-washed cells were suspended in 100 μL of TBA (5% SDS, 100 mM triethylammonium bicarbonate); triplicate samples for each experiment and control cells. Total cell extracts were submitted to the Fingerprints Proteomics Facility at the University of Dundee and processed by trypsin: μBCA (bicinchoninic acid), strap processed, quality controlled, and peptide quantified by Micro-BCA assay (Thermo Scientific). 3 μl of each sample was processed using S-Trap Micro columns (Protifi) where proteins were reduced, alkylated and digested overnight at 37°C at 1:40 enzyme-to-substrate. A second digest was repeated for 6 h the following day. For mass spectrometry analysis, digested peptides (200 ng) were run on an Astral Orbitrap Mass Spectrometer (Thermo Scientific) coupled to a Vanquish Neo UHPLC system (Thermo Scientific). Buffer conditions used Buffer A (0.1% formic acid) and Buffer B (80% acetonitrile in 0.1% formic acid). Flow was 60 µl/min and loading volume was set at automatic. Peptides were initially trapped on a PepMap Neo C18 column (5 µm, 300 µm x 5 cm) and then separated on an Easy-Spray PepMap RSLC C18 column (2 µm, 150 µm x 15 cm) (Thermo Scientific). Columns was kept at a constant temperature of 50°C and a source voltage of 2.0 kV. Full MS scan was performed in data-independent acquisition (DIA) mode with an m/z range of 380–980 with orbitrap resolution 2400000, Automatic Gain Control (AGC) target of 500% and a maximum injection (IT) of 3 ms. MS scans were followed by MS/MS DIA using the following parameters; scans of isolation window of 2.0 m/z unit and window overlap set at 0 m/z. Normalised collision energy was set to 25%. Data for MS scans were acquired in profile mode with MS/MS DIA scan events being acquired in centroid mode.

 Proteomic data analysis

We generated a spectral library based on the predicted protein sequences for T. brucei TREU927 sourced from TriTrypDB (version 51) [43]. The raw mass spectrometry data were processed using DIA-NN (version 2.2.1) [44] and analysed using default settings. Initial QC analysis, median normalisation and missing value imputation was performed with the project utility python package (https://github.com/mtinti/ProjectUtility). Differential expression analysis was performed using the limma package in R [45]. A linear model was fitted using the lmFit() function, followed by Empirical Bayes Statistics for Differential Expression computed with the ebayes() function. Adjusted p-values were calculated using the topTable function with the Benjamini & Hochberg (BH) correction method.

 High-throughput sequencing

For analysis following site saturation mutagenesis, PCR amplicons were sequenced at the Beijing Genome Institute (BGI) on a DNBseq platform with 150 base paired-end reads as described previously [16] and codon-based read counts were derived using the OligoSeeker software [46]. Whole genome sequencing data were analysed with alignment to the T. b. brucei reference genome v46 clone 427_2018 supplemented with 427 maxi and minicircle sequences [2,43]. The alignment and read counts were performed with the automated snakemake [47] pipeline myRna-seq [48]. Read coverage was extracted from the BAM files using a bin size of 150 with the bamCoverage function from deepTools (v 3.5.6). The circular visualisation was performed with the pyCirclize (1.6) Python package (https://github.com/moshi4/pyCirclize).

 Microscopy

To identify clones lacking kDNA, cells were fixed in 1% paraformaldehyde (PFA) for 15 min, washed twice in PBS and resuspended in water with 1% bovine serum albumin (BSA). Cells were attached to a 12-well 5 mm slide (Thermo Scientific) by drying overnight. After rehydration in PBS for 5 min, slides were mounted in Vectashield with DAPI and sealed under a coverslip. Cells were viewed at 63x magnification with oil immersion on a Zeiss Axiovert 200 M microscope with Zen Pro software (Zeiss). For mitochondria morphology visualisation, cells were stained with 100 nM Mitotracker red CMXRos (Invitrogen) for 5 min at 37°C prior to fixation in 3% PFA for 15 min, washed in PBS then resuspended in water with 1% BSA. Cells were attached to poly-lysine-coated coverslips for 4 h at room temperature then stained with DAPI for 30 min before mounting in Vectashield (without DAPI) and sealing to a glass slide. For wide-field microscopy, cells were imaged as z-stacks (0.2 μm) at 100x magnification with oil immersion on the same microscope described above. For super resolution microscopy, cells were imaged as z-stacks (0.1–0.2 μm) at 63x magnification on a Leica Stellaris 8 inverted confocal microscope equipped with Power HyD detectors and subjected to adaptive deconvolution using the integrated Leica LIGHTNING algorithm for super-resolution microscopy. Images were analysed using Fiji v1.5.2e.

