Ubiquinone synthesis in mitochondrial and microsomal subcellular fractions of Pneumocystis spp.: differential sensitivities to atovaquone

Abstract

The lung pathogen Pneumocystis spp. is the causative agent of a type of pneumonia that can be fatal in people with defective immune systems, such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), inhibits mitochondrial electron transport and is effective in clearing mild to moderate cases of the infection. Purified rat-derived intact Pneumocystis carinii cells synthesize de novo four CoQ homologs, CoQ7, CoQ8, CoQ9, and CoQ10, as demonstrated by the incorporation of radiolabeled precursors of both the benzoquinone ring and the polyprenyl chain. A central step in CoQ biosynthesis is the condensation of p-hydroxybenzoic acid (PHBA) with a long-chain polyprenyl diphosphate molecule. In the present study, CoQ biosynthesis was evaluated by the incorporation of PHBA into completed CoQ molecules using P. carinii cell-free preparations. CoQ synthesis in whole-cell homogenates was not affected by the respiratory inhibitors antimycin A and dicyclohexylcarbodiimide but was diminished by atovaquone. Thus, atovaquone has inhibitory activity on both electron transport and CoQ synthesis in this pathogen. Furthermore, both the mitochondrial and microsomal fractions were shown to synthesize de novo all four P. carinii CoQ homologs. Interestingly, atovaquone inhibited microsomal CoQ synthesis, whereas it had no effect on mitochondrial CoQ synthesis. This is the first pathogenic eukaryotic microorganism in which biosynthesis of CoQ molecules from the initial PHBA:polyprenyl transferase reaction has been unambiguously shown to occur in two distinct compartments of the same cell.

FIG. 1.

CoQ synthesis and atovaquone. A. Generalized scheme of CoQ biosynthesis de novo. The benzoquinone ring is derived from chorismate, which is produced by the shikimic acid pathway. The polyprenyl moiety is derived from the isoprenoid pathway. After condensation of PHBA and a polyprenyl diphosphate, several modifications at the aromatic ring moiety occur, leading to the production of the completed CoQ molecule. B. Structure of the hydroxynaphthoquinone atovaquone (566C80), an analog of ubiquinone.

FIG. 2.

HPLC separation of P. carinii CoQ homologs. A. CoQ₇, CoQ₈, CoQ₉, and CoQ₁₀ were detected in P. carinii, and the peak areas that represented these homologs were integrated in this analysis, set at 5 μA full scale. CoQ₆ was not detected in the chromatogram. The arrow denotes the elution time for authentic CoQ₆. B. The four CoQ homologs in P. carinii were detected using 1-μA full scale, in which CoQ₁₀ was too high for integration but CoQ₇ is clearly evident in the tracing. CoQ₆ was not detected even at this higher detector sensitivity. C. Authentic CoQ standards analyzed under similar HPLC conditions indicate the elution times of CoQ₆, CoQ₇, CoQ₈, CoQ₉, and CoQ₁₀.

FIG. 3.

Characterizations of [U-¹⁴C]PHBA incorporation into CoQ using P. carinii whole-cell homogenates. Effects of pH and temperature on the incorporation in vitro of [U-¹⁴C]PHBA into P. carinii ubiquinones using whole-cell homogenates are shown. A. Effects of pH. Optimal pH was detected at pH 7.5. B. Effects of temperature. Incorporation was observed over a broad temperature range; optimal temperature was 37°C, which sharply declined at temperatures higher than 40°C. There was relatively high activity at 20°C, which is the optimal growth temperature reported for in vitro axenic cultivation of the organism (43). C. First-order kinetics was exhibited with increased substrate concentration. Values are means ± SEM; n = 4 separate experiments. D. Effects of atovaquone, shown in a double reciprocal plot demonstrating competitive inhibition. Filled squares, without atovaquone; open squares, with 2 nM atovaquone; open circles, with 10 nM atovaquone. Values are means ± SEM; n = 3 separate experiments. E. Eadie-Hofstee representation of the data shown in panel D, verifying competitive inhibition kinetics of atovaquone on the incorporation of PHBA into P. carinii ubiquinones. F. Maximal inhibition attained with atovaquone was 60% of untreated controls (40% inhibition); no further inhibition was observed at concentrations up to 10 μM. Values represent means ± SEM; n = 4.

FIG. 4.

Inhibition of PHBA incorporation into P. carinii CoQ homologs by atovaquone using whole-cell homogenates. Homologs in each set are arranged from left to right and correspond to CoQ₇, CoQ₈, CoQ₉, and CoQ₁₀, respectively.

FIG. 5.

Light microscopy of P. carinii subcellular fractions treated with MitoTracker Green FM. Bar, 5 μm. A. Intact cells viewed under fluorescence optics. The mitochondria within cells and in the plane of focus are seen exhibiting intense fluorescence. B. The mitochondrial fraction showing particles similar to the size of mitochondria in intact cells was isolated and contained mitochondrial-specific lipids. C. The postmitochondrial fraction does not contain objects exhibiting fluorescence, demonstrating that this subcellular fraction is devoid of mitochondria and mitochondrial fragments.

FIG. 6.

Marker enzymes for mitochondrial and microsomal fractions. Open diamonds, mitochondrial fraction; open circles, microsomal fraction; X, soluble fraction. A. Succinate dehydrogenase activity was detected in the P. carinii mitochondrial fraction but not in the microsomal or soluble fractions. B. Glucose-6-phosphate dehydrogenase activity was predominantly found in the P. carinii microsomal fraction. A low level of enzyme activity was detected in the soluble fraction; no activity was detected in the mitochondrial fraction.

FIG. 7.

Effects of atovaquone on [U-¹⁴C]PHBA incorporation into CoQ in P. carinii mitochondrial and microsomal fractions. Circles, mitochondrial fraction; squares, microsomal fraction. Incorporation into the microsomal fraction was inhibited, whereas the drug had no effect on CoQ synthesis in the mitochondrial fraction. Each point represents the mean ± SEM; n = 3.