The apicoplast: A novel organelle in apicomplexan parasites is a target for clindamycin and other antibiotics

Lincosamide and macrolide antibiotics are effective chemotherapeutic agents against Toxoplasma gondii and other protozoan parasites, both in vitro and in vivo. These agents are well-known inhibitors of transpeptidation in prokaryotes, but fail to inhibit cytoplasmic protein synthesis or impair mitochondrial function in Toxoplasma. Their mechanism of action in Toxoplasma has therefore been unclear. The research described in this thesis was undertaken to characterize the in vitro effect of clindamycin on T. gondii replication. High concentrations of clindamycin have no immediate effect on parasite replication, but growth rates are reduced after parasites lyse out of the infected cell where they are treated and establish a new parasitophorous vacuole within the next host cell. This \u27delayed death\u27 phenotype is also exhibited by the structurally unrelated antibiotics azithromycin and chloramphenicol. Toxoplasma is not a prokaryotic organism but sequence analysis of ribosomal genes from its 35 kb episomal genome suggested that this may be the functional target for clindamycin. This mysterious genome is associated with an endosymbiotic organelle of unknown function--the apicomplexan plastid, or apicoplast. It has not been possible to directly test the apicoplast as a target for clindamycin. While the apicoplast genome is transcribed, it has not been possible to demonstrate synthesis of plastid proteins. Furthermore, no transpeptidation-domain mutations have been identified on the plastid genome in any clindamycin-resistant parasites. To test whether the apicoplast is an essential organelle, the prokaryotic gyrase inhibitor ciprofloxacin was employed to block replication of the circular plastid genome. Quantitative analysis of plastid vs. nuclear DNA in ciprofloxacin-treated parasites revealed a selective reduction in genome copy. Ciprofloxacin also displays the \u27delayed death\u27 phenotype--demonstrating a link between this peculiar effect and the plastid. Moreover, clindamycin, azithromycin and chloramphenicol treatment cause a decline in apicoplast DNA within the first host cell, providing the earliest manifestation of treatment yet observed. These data directly link apicoplast function with parasite survival, validating this organelle as an effective target for parasiticidal drug design