 MitoTracker staining and flow cytometry

Live T. brucei cells (1x106/ml) were incubated with 100 nM MitoTracker Red CMXROS (Molecular Probes) at 37°C for 5 min. Cells were fixed with 1% of paraformaldehyde (PFA) at 37°C for 15 min, then washed and resuspended in cold PBS and stored at 4 °C. MitoTracker fluorescence intensity was measured using a CytoFlex S flow cytometer (Beckman Coulter) and analysed using FlowJo v10.10. Forward scatter area (FSC-A) versus forward scatter height (FSC-H) was used to exclude cell aggregates.

 Supporting information Dose-response curves. (A)

For oligomycin, measured in duplicate. (B) For acriflavine, measured in duplicate.

(TIF)

 Proteomics analysis of homozygous γATPase M<sup>282</sup>F mutants with and without kDNA.

Subunits of the F1 and Fo γATPase components are highlighted. Averages from three replicates; n = 6768 proteins; clone 1.2 is shown in Fig 5A.

(TIF)

 Gallery of super resolution microscopy images showing wild-type <italic>T. b. brucei</italic> and homozygous γATPase M<sup>282</sup>F mutant cells lacking kDNA.

Other details as in Fig 5B.

(TIF)

 Oligonucleotides and proteomics data.

Sheet 1: Oligonucleotides and primers used in this study. Sheet 2: Proteomics data – M282F kDNA positive parent v wild-type. Sheet 3: Proteomics data – M282F kDNA negative clone 1.1 v kDNA positive parent. Sheet 4: Proteomics data – M282F kDNA negative clone 1.2 v kDNA positive parent. Sheet 5: Proteomics data – M282F kDNA negative clone 2.1 v kDNA positive parent. Sheet 6: Proteomics data – M282F kDNA negative clone 2.2 v kDNA positive parent.

(XLS)

We thank Gustavo Bravo Ruiz for assistance with visualising GO-term profiles.

