title: An rRNA mutation identifies the apicoplast as the target for clindamycin in Toxoplasma gondii.
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.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124, USA.
authors.forename: Manel
authors.initials: M
authors.lastname: Camps
authors.forename: Gustavo
authors.initials: G
authors.lastname: Arrizabalaga
authors.forename: John
authors.initials: J
authors.lastname: Boothroyd
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002981:Clindamycin / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics; D008854:Microscopy, Electron; D021261:Parasitic Sensitivity Tests; D018087:Plastids / Q000187:drug effects* / Q000235:genetics; D017354:Point Mutation; D012335:RNA, Ribosomal / Q000235:genetics*; D014122:Toxoplasma / Q000187:drug effects* / Q000254:growth & development
chemical_list: D000900:Anti-Bacterial Agents; D012335:RNA, Ribosomal; D002981:Clindamycin