Fig. 3

Azithromycin inhibits P. falciparum merozoite invasion. (a) The potency of azithromycin in ethanol or DMSO as vehicle was compared for invasion inhibition (unbroken line, 10 minute merozoite treatment, parasitaemia measured 40 hours later) and 1 cycle growth inhibition assays (broken line, treatment rings to trophozoites’ next cycle). The invasion inhibitory IC₅₀ of azithromycin prepared in ethanol (blue, IC₅₀ 10 μM) was similar to that for growth inhibition assays (IC₅₀ 7 μM; P = 0.0743, Log IC₅₀ same between data sets, extra sum of squares F-test). The invasion inhibitory activity of azithromycin in DMSO (red, IC₅₀ 38 μM) was 5-fold higher than 1 cycle growth assays (IC₅₀ 7 μM; P <0.0001, Log IC₅₀ different between data sets). (b) Inhibition profiles for pretreated erythrocytes (RBC Pre), merozoite treatment (T = 0; drug added at time zero) and rings treated for <1 hour (T = 20; drug added 20 minutes post-invasion) were identical between azithromycin (in DMSO) and the invasion inhibitor heparin (IC₈₀ concentration). (c) Increasing the concentration of azithromycin (in ethanol) to 10 × IC₈₀ (380 μM) did not result in substantial inhibition of invasion into pretreated cells compared to treatment of merozoites. (d) Flow cytometry and microscopy assessments confirmed that azithromycin (IC₈₀ in ethanol) and heparin, but not the trophozoite-targeting antimalarial halofantrine (2 × IC₈₀ ring-stage treatment (46 nM) [13]), inhibit merozoite invasion and establishment of ring stages in erythrocytes. Representative (e) flow cytometry plots (GFP high and EtBr low ring-stage parasites represented by square gate) and (f) microscopy thin smears (rings highlighted by green arrows) show absence of ring-stage parasites for azithromycin and heparin compared to non-invasion inhibitory controls (labeling as per Fig. 3d). Experiments represent the mean and SEM of three or more experiments. Significance was tested using an unpaired t-test (*P = 0.01–0.05, **P ≤0.01, ***P ≤0.001)