 References Cooper S, Wadsworth ES, Schnaufer A, Savill NJ. Organization of minicircle cassettes and guide RNA genes in Trypanosoma brucei. RNA. 2022;28(7):972–92. doi: 10.1261/rna.079022.121 35414587 Zhao X, He Y, Zhang F, Aphasizheva I, Aphasizhev R, Zhang L. Comparative mitochondrial genome and transcriptome analyses reveal strain-specific features of RNA editing in Trypanosoma brucei. Nucleic Acids Res. 2025;53(13):gkaf661. doi: 10.1093/nar/gkaf661 40671526 Zikova A. Mitochondrial adaptations throughout the Trypanosoma brucei life cycle. J Eukaryot Microbiol. 2022;69(6):e12911. Epub 2022/03/25. doi: 10.1111/jeu.12911 35325490 Gahura O, Hierro-Yap C, Zíková A. Redesigned and reversed: architectural and functional oddities of the trypanosomal ATP synthase. Parasitology. 2021;148(10):1151–60. doi: 10.1017/S0031182021000202 33551002 Gahura O, Mühleip A, Hierro-Yap C, Panicucci B, Jain M, Hollaus D, et al. An ancestral interaction module promotes oligomerization in divergent mitochondrial ATP synthases. Nat Commun. 2022;13(1):5989. doi: 10.1038/s41467-022-33588-z 36220811 Baker N, Hamilton G, Wilkes JM, Hutchinson S, Barrett MP, Horn D. Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes. Proc Natl Acad Sci U S A. 2015;112(29):9112–7. doi: 10.1073/pnas.1505411112 26150481 Gould MK, Schnaufer A. Independence from Kinetoplast DNA maintenance and expression is associated with multidrug resistance in Trypanosoma brucei in vitro. Antimicrob Agents Chemother. 2014;58(5):2925–8. doi: 10.1128/AAC.00122-14 24550326 Carnes J, Anupama A, Balmer O, Jackson A, Lewis M, Brown R, et al. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty. PLoS Negl Trop Dis. 2015;9(1):e3404. doi: 10.1371/journal.pntd.0003404 25568942 Oldrieve GR, Venter F, Cayla M, Verney M, Hébert L, Geerts M, et al. Mechanisms of life cycle simplification in African trypanosomes. Nat Commun. 2024;15(1):10485. doi: 10.1038/s41467-024-54555-w 39622840 Dewar CE, MacGregor P, Cooper S, Gould MK, Matthews KR, Savill NJ, et al. Mitochondrial DNA is critical for longevity and metabolism of transmission stage Trypanosoma brucei. PLoS Pathog. 2018;14(7):e1007195. doi: 10.1371/journal.ppat.1007195 30020996 Schnaufer A, Domingo GJ, Stuart K. Natural and induced dyskinetoplastic trypanosomatids: how to live without mitochondrial DNA. Int J Parasitol. 2002;32(9):1071–84. doi: 10.1016/s0020-7519(02)00020-6 12117490 Dean S, Gould MK, Dewar CE, Schnaufer AC. Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes. Proc Natl Acad Sci U S A. 2013;110(36):14741–6. doi: 10.1073/pnas.1305404110 23959897 Eze AA, Gould MK, Munday JC, Tagoe DNA, Stelmanis V, Schnaufer A, et al. Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase. PLoS Negl Trop Dis. 2016;10(8):e0004791. doi: 10.1371/journal.pntd.0004791 27518185 Lai D-H, Hashimi H, Lun Z-R, Ayala FJ, Lukes J. Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei. Proc Natl Acad Sci U S A. 2008;105(6):1999–2004. doi: 10.1073/pnas.0711799105 18245376 Schnaufer A, Clark-Walker GD, Steinberg AG, Stuart K. The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function. EMBO J. 2005;24(23):4029–40. doi: 10.1038/sj.emboj.7600862 16270030 Altmann S, Rico E, Carvalho S, Ridgway M, Trenaman A, Donnelly H, et al. Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids. Nucleic Acids Res. 2022;50(14):e79. doi: 10.1093/nar/gkac319 35524555 Symersky J, Osowski D, Walters DE, Mueller DM. Oligomycin frames a common drug-binding site in the ATP synthase. Proc Natl Acad Sci U S A. 2012;109(35):13961–5. doi: 10.1073/pnas.1207912109 22869738 Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–9. doi: 10.1038/s41586-021-03819-2 34265844 Arsenieva D, Symersky J, Wang Y, Pagadala V, Mueller DM. Crystal structures of mutant forms of the yeast F1 ATPase reveal two modes of uncoupling. J Biol Chem. 2010;285(47):36561–9. doi: 10.1074/jbc.M110.174383 20843806 Wang J, Pappas-Brown V, Englund PT, Jensen RE. TbKAP6, a mitochondrial HMG box-containing protein in Trypanosoma brucei, is the first trypanosomatid kinetoplast-associated protein essential for kinetoplast DNA replication and maintenance. Eukaryot Cell. 2014;13(7):919–32. doi: 10.1128/EC.00260-13 24879122 Jetishi C, Aeschlimann S, Schimanski B, Käser S, Mullner R, Oeljeklaus S, et al. Connecting basal body and mitochondrial DNA: TAC53 and the tubular organization of the tripartite attachment complex. PLoS Pathog. 2025;21(9):e1013521. doi: 10.1371/journal.ppat.1013521 40953125 Hines JC, Ray DS. A second mitochondrial DNA primase is essential for cell growth and kinetoplast minicircle DNA replication in Trypanosoma brucei. Eukaryot Cell. 2011;10(3):445–54. doi: 10.1128/EC.00308-10 21257796 Delzell SB, Nelson SW, Frost MP, Klingbeil MM. Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Polymerase Domain Insertion That Confers Dominant Exonuclease Activity. Biochemistry. 