Parasiticidal effect of clindamycin on Toxoplasma gondii grown in cultured cells and selection of a drug-resistant mutant

Abstract

Clindamycin, which has been reported to have no significant in vitro activity against Toxoplasma gondii, actually markedly inhibits the growth of this parasite in infected human fibroblasts. When measured 3 days after treatment, the concentration required to reduce parasite growth by 50%o is about 1 ng/ml. Some observers failed to note this inhibition because of its markedly delayed onset. At 6 ng/ml, clindamycin is parasiticidal, and the rate and extent of parasite killing increase with higher drug concentrations. With the aid of chemical mutagenesis, we isolated a parasite mutant that is approximately 100-fold more resistant to clindamycin than is the wild type. Lincomycin inhibits T. gondii at a higher 50% inhibitory concentration, about 100 ng/ml. The clindamycin-resistant mutant is partially cross-resistant to lincomycin.While Derouin et al. (3) observed that clindamycin concentrations as low as 0.5 ng/ml inhibited the growth of Toxoplasma gondii, at least three other laboratories have reported that the drug has no in vitro activity (5,6,8). These latter observations are in marked contrast both to the well-established efficacy of clindamycin phosphate against toxoplasmosis in infected mice (1, 6) and to the preliminary evidence for the successful use of clindamycin phosphate to treat toxoplasmic encephalitis in AIDS patients (2). We show here that clindamycin is actually a potent parasiticidal agent in cultured cells infected with T. gondii, with a 50% inhibitory concentration (IC50) that is among the lowest reported for any antitoxoplasma drug. This activity previously escaped detection by several laboratories because the drug has little effect on parasite multiplication until many cell divisions have occurred. MATERIALS AND METHODSThe host cells for all experiments were confluent cultures of human fibroblasts that were maintained as previously described (13), except that the medium for growth contained 10% calf serum and that for infection contained 1% fetal bovine serum. The parasites were our cloned line (13) of the RH strain or mutants derived from this clone. The parasites were maintained by serial subculturing in human fibroblasts. Infected cells were disrupted by forced extrusion through a 25-gauge needle to release intracellular parasites. The newly released parasites were assayed by a plaque procedure (13). In all final experiments, this procedure was carried out with 25-cm2 flasks and the plaques were counted without staining. For preliminary plaque assays with multiwell trays, the monolayer was first fixed with 0.3 N trichloroacetic acid and then stained with Coomassie blue. Parasites were cloned by the detection of a single plaque 6 days after infection of 0.15-cm2 wells with suitably diluted suspensions of extracellular T. gondii.

In vitro assays elucidate peculiar kinetics of clindamycin action against Toxoplasma gondii

“…We have confirmed the delayed death phenotype observed when these drugs were used against T. gondii in vitro (20,21) and examined the kinetics of this effect in detail, by directly measuring parasite replication as a function of the drug concentration and the duration and timing of drug exposure. Our studies indicate that even concentrations of clindamycin far in excess of the 50% inhibitory concentration have no immediate effect on parasite multiplication.…”

“…These compounds are known to block protein synthesis in bacteria by interacting with the peptidyl transferase domain of 23S rRNA (5), but their target in T. gondii and related parasites remains unclear (1). Early difficulties in establishing a functional in vitro system to study clindamycin and macrolide action against T. gondii (4, 9, 17) can now be explained by the long lag period between drug administration and effect (20,21). Nanomolar drug concentrations block parasite replication, but only 2 to 3 days after treatment-a remarkable delay, considering that the tachyzoite undergoes ϳ8 generations in this time.…”

Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii

“…At least some of its functions are essential for the parasite and are responsible for its conservation during evolution. Macrolide (and other) antibiotics targeting apicoplast ribosomes (30)(31)(32) and fluoroquinolone antibiotics eliminating the apicoplast genome (33) kill the parasite. Thiolactomycin, an inhibitor of fatty acid elongation specific for the multisubunit enzyme found in the apicoplast, has been shown to prevent the growth of P. falciparum (5).…”

Inhibition of cytoplasmic and organellar protein synthesis in Toxoplasma gondii. Implications for the target of macrolide antibiotics.

“…Extremely low concentrations of clindamycin or azithromycin (10 and 30 ng/ml, respectively) are sufficient to inhibit parasite growth in extended tissue culture assays (2,3), and it has been suggested that the activity of azithromycin against Toxoplasma results from the inhibition of total protein synthesis (4). The observation that > 100-fold higher drug concentrations are needed to even partially inhibit total protein synthesis in extracellular parasites (3) has been taken to imply that parasites growing inside the parasitophorous vacuole must concentrate these compounds extensively within the parasite cytoplasm (4).…”