Azithromycin disrupts apicoplast biogenesis in replicating and dormant liver stages of the relapsing malaria parasites Plasmodium vivax and Plasmodium cynomolgi

The control and elimination of malaria caused by Plasmodium vivax is hampered by the threat of relapse infection resulting from the activation of dormant hepatic hypnozoites. Currently, only the 8-aminoquinolines, primaquine and tafenoquine, have been approved for the elimination of hypnozoites, though their use is hampered by potential toxicity. Therefore, an alternative radical curative drug that safely eliminates hypnozoites is a pressing need. In this study, we assessed the potential hypnozoiticidal activity of the antibiotic azithromycin, which is thought to exert antimalarial activity by inhibiting prokaryote-like ribosomal translation, within the apicoplast, an indispensable organelle. Our results show that azithromycin inhibited apicoplast development during P. vivax and P. cynomolgi liver-stage schizogony leading to impaired parasite maturation. More importantly, we show that azithromycin is likely to impair the hypnozoite's apicoplast resulting in the loss of this organelle. Subsequently, by using a recently developed long-term hepatocyte culture system, we found that this loss probably induces a delay in the hypnozoite activation rate and that those parasites that do proceed to schizogony display liver stage arrest prior to differentiating into hepatic merozoites, thus, potentially preventing relapses. Altogether, this work provides evidence for the potential use of azithromycin for the radical cure of relapsing malaria and identifies apicoplast functions as potential drug targets in quiescent hypnozoites.