2022;61(23):2751–65. doi: 10.1021/acs.biochem.2c00392 36399653 Aphasizheva I, Yu T, Suematsu T, Liu Q, Mesitov MV, Yu C, et al. Poly(A) binding KPAF4/5 complex stabilizes kinetoplast mRNAs in Trypanosoma brucei. Nucleic Acids Res. 2020;48(15):8645–62. doi: 10.1093/nar/gkaa575 32614436 Huang G, Docampo R. The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans. mBio. 2020;11(2):e00268-20. doi: 10.1128/mBio.00268-20 32184243 Archer SK, Luu V-D, de Queiroz RA, Brems S, Clayton C. Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle. PLoS Pathog. 2009;5(8):e1000565. doi: 10.1371/journal.ppat.1000565 19714224 Panicucci B, Gahura O, Zíková A. Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite. PLoS Negl Trop Dis. 2017;11(4):e0005552. doi: 10.1371/journal.pntd.0005552 28414727 Grewal JS, McLuskey K, Das D, Myburgh E, Wilkes J, Brown E, et al. PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast. J Biol Chem. 2016;291(18):9492–500. doi: 10.1074/jbc.M116.714972 26940875 Klebanov-Akopyan O, Mishra A, Glousker G, Tzfati Y, Shlomai J. Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection. Nucleic Acids Res. 2018;46(15):7757–71. doi: 10.1093/nar/gky597 30007364 Aphasizheva I, Alfonzo J, Carnes J, Cestari I, Cruz-Reyes J, Göringer HU, et al. Lexis and Grammar of Mitochondrial RNA Processing in Trypanosomes. Trends Parasitol. 2020;36(4):337–55. doi: 10.1016/j.pt.2020.01.006 32191849 Pyrih J, Hammond M, Alves A, Dean S, Sunter JD, Wheeler RJ, et al. Comprehensive sub-mitochondrial protein map of the parasitic protist Trypanosoma brucei defines critical features of organellar biology. Cell Rep. 2023;42(9):113083. doi: 10.1016/j.celrep.2023.113083 37669165 Hoffmann A, Käser S, Jakob M, Amodeo S, Peitsch C, Týč J, et al. Molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei. Proc Natl Acad Sci U S A. 2018;115(8):E1809–18. doi: 10.1073/pnas.1716582115 29434039 Colasante C, Peña Diaz P, Clayton C, Voncken F. Mitochondrial carrier family inventory of Trypanosoma brucei brucei: Identification, expression and subcellular localisation. Mol Biochem Parasitol. 2009;167(2):104–17. doi: 10.1016/j.molbiopara.2009.05.004 19463859 Schneider A. Evolution of mitochondrial protein import - lessons from trypanosomes. Biol Chem. 2020;401(6–7):663–76. doi: 10.1515/hsz-2019-0444 32142472 Kaurov I, Vancová M, Schimanski B, Cadena LR, Heller J, Bílý T, et al. The Diverged Trypanosome MICOS Complex as a Hub for Mitochondrial Cristae Shaping and Protein Import. Curr Biol. 2018;28(21):3393-3407.e5. doi: 10.1016/j.cub.2018.09.008 30415698 von Känel C, Aeschlimann S, Husová M, Oeljeklaus S, Stettler P, Schnaufer A, et al. TbTim20 facilitates protein import at a low membrane potential in trypanosomes lacking the mitochondrial genome. FEBS J. 2026;293(6):1771–84. doi: 10.1111/febs.70297 41129280 Niemann M, Wiese S, Mani J, Chanfon A, Jackson C, Meisinger C, et al. Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology. Mol Cell Proteomics. 2013;12(2):515–28. doi: 10.1074/mcp.M112.023093 23221899 Iyer A, Niemann M, Serricchio M, Dewar CE, Oeljeklaus S, Farine L, et al. The endoplasmic reticulum membrane protein complex localizes to the mitochondrial - endoplasmic reticulum interface and its subunits modulate phospholipid biosynthesis in Trypanosoma brucei. PLoS Pathog. 2022;18(5):e1009717. doi: 10.1371/journal.ppat.1009717 35500022 Schnaufer A. Evolution of dyskinetoplastic trypanosomes: how, and how often?. Trends Parasitol. 2010;26(12):557–8. doi: 10.1016/j.pt.2010.08.001 20801716 Domingo GJ, Palazzo SS, Wang B, Pannicucci B, Salavati R, Stuart KD. Dyskinetoplastic Trypanosoma brucei contains functional editing complexes. Eukaryot Cell. 2003;2(3):569–77. doi: 10.1128/EC.2.3.569-577.2003 12796302 Šubrtová K, Panicucci B, Zíková A. ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic trypanosomes. PLoS Pathog. 2015;11(2):e1004660. doi: 10.1371/journal.ppat.1004660 25714685 Rühle T, Leister D. Assembly of F1F0-ATP synthases. Biochim Biophys Acta. 2015;1847(9):849–60. doi: 10.1016/j.bbabio.2015.02.005 25667968 Shanmugasundram A, Starns D, Böhme U, Amos B, Wilkinson PA, Harb OS, et al. TriTrypDB: An integrated functional genomics resource for kinetoplastida. PLoS Negl Trop Dis. 2023;17(1):e0011058. doi: 10.1371/journal.pntd.0011058 36656904 Demichev V, Messner CB, Vernardis SI, Lilley KS, Ralser M. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat Methods. 2020;17(1):41–4. doi: 10.1038/s41592-019-0638-x 31768060 Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47. doi: 10.1093/nar/gkv007 25605792 Tinti M. OligoSeeker. https://doiorg/105281/zenodo15011916 2025. Mölder F, Jablonski KP, Letcher B, Hall MB, van Dyken PC, Tomkins-Tinch CH, et al. Sustainable data analysis with Snakemake. F1000Res. 2021;10:33. doi: 10.12688/f1000research.29032.3 34035898 Tinti M. myRna-seq. https://github.com/mtinti/myRna-seq 2025. 10.1371/journal.ppat.1013846.r001 Decision Letter 0 He Cynthia Academic Editor 2026 Cynthia HeCynthia He This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

