Second cycle PRDD schizonts cannot efficiently segment or egress from their host RBC. A) Giemsa blood smears show that PRDD stall at the schizont stage and are unable to establish a third cycle of infection. B) To test parasites egress, an assay was performed with parasites transfected with an exported Nanoluciferase (Nluc). Upon RBC lysis, Nluc is released into the supernatant, which is collected. The NanoGlo substrate is added, with the relative light released used as a proxy for egress. Parasite egress can be reversibly blocked with the inhibitor compound 1 (C1). C) Purified schizonts were plated out in the presence of either DMSO vehicle or C1 at 4 μMand the supernatant collected after 4 or 8 hours. PRDD parasites showed egress comparable to incubations with C1. Each dot represents a technical replicate normalised to the DMSO 100% control, with 3 technical replicates in each of the 3 biological experiments. Error bars = SD. Unpaired t-test, p value < 0.05. D) IFAs showing persistence of the parasitophorous vacuole membrane (PVM) structure as seen by maintained EXP2 labelling. The egress defect of PRDD schizonts involves an inability to completely rupture their PVM long after typical egress occurs. BF, bright field E) These schizonts are also unable to segment appropriately, with new inner membrane complex (IMC) forming around only some nuclei as seen by labelling of the IMC protein GAP45. Nuclei stained with DAPI (blue). Images represent single Z stacks. Scale bar = 5 μm. F) Electron microscopy shows that while some nuclei (Nu) are not successfully segregated and enveloped by a new parasite plasma membrane (PPM), apparently normal rhoptry formation still occurs in PRDD parasites. Hpi, hours post invasion; M, merozoite; R, rhoptry; S PPM, schizont parasite plasma membrane;MPPM, merozoite parasite plasma membrane. Scale bar = 1 μm. Bulloch MS, Huynh LK, Kennedy K, Ralton JE, McConville MJ, Ralph SA. Apicoplast-derived isoprenoids are essential for biosynthesis of GPI protein anchors, and consequently for egress and invasion in Plasmodium falciparum. PLoS Pathog. 2024 20(9):e1012484.