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Updated: Oct 2, 2026
Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay
Published on: April 29, 2020
An rRNA mutation identifies the apicoplast as the target for clindamycin in Toxoplasma gondii
Manel Camps1, Gustavo Arrizabalaga, John Boothroyd
1Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124, USA.
Abstract:
Toxoplasma gondii is a protozoan sensitive to several inhibitors of prokaryotic translation (e.g. clindamycin, macrolides and tetracyclines). A priori, two prokaryotic-like organelles, the 'apicoplast' (a non-photosynthetic plastid) and the mitochondrion, are likely targets for these drugs. Without using overt mutagenesis, we selected two independent clones (ClnR-4 and ClnR-21) with strong and stable clindamycin resistance. Several lines with substantial but lower levels of resistance were also isolated with (XR-46) or without (ClnR-23) overt mutagenesis. The ClnR-4 and ClnR-21 mutants uniquely possess a G-->U point mutation at position 1857 of the apicoplast large-subunit rRNA, whereas no mutation was identified in this region for ClnR-23 or XR-46. Position 1857 corresponds to position 2061 in Escherichia coli where it is predicted to bind clindamycin. The mutation is present in all the apicoplast rDNA copies (an estimated 12 per organelle), indicative of a strong selective advantage in the presence of clindamycin. In the absence of drug, however, such a mutation is unlikely to be neutral, as the G is a critical contributor to the transpeptidation reaction and absolutely conserved in all kingdoms. This may explain why ClnR-4 shows a slight growth defect in vitro. These mutants provide direct genetic evidence that apicoplast translation is the target for clindamycin in Toxoplasma. Further, their sensitivity profiles to other antibiotics specific for the large ribosomal subunit (macrolides and chloramphenicol) and, intriguingly, the small subunit (doxycycline) argue that these drugs also target the apicoplast ribosome.
Insights
Toxoplasma gondii drug resistance reveals the apicoplast ribosome as the target for clindamycin and other antibiotics. This finding provides genetic evidence for apicoplast translation inhibition in this protozoan parasite.
Area of Science:
- Molecular Parasitology
- Antimicrobial Resistance
- Organelle Biology
Background:
- Toxoplasma gondii, a protozoan parasite, exhibits sensitivity to prokaryotic translation inhibitors like clindamycin.
- The apicoplast and mitochondrion are potential targets for these drugs due to their prokaryotic-like ribosomes.
- Understanding drug targets is crucial for developing effective treatments against toxoplasmosis.
Purpose of the Study:
- To genetically identify the specific target of clindamycin in Toxoplasma gondii.
- To investigate the role of the apicoplast ribosome in parasite drug sensitivity.
- To explore the mechanism of resistance to clindamycin and related antibiotics.
Main Methods:
- Selection of clindamycin-resistant Toxoplasma gondii clones through mutagenesis and non-mutagenesis approaches.
- Genetic analysis, including sequencing of apicoplast ribosomal RNA (rRNA) genes.
- Characterization of drug sensitivity profiles for resistant mutants.
Main Results:
- Two resistant clones (ClnR-4 and ClnR-21) exhibited a specific G to U point mutation at position 1857 in the apicoplast large-subunit rRNA.
- This mutation site corresponds to the clindamycin binding site in Escherichia coli ribosomes.
- Resistant mutants showed altered sensitivity to other antibiotics targeting both large and small ribosomal subunits, implicating the apicoplast ribosome.
Conclusions:
- The identified mutation provides direct genetic evidence that the apicoplast ribosome is the primary target for clindamycin in Toxoplasma gondii.
- Antibiotics targeting prokaryotic ribosomes, including macrolides, chloramphenicol, and doxycycline, likely exert their effects via the apicoplast.
- These findings have significant implications for understanding antiparasitic drug action and developing new therapeutic strategies.