29 Jan 2026

Origins and consequences of kinetoplast loss in trypanosomes

PLOS Pathogens

Dear Dr. Horn,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Mar 30 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Cynthia Y. He

Academic Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

All three reviewers agreed that the work presented interesting new data that may contribute to the understanding of kDNA loss and mitochondrial biology in general. The main critique is that the mutants weren’t functionally or morphologically characterized, and the proteomics results lack experimental confirmation. Additional studies to characterize mitochondrial morphology and functions in the mutants are suggested. Corroborating changes in targeted pathways and the formation of protein complexes experimentally would enhance the manuscript.

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Melanie Ridgway, Douglas Escrivani, Marketa Novotna, Amy Wood, Michele Tinti, Achim Schnaufer, and David Horn. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plospathogens/s/authorship#loc-author-contributions

2) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines:

https://journals.plos.org/plospathogens/s/figures

3) We have noticed that you have uploaded Supporting Information files, but you have not included a list of legends. Please add a full list of legends for your Supporting Information files after the references list.

4) Some material included in your submission may be copyrighted. According to PLOSu2019s copyright policy, authors who use figures or other material (e.g., graphics, clipart, maps) from another author or copyright holder must demonstrate or obtain permission to publish this material under the Creative Commons Attribution 4.0 International (CC BY 4.0) License used by PLOS journals. Please closely review the details of PLOSu2019s copyright requirements here: PLOS Licenses and Copyright. If you need to request permissions from a copyright holder, you may use PLOS's Copyright Content Permission form.

Please respond directly to this email and provide any known details concerning your material's license terms and permissions required for reuse, even if you have not yet obtained copyright permissions or are unsure of your material's copyright compatibility. Once you have responded and addressed all other outstanding technical requirements, you may resubmit your manuscript within Editorial Manager.

Potential Copyright Issues:

i) Figure 1A. Please confirm whether you drew the images / clip-art within the figure panels by hand. If you did not draw the images, please provide (a) a link to the source of the images or icons and their license / terms of use; or (b) written permission from the copyright holder to publish the images or icons under our CC BY 4.0 license. Alternatively, you may replace the images with open source alternatives. See these open source resources you may use to replace images / clip-art:

- https://commons.wikimedia.org

- https://openclipart.org/.

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This manuscript investigates how mutations in the gamma subunit of the mitochondrial F₁F₀-ATP synthase (gATPase) facilitate survival of Trypanosoma brucei in the absence of the kinetoplast DNA (kDNA). The study builds on the observation that certain gATPase mutations render T. brucei resistant to drugs targeting mitochondrial functions and enable life without kDNA. By employing targeted gene editing and selection with the ATP synthase inhibitor oligomycin, the authors isolate and characterize novel and known gATPase mutations (M282F, M282W, M282Y, L262P, A273P) and analyze their biochemical, proteomic, and cellular consequences.

This work provides an important contribution to our understanding of mitochondrial genome independence in eukaryotes and explores the functional consequences of mitochondrial genome loss, which has implications for parasite biology, transmission, and drug resistance. However, some aspects of the mechanistic interpretation and data presentation require further clarification and experimental support.

Major Claims by the Authors

1. Mutations in the gamma subunit enable viability without kDNA including the newly found M282F homozygous mutant

2. Study aims to explore the origins and consequences of kDNA loss

3. Use of oligomycin for enrichment of gamma ATPase mutants following oligo-directed mutagenesis

4. Homozygous M282F mutants resist acriflavine and survive kDNA loss

5. Proteomic analyses show mitochondrial remodeling associated with kDNA loss

o Pre-kDNA loss: Specific depletion of ATP synthase-associated proteins (excluding F₁ subunits).

o Post-kDNA loss: Loss of kDNA-binding proteins and mitochondrial RNA processing factors; increase in mitochondrial membrane-associated transporters.

6. Conclusion

o Cells with homozygous M282F mutations assemble a remodeled ATP synthase, tolerate kDNA loss, and undergo substantial mitochondrial proteome remodeling.

Reviewer #2: While the flexible mitochondrial biology of Trypanosoma brucei has been studied in detail, knowledge about how mutations that render the cells susceptible to kDNA loss and their full impact during drug treatments are understudied. In this body of work, the authors use a rapid and specific precision editing tool based upon oligo-targeting first reported in the field by the Horn lab. They combined this with a selection using 200 nm oligomycin. The authors generated mutants against known gamma ATPase mutations associated with kDNA dispensability and recover new mutations at the position M282. The methodology is robust with good internal controls to distinguish heterozygote and homozygous mutants.

The authors are laying the foundational tools for futures studies on analyzing mutations that occur in the wild and could drive drug resistance. There is a lot of interesting data that was generated but perhaps not fully analyzed. The heterogyzous mutant seems important for the trajectory of the population becoming kDNA dispenable but was not fully analyzed.

Reviewer #3: This study demonstrates that specific mutations in the nuclear-encoded γ subunit of the mitochondrial F₁F₀-ATP synthase enable Trypanosoma brucei to tolerate complete loss of kinetoplast DNA. Using precision oligo-targeting, the authors identify a novel homozygous γATPase M282F mutation that confers reduced sensitivity to both oligomycin and the kDNA-targeting drug acriflavine, and show that this mutation permits acriflavine-induced elimination of the mitochondrial genome. Quantitative proteomics reveals that γATPase mutation leads to selective remodelling of the ATP synthase complex prior to kDNA loss, followed by extensive mitochondrial proteome reprogramming after kDNA loss, including depletion of kDNA-binding and RNA-processing factors and upregulation of mitochondrial membrane transport systems.

