(A) Live epifluorescence microscopy of the PfMev CLD-PyrKII parasite line after Shield1 treatment. This parasite line expresses api-SFG (green) and is also stained with MitoTracker (red) and DAPI (blue). Representative images show the state of the apicoplast organelle at the corresponding time points. Microscopy images represent fields that are 10 µM long by 10 µM wide. (B) Graph of the percentage of disrupted apicoplast organelles in synchronized PfMev CLD-PyrKII parasites after Shield1 treatment. At each time point, a minimum of 15 microscopy images were taken to determine apicoplast morphology (intact or disrupted) based on api-SFG fluorescence. The percentage of parasites containing an intact apicoplast dropped precipitously between the second and third growth cycle of the parasite, corresponding to the 64 and 88 hr time points. Error bars represent the standard deviation from three independent experiments with at least 20 observations each. (C) Synchronized ring-stage PfMev CLD-PyrKII parasites were treated with Shield1, with samples collected every 8 hr for microarray analysis (biological triplicates were collected). Nuclear and mitochondrial mRNA levels (blue) were compared to an untreated control and plotted as the fold difference. The same analysis for apicoplast genes (orange) shows that there is a sharp reduction in RNA levels in the treated parasites between 24 and 40 hr after Shield1 treatment. (D) Synchronized ring-stage PfMev CLD-PyrKII api-luciferase parasites were prepared for a 48 hr time course under four conditions, each of which contained 500 nM aTc: exchange into glucose-free medium (CHO-, green), 500 nM Shield1 treatment (Shield1, black), 50 µM fosmidomycin treatment (Fos, red), and no treatment (Control, blue). Samples were collected every 8 hr for analysis of luciferase activity (biological triplicates were collected, each with three technical replicates). We observed a significant reduction in luminescence ~8–24 hr after triggering mislocalization of PyrKII with Shield1. For parasites treated with fosmidomycin, luminescence levels did not meaningfully decrease until 24–40 hr, while levels in parasites cultured in glucose-free medium dropped within 8 hr. For all treatment groups, luminescence levels were compared to an untreated parasite control. Error bars represent the standard error of the mean. (E) Proposed progression of events leading from loss of PyrKII to parasite death.