Solanesyl diphosphate synthase, an enzyme of the ubiquinone synthetic pathway, is required throughout the life cycle of Trypanosoma brucei

Abstract

Ubiquinone 9 (UQ9), the expected product of the long-chain solanesyl diphosphate synthase of Trypanosoma brucei (TbSPPS), has a central role in reoxidation of reducing equivalents in the mitochondrion of T. brucei. The ablation of TbSPPS gene expression by RNA interference increased the generation of reactive oxygen species and reduced cell growth and oxygen consumption. The addition of glycerol to the culture medium exacerbated the phenotype by blocking its endogenous generation and excretion. The participation of TbSPPS in UQ synthesis was further confirmed by growth rescue using UQ with 10 isoprenyl subunits (UQ10). Furthermore, the survival of infected mice was prolonged upon the downregulation of TbSPPS and/or the addition of glycerol to drinking water. TbSPPS is inhibited by 1-[(n-oct-1-ylamino)ethyl] 1,1-bisphosphonic acid, and treatment with this compound was lethal for the cells. The findings that both UQ9 and ATP pools were severely depleted by the drug and that exogenous UQ10 was able to fully rescue growth of the inhibited parasites strongly suggest that TbSPPS and UQ synthesis are the main targets of the drug. These two strategies highlight the importance of TbSPPS for T. brucei, justifying further efforts to validate it as a new drug target.

FIG 1

Northern and Western blot analyses of the TbSPPS T. brucei RNAi cell lines. (A) Effects of TbSPPS RNAi on mRNA and protein levels in the procyclic stage (clone 4). Total RNA and protein were extracted from parental 29-13 cells (lane 1), noninduced cells (lane 2), and procyclic cells on day 6 after RNAi induction (lane 3). The two upper panels show Northern blot analysis results with the full-length TbSPPS gene used as a probe. Ethidium bromide-stained rRNAs was used as a loading control. The two lower panels show Western blot analysis results showing expression of the TbSPPS protein in the same cell lines as in the RNA panels. The target protein was detected with specific polyclonal anti-TbSPPS antibodies. Antibody against RBP16 was used as a loading control. (B) Effects of TbSPPS RNAi on mRNA and protein levels in the bloodstream stage (clone 6) on day 3 after RNAi induction. Total protein was extracted from parental SM cells (lane 1), noninduced cells (lane 2), and bloodstream cells 3 days after RNAi induction (lane 3). TbSPPS and RBP16 were detected as described for panel A.

FIG 2

Effects of TbSPPS RNAi on cell growth of the procyclic form (A) and bloodstream form (B) of T. brucei. Cell densities (in cells ml⁻¹) of procyclic forms and bloodstream forms were measured and diluted as described in the text. The total cell numbers were calculated and plotted on a logarithmic scale on the y axis over 14 days (A) or 6 days (B). Clonal procyclic form (A) and bloodstream form (B) parasite growth in the absence or presence of 1 mg ml⁻¹ tetracycline, which induces RNAi, is indicated in the graphs. The growth data for parental procyclic (strain 29-13 [A]) and bloodstream (SM [B]) forms are also shown.

FIG 3

Effects of TbSPPS RNAi on the oxygen consumption rate in the procyclic (A) and bloodstream (B) forms of cells. (A) For procyclics, the relative contributions of the alternative pathway via TAO and of the cytochrome-mediated pathway (OXPHOS) were measured in parental 29-13 cells, noninduced cells (-), and cells at 2, 6, and 10 days after RNAi induction. The amount of O₂ consumption inhibited by KCN (0.1 mM) reflected the capacity of the cytochrome-mediated pathway, while the amount inhibited by SHAM (0.03 mM) represented the TAO activity. The noninhibited residual oxygen consumption was taken as zero. The means and standard deviation values of three experiments are shown. (B) In the absence of the cytochrome-mediated pathway in the bloodstream-form cells, all respiration is mediated by TAO. Oxygen consumption was measured in parental SM cells, noninduced cells (-), cells after 1 or 3 days RNAi induction, cells after inhibition by compound 1 (1 μM for 24 h), and an RNAi cell group supplied with UQ10. Statistical significance levels compared to the control group are indicated by asterisks: *, P < 0.05; ***, P < 0.0005; ****, P < 0.00005.

FIG 4

Growth curve of bloodstream transfectant cells in the presence of glycerol or compound 1. (A) The addition of 4 mM glycerol to the medium had an inhibitory effect on TbSPPS knockdown after RNAi induction (+, glycerol), while only a mild effect was observed for the SM parental cells (SM, glycerol) and noninduced cells (-, glycerol). The same cell lines were grown in the absence of glycerol as controls. (B) Chemical structure of 1-[(n-oct-1-ylamino)ethyl] 1,1-bisphosphonic acid (compound 1), a potent inhibitor of the enzymatic activity of TcSPPS. (C) The addition of 1 μM compound 1 to the medium was lethal for the RNAi-induced TbSPPS knockdown cells (+, Comp1), while just a very small effect was observed for the SM parental cells (SM, Comp1) and the noninduced cells (-, Comp1). The same cell lines were grown in the absence of compound 1 as controls. The experiment was repeated three times, and a representative curve is shown.

FIG 5

Generation of reactive oxygen species (A) and paraquat treatment (B) in TbSPPS procyclic cells. Experiments were performed at least twice with triplicate samples. (A) Parental 29-13 cells (area under black line), noninduced cells (gray area), and procyclics 6 days after RNAi induction (area under gray line) were incubated in the presence of 5 mg ml⁻¹ dihydroethidium for 30 min. The fluorescence distributions, measured by flow cytometry, were plotted as frequency histograms. (B) Growth of procyclic form noninduced cells (clone 4) or cells induced by RNAi for 5 days (clone 4 Ind), incubated for 3 additional days in the presence of 0.2, 0.5, 1, or 2 μM paraquat. The growth of cells in the absence of paraquat, either noninduced or RNAi induced, was considered to be 100%. Statistical significance levels for comparisons between the two groups are indicated by asterisks: ***, P < 0.0005; ****, P < 0.00005.

FIG 6

Survival of mice infected with TbSPPS RNAi transfectant cells was prolonged upon the addition of glycerol and doxycycline. Drinking water available to four groups of mice, each consisting of five individuals, was either pure (water) or supplemented with 1 mg ml⁻¹ doxycyline sweetened with 50 mg ml⁻¹ of sucrose, 5% glycerol, or both doxycycline and glycerol. The survival of mice was followed on a daily basis.

FIG 7

Metabolic effects of the inhibition by compound 1 on wild-type parasites. (A) Measurement of the UQ pool. HPLC representative runs for untreated and treated bloodstreams are shown. The positions of the calibration standards are indicated by arrows. (B) The ΔΨ_m displayed by procyclics treated with different concentrations of compound 1. Statistical analysis results and a representative experiment are presented. Asterisks indicate significant differences in comparison to the control group (untreated parasites). The arrowhead represents the position of the depolarized membrane control, CCCP. (C) ROS level in procyclics treated for 72 h with 50 μM compound 1. The data are the means ± standard deviations of at least two independent experiments. (D) Rescue of parental SM bloodstream cells. The addition of 10 μM compound 1 to the medium was lethal within 3 days (SM + Comp1). Further addition of 20 μM UQ10 fully rescued cell growth. Nontreated (SM) and cells treated only with UQ10 (SM + UQ10) were used as controls.