While the study does not provide fundamentally new mechanistic insight into how γATPase mutations uncouple the ATP synthase, the proteomic analyses generate a valuable dataset that may be useful for further exploration of cellular adaptations to mitochondrial DNA loss.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. Oligomycin Resistance and Wild-Type gamma ATPase Contribution

The manuscript should explore whether the level of oligomycin resistance correlates with residual wild-type gamma ATPase expression in heterozygous strains. Such analysis would provide insights into the dosage sensitivity and functional threshold of the mutant protein.

2. Mitochondrial Morphology and Organelle Interaction

How do these mutations affect:

• Mitochondrial ultrastructure?

• ER-mitochondria contact sites?

• Electrochemical potential across the mitochondrial membrane?

These aspects are central to mitochondrial function and may be altered in kDNA-lacking cells. The authors should consider EM or confocal imaging, and membrane potential assays.

3. ATP Synthase Assembly Pathway

The authors suggest that assembly is arrested post-F₁–c-ring attachment, in analogy to other eukaryotes. This hypothesis is testable using blue native PAGE. Inclusion of such data or acknowledgment of this as a future direction would strengthen mechanistic claims.

4. TAC Components and kDNA Loss

Given the complete loss of kDNA in homozygous mutants, the status of TAC (tripartite attachment complex) proteins should be addressed. Are these proteins depleted? Their inclusion in the proteomic analysis (especially in Figure 5) would provide a useful internal control and functional insight.

5. Mitochondrial Import Machinery

The increase in membrane-associated transporters is noted, but specific components and the extent of change should be detailed in the main text (not solely in figures). Which import components are increased, and by how much?

Figure-Specific Critiques

Figure 1D

• Structural labeling is incomplete. Clearly label F₁ and F₀ domains.

• Mutation positions (e.g., M282F) should be mapped explicitly also in the stick figure

• Color coding lacks explanation; provide a legend or rationale.

Figure 3

• Clarify the meaning of n in the legend; does not refer to replicates?

• Justify inclusion of both log₂ fold-change and –log₁₀ FDR. Are both dimensions necessary?

• Consider whether this figure is redundant with Figure 5. If retained, explain the complementary value.

Figure 4A

• Discrepancy noted between DNA channel and merged image.

• A single-cell image is insufficient to claim kDNA loss. Include a field with multiple cells for representativeness.

Figure 5

• The presentation is inconsistent and cluttered. Multiple visualization types could/should be unified.

• Cohort terms (Figure 5E) require definition. 400+ proteins for "kinetoplast" are a lot? Is that real? Are functional overlaps considered, i.e. the ribosome etc.?

• Indicate what red/blue colors represent.

• Include TAC proteins to assess stability of mitochondrial structural elements.

• Revise labeling of "increased" and "decreased" with fold enrichment to improve clarity.

• PUF9 Target 1 Speculation: The discussion suggests that kDNA loss disrupts kDNA–nuclear genome communication. This needs clarification. What is the functional relevance of PUF9 targets in this context? How does their regulation support the proposed communication axis?

• Scope and Novelty: While the experiments are technically sound and well-conceived, the manuscript would benefit from a clearer articulation of what new insights this study adds over prior work. Specifically:

o What do the newly identified mutations (e.g., M282F) reveal beyond earlier L262P and A273P studies?

o How does this work advance our understanding of the origins (not just consequences) of kDNA loss?

• Comparative Context: The authors may consider placing their findings in the broader context of mitochondrial genome loss in other systems (e.g., Saccharomyces cerevisiae “petite” mutants) to highlight evolutionary or mechanistic parallels.

Reviewer #2: 1. In the current study it appears that the kDNA loss does not occur until after drug pressure from acriflavine. This is not a compound that would be used in practice though. If one is considering what is driving kDNA loss and drug resistance in the field perhaps using a drug more relevant to human treatments is warranted. With proof of principle in place with acriflavine, the authors should use a drug(s) in use and evaluate the outcome of their homozygous M282F mutant (perhaps even the hetrozygote).

2. Authors should provide more details on the proteomics experiment of homozygous M282F mutant vs WT. There is clearly a subset of proteins other than F0 or F1 subunits that are also impacted when generating this mutant cell line. Did the authors perform proteomics on the M282F hetrozygote to identify significant changes form WT that were emerging to drive the population toward becoming kDNA dispensible? This progression would be then interesting to correlate with the different responses to acriflavine and oligomycin dose-response data.

3. Similarly because selection of the ATPase mutants is already an experiment – the authors should report percent loss of kDNA compared to WT for each of the precision oligo edited clones that were generated (ie. prior to sub-lethal treatment with acriflavine or other drug).

