Tetracyclines are effective but slow-acting antimalarial drugs whose mechanism of action remains uncertain. To characterize the antimalarial mechanism of tetracyclines, we evaluated their stage-specific activities, impacts on parasite transcription, and effects on two predicted organelle targets, the apicoplast and the mitochondrion, in cultured Plasmodium falciparum. Antimalarial effects were much greater after two 48-h life cycles than after one cycle, even if the drugs were removed at the end of the first cycle. Doxycycline-treated parasites appeared morphologically normal until late in the second cycle of treatment but failed to develop into merozoites. Doxycycline specifically impaired the expression of apicoplast genes. Apicoplast morphology initially appeared normal in the presence of doxycycline. However, apicoplasts were abnormal in the progeny of doxycycline-treated parasites, as evidenced by a block in apicoplast genome replication, a lack of processing of an apicoplast-targeted protein, and failure to elongate and segregate during schizogeny. Replication of the nuclear and mitochondrial genomes and mitochondrial morphology appeared normal. Our results demonstrate that tetracyclines specifically block expression of the apicoplast genome, resulting in the distribution of nonfunctional apicoplasts into daughter merozoites. The loss of apicoplast function in the progeny of treated parasites leads to a slow but potent antimalarial effect.Plasmodium falciparum causes an estimated half billion cases of malaria, resulting in over a million deaths, each year (5, 33). Tetracyclines are effective, albeit slow-acting, antimalarials that are used in combination with a more rapidly acting drug to treat malaria (1) and for antimalarial chemoprophylaxis (29). The antimalarial mechanism of action of tetracyclines remains undefined. Consistent with their slow clinical activity, tetracyclines exert in vitro antimalarial effects slowly, requiring incubation with cultured P. falciparum beyond a single 48-h asexual cycle for maximal effects (16). Tetracyclines exert antibacterial activity by inhibiting prokaryotic translation (41). However, in the eukaryote P. falciparum, protein synthesis is broadly inhibited by tetracyclines only at much higher concentrations than those required to block parasite growth (6), suggesting that their antimalarial effects are due to action against a target other than cytosolic ribosomes.Early studies of the mechanism of action of tetracyclines against P. falciparum focused on the parasite's single mitochondrion as the likely target (18, 26). However, these studies preceded the identification of the apicoplast, an organelle of uncertain function that is related to the chloroplast of plant cells (20,42). The mitochondrion and the apicoplast each contain their own genome, encoding prokaryote-like ribosomal RNAs, tRNAs, and some proteins (12, 45). In addition, several hundred nuclear genes encode proteins targeted to the apicoplast or the mitochondrion (2,14,15,28,39). The apicoplast houses enzy...
