Inhibitory effect of aureobasidin A on Toxoplasma gondii

Abstract

The apicomplexan parasite Toxoplasma gondii is a leading opportunistic pathogen associated with AIDS and congenital birth defects. Due to the need for identifying new parasite-specific treatments, the possibility of targeting sphingolipid biosynthesis in the parasite was investigated. Aureobasidin A, an inhibitor of the enzyme synthesizing the sphingolipid inositol phosphorylceramide, which is present in fungi, plants, and some protozoa but absent in mammalian cells, was found to block in vitro T. gondii replication without affecting host cell metabolism. Aureobasidin A treatment did not induce tachyzoite to bradyzoite stage conversion in T. gondii but resulted in a loss of intracellular structures and vacuolization within the parasite. In addition, aureobasidin A inhibited sphingolipid synthesis in T. gondii. Sphingolipid biosynthetic pathways may therefore be considered targets for the development of anti-T. gondii agents.

FIG. 1.

Sphingolipid synthesis in eukaryotic cells. Schematic representation of the synthetic pathways and site of action of the inhibitor aureobasidin A.

FIG. 2.

Effect of aureobasidin A on T. gondii replication after 24 h of drug treatment. (A) HFF monolayers were infected for 2 h with T. gondii, washed to remove extracellular parasites, and subsequently treated with the indicated concentrations of aureobasidin A (AbA). After 24 h of drug treatment, cells were fixed and permeabilized, and intracellular parasites were quantified by staining with a polyclonal anti-T. gondii tachyzoite serum, followed by a fluorescein-conjugated secondary antibody. The distributions of the parasite numbers in single vacuoles are expressed as percentages of the total vacuoles examined (n > 30). (B) Immunofluorescence analysis of intracellular parasites treated with vehicle (control) or 10 μg/ml of aureobasidin A (AbA) by the use of anti-T. gondii tachyzoite serum (α-T. gondii), followed by fluorescein-conjugated secondary antibodies and DAPI staining. (C) Immunofluorescence analysis of intracellular parasites treated with 10 μg/ml of aureobasidin A (AbA) by the use of anti-T. gondii tachyzoite serum, followed by fluorescein-conjugated secondary antibodies. N, normal parasites; A, aberrant parasites. Bar = 5 μm.

FIG. 3.

Effect of aureobasidin A on T. gondii replication after 48 h of drug treatment. (A) HFF monolayers were infected for 2 h with T. gondii, washed to remove extracellular parasites, and subsequently treated with the indicated concentrations of aureobasidin A (AbA). After 48 h of drug treatment, intracellular parasites were quantified by a colorimetric assay measuring the amount of parasite-expressed β-galactosidase. Results are presented as percentages of the control (vehicle-treated cells) values ± standard errors (n = 3). Results of a representative experiment (out of three) are shown. (B) HFF monolayers were infected for 2 h with T. gondii, washed to remove extracellular parasites, and treated with the indicated concentrations of aureobasidin A immediately after infection (AbA day 1) or 24 h postinfection (AbA day 2). Intracellular parasites were quantified at 48 h postinfection by a colorimetric assay measuring the parasite-expressed β-galactosidase, and data are expressed as described for panel A. (C) Metabolic activity of host cells treated for 48 h with vehicle (control), 10 μg/ml of aureobasidin A, or 20% sodium dodecyl sulfate (SDS), assessed by measuring AlamarBlue reduction (Biosource).

FIG. 4.

Reversibility of aureobasidin A-mediated inhibition of T. gondii replication. HFF monolayers were infected for 2 h with T. gondii, washed to remove extracellular parasites, and treated with 10 μg/ml of aureobasidin A for 24 or 48 h, followed by an additional 24- or 48-h incubation without the drug, respectively. After cell fixation and permeabilization, intracellular parasites were visualized with an antiserum against T. gondii tachyzoites followed by fluorescein-conjugated secondary antibodies. Parasites were considered viable when present in at least eight units per vacuole after 24 or 48 h of incubation in the absence of the drug. Data are expressed as percentages of vacuoles containing viable or nonviable parasites (total number of vacuoles examined, >50) ± standard errors of a representative from three experiments done in duplicate.

FIG. 5.

Analysis of bradyzoite stage conversion. HFF monolayers were infected for 2 h with T. gondii, washed to remove extracellular parasites, and treated either for 6 days with 10 μg/ml of aureobasidin A (AbA) or for 4 days with alkaline medium and ambient CO₂ (pH 8.1). After cell fixation in 4% paraformaldehyde and permeabilization in 0.2% Triton X-100, intracellular parasites were visualized with an antiserum against T. gondii tachyzoites, followed by fluorescein-conjugated secondary antibodies (α-T. gondii), or with an antiserum raised against the bradyzoite-specific antigen BAG5, followed by Cy3-conjugated secondary antibodies (α-BAG5). Bar = 5 μm.

FIG. 6.

Analysis of T. gondii ultrastructure upon aureobasidin A treatment. HFF monolayers were infected for 2 h with T. gondii, washed to remove extracellular parasites, and treated for 24 h with a vehicle (control) or 10 μg/ml of aureobasidin A (AbA). Samples were fixed and processed for transmission electron microscopy analysis as described in Materials and Methods. Bar = 2 μm.

FIG. 7.

Analysis of sphingolipid synthesis upon aureobasidin A treatment. (A) Representative thin-layer chromatography (TLC) results for inositol-labeled lipids extracted from T. gondii. HFF monolayers were infected with T. gondii and labeled with myo-[³H]inositol in the presence of vehicle (control) or 10 μg/ml of aureobasidin A (AbA) for 24 h. After washing of the cells, T. gondii tachyzoites were harvested, lipids were extracted from 10⁸ parasites, and aliquots were processed by mild alkaline hydrolysis (NaOH) and resolved by TLC analysis in solvent system A. (B) Representative TLC of palmitate-labeled lipids extracted from T. gondii or host cells. Purified parasites (10⁸) or 10⁷ host cells were labeled with [³H]palmitate in the presence of vehicle (Cntl) or 10 μg/ml of aureobasidin A (AbA) for 7 h. Lipids were extracted and resolved by TLC analysis in solvent system B. Cer, ceramide; SM, sphingomyelin. (C) Densitometric quantification of newly synthesized ceramide versus sphingomyelin ratio after labeling with [³H]palmitate. Data are expressed as percentages of newly synthesized ceramide/sphingomyelin in vehicle (Cntl)-treated parasites or host cells.