4. Could the authors please rewrite the abstract. The authors’ summary was more informative/clear than the abstract (just lacked some specifics). For example in the abstract pre-kDNA-loss is introduced out of context; proposed a remodelled ATP suynthase complex – was this isolated and characterized? Rewriting would provide better clarity.

5. It would be beneficial if the authors could focus the discussion more on how homozygous vs heterozygous mutations could drive events in the wild. This is interesting and directly related to the problem of emerging subspecies and drug resistance….and related this to current drug classes that are used or overused in practice.

Reviewer #3: Although the genetic and proteomic analyses strongly suggest altered ATP synthase function and mitochondrial physiology in the gamma ATPase mutants, the study remains largely descriptive. The manuscript would be strengthened by direct functional measurements, such as mitochondrial membrane potential, ATP levels, or ATPase activity, before and after kDNA loss. Such data would more firmly link the observed molecular changes to mitochondrial function.

The quantitative proteomics provide a rich dataset documenting extensive mitochondrial remodelling following kDNA loss. However, the functional significance of many of the observed changes remains unclear (for example, increased abundance of TOM, TIM, MICOS complexes, and various mitochondrial carrier proteins). Experimental validation of selected pathways or protein complexes highlighted in the proteomic analysis would strengthen conclusions regarding their adaptive or mechanistic roles.

The growth defect observed in kDNA-negative mutants is noted but not analysed further. Additional characterization of this phenotype could provide important insight into the biological costs associated with kDNA loss and help contextualize the adaptive significance of these mutations.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: 1. Please define kinetoplast more clearly. In some cases it sounds as if this is a separate organelle, in other instances it is stated “loss of the kinetopalst” – so loss of an organelle or loss of the DNA? Throughout the manuscript there are instances where kineoplast loss is used and other where kDNA loss is preferred.

2. eliminate use of “approx.”

3. Discussion of multidrug resistant – please be more precise by indicating which two classes of drugs are involved with the resistance, or are these parasites resistant to all drugs in use for treatments?

4. abbreviation for kinetoplast DNA is defined in Introduction – it is only needed once but is reintroduced several times.

5. Please state the doubling times of the different clones that were generated.

6. Authors should acknowledge TritrypDB for information they access – you reference the database in figure 5 legend – full acknowledgment is appropriate.

7. Figure 1D – the authors indicate that there are predicted conformational changes due to the precision editing mutants. Two are obvious a third is less obvious. Could the authors provide additional text to describe the difference since it is hard to see this in a status 2D image.

8. Could the authors link the conformational changes to M282F proteomic outcomes – are there similarities/differences that arise from the L262P and A273P structural changes when compared the current dataset for M282F? Reviewer recognizes that this data might not have been collected in the previous studies and does not think this type of additional proteomics studies of the other mutants are warranted in the current study.

9. awkward wording that described which strain was used for sequencing – just state the strain.

10. End of paragraph related to Fig3 – statements was…. Thus, proteomic analysis revealed highly specific depletion of subunits of the FO component of the T. b. brucei ATP synthase in homozygous M282FTTT mutants pre kDNA loss. Perhaps it would be better to state it as prior to acriflavine treatment?

11. Could the authors report how long it took to achieve stable populations following the sublethal treatment with acriflavine and the dilution cloning process – they mentioned slower doubling times….were the cells considered fully recovered after dilution cloning?

Comments on Figure and Legends:

1. Fig1A: - Nice diagram outlining approach, however in the flask designated oligomycin-resistant cell, why are all the cells blue. As diagramed the approach should yield red and green resistant cells from the precision editing.

2. Fig1B; what does the red outline square represent?

3. Fig1D: what does the vertical line represent in the alphafold portion of the figure?

4. Fig4A: authors should outline the cells or provide phase images. Current images look as if the nucleus is outside of the cell body.

5. Fig4B: the labelling of the Mini sequence data is a little misleading – is their nomenclature that can be added to indicate those are indeed different classes of sequences?

6. Fig4B legend: please specify exactly which clone was used for the sequence analysis.

7. Fig. 4C: it is very difficult to see data because of the grey scale chosen. Please improve by using a colored scale. It looks like there might be some maxicircle sequence retained in one of the clones???

8. Fig5E: The boxes and whiskers are very difficult to see in the current figure.

9. Figure5E legend: indicate what the two colors represent.

Reviewer #3: The title appears more suitable for a review article than for a primary research paper. In particular, the term “origins” may imply a broad evolutionary origin of organisms lacking mitochondrial DNA, whereas this study focuses on the identification of three γATPase mutations and experimental validation of one of them. Similarly, the term “consequences” may be somewhat overstated. The consequences described here are largely inferred from proteomic datasets. While valuable, these data provide limited direct insight into how cells functionally adapt to the loss of mitochondrial DNA.

