Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes

Abstract

Viability of the tsetse fly-transmitted African trypanosome Trypanosoma brucei depends on maintenance and expression of its kinetoplast (kDNA), the mitochondrial genome of this parasite and a putative target for veterinary and human antitrypanosomatid drugs. However, the closely related animal pathogens T. evansi and T. equiperdum are transmitted independently of tsetse flies and survive without a functional kinetoplast for reasons that have remained unclear. Here, we provide definitive evidence that single amino acid changes in the nuclearly encoded F1FO-ATPase subunit γ can compensate for complete physical loss of kDNA in these parasites. Our results provide insight into the molecular mechanism of compensation for kDNA loss by showing FO-independent generation of the mitochondrial membrane potential with increased dependence on the ADP/ATP carrier. Our findings also suggest that, in the pathogenic bloodstream stage of T. brucei, the huge and energetically demanding apparatus required for kDNA maintenance and expression serves the production of a single F1FO-ATPase subunit. These results have important implications for drug discovery and our understanding of the evolution of these parasites.

Keywords: RNA editing; dourine; dyskinetoplastic; mitochondrial DNA; surra.

Fig. 1.

Mutations in ATPase γ allow BF T. b. brucei to survive kDNA loss. (A) Acr sensitivity of γL262P-expressing and control BF trypanosomes given as EC₅₀ values. Error bars are SEM; n ≥ 3. (B) Cumulative growth in 20 nM Acr of cells ectopically expressing WT F₁γ or an L262P, A273P, A281del, or M282L mutated copy in an sKO background. Numbers indicate independent clones. Parental WT strain 427, T. b. brucei 164DK, and T. evansi Antat 3/3 were also analyzed. Fig. S2 A and B shows growth curves without Acr and for dKO cells. (C) Differential interference contrast (DIC) and fluorescence microscopy of DAPI-stained dKO + γL262P trypanosomes before and after exposure to 20 nM Acr. White arrowhead in pre-Acr exposure images indicate the kinetoplast. (Scale bars: 5 µm.) (D) PCR assay for presence of kDNA-encoded genes A6, ND4, ND7, and ND5 in dKO + γL262P cells before and after Acr exposure. The faint band observed with ND4 primers post-Acr treatment is a result of nonspecific amplification, which is shown by its larger size. The nuclearly encoded dihydrolipoamide dehydrogenase gene (LipDH) was assayed as a positive control. (E) Cumulative growth in the presence of 20 nM Acr of previously Acr-treated (and therefore, DK) γA281del clones 2 and 9 after they had been allowed to recover in Acr-free medium (dashed lines; B and Fig. S2B show the initial response of these clones to Acr exposure). The same clones but without any prior Acr exposure were included in the analysis (solid lines). The parental T. b. brucei 427 strain was assayed for comparison.

Fig. 2.

Viability of DK trypanosomes depends on expression of a mutated F₁γ. (A) Cumulative growth of T. evansi with Tetracycline (Tet) -inducible ectopic expression of a WT (circles) or L262P-mutated (squares) subunit γ (dashed lines and open symbols, + Tet; solid lines and closed symbols, −Tet). (B) A Tet-inducible γL262P was expressed in T. b. brucei cells, and kDNA loss was triggered by exposure to 20 nM Acr for 7 d. The culture was split, and expression of γL262P in one subculture was repressed by transfer to Tet-free medium (0 h). Cumulative cell growth in the presence (dashed line and open circles) or absence (solid line and closed circles) of Tet was determined.

Fig. 3.

The L262P mutation allows BF T. b. brucei to survive inhibition of mitochondrial gene expression. (A) Cumulative growth of T. b. brucei after RNAi-mediated knockdown of REL1 (dashed lines). Cells expressed either γWT (open circles) or γL262P (closed squares). RNAi was induced with 1 μg/mL Tet; uninduced control cultures are shown as solid lines. (B) Western analysis of REL1 protein expression from whole-cell lysates taken at the 72-h time point in A; α-tubulin was used as a loading control (Lower).

Fig. 4.

Subunit γ mutations that can compensate for kDNA loss in BF T. b. brucei uncouple F₁ and F_O and prevent ΔΨm loss. (A and B) ΔΨm of BF cells continuously cultured with 20 nM Acr and expressing either ectopic (A) γWT or (B) γL262P in a γ dKO background. ΔΨm was assessed using rhodamine 123 and flow cytometry. Baseline fluorescence was determined by preincubation with the protonophore trifluorocarbonylcyanide phenylhydrazone (FCCP) (0 h + FCCP). In A, insufficient cells survived beyond 48 h to accurately determine ΔΨm. (C) Oligomycin sensitivity (ED₅₀ values) of trypanosomes expressing either γWT or γL262P in a γ dKO background. The Acr-induced DK form of the γL262P-expressing cell line was assayed in parallel along with the parental WT 427 strain. Error bars are SEM; n ≥ 3. (D) The same as in C, except assessing bongkrekic acid sensitivity (given as EC₅₀ values).