The authors state that the homozygous M282F mutant is resistant to acriflavine, yet kDNA loss is induced using this same drug. Given that similar EC₅₀ values are observed for heterozygous L262P and A273P mutants, it appears that this is how “resistance” is defined in this context. However, acriflavine is then used to induce kDNA loss in an acriflavine-resistant strain, which is difficult to reconcile. Clarification of the definition of “resistance” would therefore be helpful. In addition, it would be informative to indicate the acriflavine sensitivity of naturally occurring T. evansi and T. equiperdum, which are expected to be kDNA-independent.

Can the whole-genome sequencing data be used to assess whether compensatory mutations arise in the nuclear genome following kDNA loss? A brief comment on this possibility would strengthen the manuscript.

The statement that doubling time increased by 47% is somewhat abstract. Reporting the actual doubling times and stating that growth is slowed approximately 1.5-fold would be clearer and more intuitive.

The strain labels used in the supplementary tables (e.g. S22, S24, S312, S315) do not clearly correspond to the strain descriptions in the main text or figures, which use +kDNA and −kDNA designations. Clarification of this nomenclature is needed.

Which specific mitochondrial carrier proteins are upregulated? Can any functional adaptations be inferred from these changes?

Several sections refer to decreased mitochondrial membrane potential in −kDNA cells. Can this be experimentally measured to directly support this conclusion?

The depletion of ATP synthase Fo components prior to kDNA loss is a key observation. Is this more likely due to protein instability (e.g. degradation of unassembled subunits) or to regulated changes in gene expression? Clarification or discussion of this point would be helpful.

The authors suggest that F₁ remains attached to the c-ring in the absence of other Fo components. Could this be tested experimentally, for example using blue native PAGE to assess ATP synthase subcomplexes?

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy ..

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure resubmission:

Reproducibility:

?>

 10.1371/journal.ppat.1013846.r002 Author response to Decision Letter 1 Submission Version 1

16 Feb 2026

Submitted filename: Ridgway 03269 Reviewer responses.pdf

 10.1371/journal.ppat.1013846.r003 Decision Letter 1 He Cynthia Academic Editor 2026 Cynthia HeCynthia He This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

14 Mar 2026

Dear Dr. Horn,

We are pleased to inform you that your manuscript 'Genetic origins and proteomic consequences of kinetoplast loss in trypanosomes' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests. We would also like you to consider the minor changes suggested by Reviewer 2.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Cynthia Y. He

Academic Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: (No Response)

Reviewer #2: For this resubmission, the authors provide evidence that specific mutations in the nuclear-encoded γ subunit of the mitochondrial F₁F₀-ATP synthase enable Trypanosoma brucei to tolerate complete loss of kinetoplast DNA. Specifically, they use precision oligo-targeting, to generate a novel heterozygous and homogyzous γATPase M282F mutation. Only the homozygous mutant confers reduced sensitivity to the kDNA targeting drug acriflavine and demonstrate the homozygous mutation permits acriflavine-induced elimination of the kDNA. The authors use high resolution quantitative proteomics to further demonstrate selective remodelling of the ATP synthase complex prior to kDNA loss, followed by extensive mitochondrial proteome reprogramming after kDNA loss.

Reviewer #3: I appreciate the authors’ efforts to address the reviewers’ comments and have no further remarks.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: No Major issues identified.

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: The authors address essentially all of the reviewers’ comments by adding additional functional data to assess mitochondrial membrane potential under the various WT and mutant conditions and modified images. Additionally, the authors added clarifying language to the text especially related to the impact on known protein complexes. They have made a clear statement on the preservation of the TAC complex (except for p166), clarified language surrounding a hypothesized ATP synthase assembly pathway and highlight additional ATPas associated proteins in their proteomic analyses.

Lastly, two reviewers thought the heterozygous mutant was significant. The authors briefly addressed the heterozygosity issue with the following statement in the manuscript ”Since differential expression of mutant alleles could impact the behaviour of heterozygous mutants, we favoured the analysis of homozygous mutants.”

It would be beneficial if the authors could add additional content especially related to their more extended explanation to reviewers: “Even if we were able to determine the relative levels of wild-type and mutant gATPase protein in the heterozygous strains, we would be unable to determine relative levels in individual cells, which could impact oligomycin resistance and other phenotypes.”

Can the authors indicate what is known in trypanosomes about expression from different alleles (in this case a WT vs mutant allele) or is this an area that is completely unexplored in their field? An expanded discussion on this would be ideal.

Reviewer #3: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy ..

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

 10.1371/journal.ppat.1013846.r004 Acceptance letter He Cynthia Academic Editor 2026 Cynthia HeCynthia He This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Dear Dr. Horn,

We are delighted to inform you that your manuscript, "Genetic origins and proteomic consequences of kinetoplast loss in trypanosomes," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

For Research Articles, you will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064