Discovery of apicoplast membrane proteins

(A-B) Activity test of biotinylation of proximal proteins by APT1-TurboID-4Ty. Parasites were grown in 500 μM D-biotin for 90 min, followed by detection of biotinylated proteins (green) by streptavidin reagents on IFA (A), and on Western blots (B). Actin served as the loading control. Scale = 5 μm.

(C) Volcano plot analysis comparing the TurboID fusion to the parental line. Three replicate mass-spectrometry experiments were analyzed by the student t-test. Known apicoplast proteins (red) and candidates with different numbers of TMDs (deep and light blue were indicated, and novel apicoplast proteins were pointed out. See Dataset S1.

(D) Workflow of the discovery of novel apicoplast membrane proteins. Candidates from hyperLOPIT (Barylyuk et al., 2020), ACP-BirA (Boucher et al., 2018) and APT1-TurboID (this study) were screened by epitopte tagging and IFA.

(E) Confocal co-localization of HAP-6Ty with the apicoplast marker ACP, showing confocal imaging of HAP-6Ty (red) and ACP (green), and Pearson Corelation Coefficiency (PCC). The PCC values over the merged fluoresent foci for candidate proteins and ACP were analyzed by a co-localization and ROI intensity analysis module in the NIS Elements AR software and shown with mean ± SEM (N=6 for each). Scale = 5 μm.

(F-H) Summary and comparison of the screening from three datasets. The putative transporters are grouped into diverse types (F) and identified from three different datasets (G), which shared three novel proteins, as shown by the venn diagram (H). Three independent experiments were performed with similar outcomes (A, B, E).

Conservation analysis of putative transporters

(A-B) Heatmap indicating conservation of known apicoplast proteins and newly identified proteins among chromalveolates (A), red alga, green alga, plants, fungi, metazoa and bacteria (B). The conservation was scaled by a heat bar (red) with E-values for putative orthologues of the listed proteins (see Dataset S2). ToxoDB protein numbers (left) and protein (temporary) names (right) are shown. Their sequences were searched for orthologues using the Hidden Markov Model (HMM) (E value<E-7) and the best orthologue was used for the heatmap analysis in ComplexHeatmap package in R. Parasite fitness scores were shown on the right with the green heat bar for T. gondii (ToxoDB)(Sidik et al., 2016) and the blue bar for P. falciparum (PlasmoDB)(Zhang et al., 2018). The tree dendrogram across the genes were clustered according to the euclidean distance using UPGMA method. The species tree (top) shows phylogenetic relationships and major clades: Theil., Theileria; Babes, Babesia; Crypt., Cryptosporidium; Proteo, Proteobacteria.

AMT1 and AMT2 are localized at the apicoplast membrane and essential for parasite growth in vitro

(A) Western blot detection of protein depletion in the AID lines. The lines were induced by auxin (IAA) for different hours (hrs) as indicated. Western blots detected immatured (p) and matured (m) forms of the protein fusions in the non-induced lanes. Actin served as the control.

(B) IFA detection of protein depletion in the AID lines. Parasites were grown in IAA for 24 hours, followed by IFA with antibodies against Ty or HA (red) and IMC1 (green). Scale = 5 μm.

(C-E) Plaque formation by the TIR1 and AID lines on HFF monolayers in ± IAA for 7 days. Numbers (D) and sizes (E) of the plaques were measured by ImageJ. Scale = 0.5 cm. Two independent experiments with triplicates were performed. Data are shown as a mean ± SEM.

(F) Parasite replication of the TIR1 and AID lines grown in IAA. The parasites were grown in IAA for 24 hours, followed by scraping, harvesting and infection for the 2nd and 3rd rounds of parasite growth in IAA. The parasites stained with GFP45 were counted in vacuoles (at least 150 vacuoles in each replicate). In comparing to TIR1 (1st, 2nd and 3rd), p<0.0001 for the AID lines with 2 and 8 parasites/vacuole in the 1st round, and for the AID lines with 1-8 parasites/vacuole in the 2nd round, p<0.0001 for the AID lines with 1 and 4 parasites/vacuoles in the 3rd round.

(G-H) ACP diffusion in the TIR1 and AID lines grown in IAA. Parasites were grown in IAA for the 1st, 2nd and 3rd lytic cycles, as described in the parasite replication assay (intracellular parasites). Those parasites in the 1st and 2nd round of growth were forced to egress for the same analysis (extracellular parasites). Vacuoles or single parasites were scored (n>150 for each replicate).

Fields/images were selected blind and all parasites/vacuoles were scored on the same fields/images (F-H). Three independent experiments were performed (A, B, F, G and H), data are shown as a mean ± SEM with two-way ANOVA with Tukey’s multiple comparisons (compared with the TIR1). ***, p<0.0001.

Depletion of AMT1 and AMT2 caused defects of MEP and FASII pathways (A-C) The MEP activity was strongly affected in the AID lines. GFP transport to the VAC was assayed by growning parasties in GFP expressing HFF cells (HFF-GFP). LHVS (10 μM) was added to inhibit GFP digestion in the VAC (Dou et al., 2014). The isoprenoid synthesis in host cells and parasites was inhibited by IC50 concentrations of atorvastatin (10 μM) and zoledronate (0.3 μM)(Li et al., 2013), respectively (A). The inhibition over the TIR1 line using two of the drugs was used as a positive control (B). The AID parasites were grown in HFF-GFP cells in the presence of LHVS for 36 hours in total, with the addition of atorvastatin, zoledronate or IAA as stated for different experiments in the figure in the last 12 hours. Parasites were immediately harvested, adhered and scored, as shown in the example images in B. Percentages of parasites with GFP foci were plotted (C). Fields/images were selected blind and all parasites/vacuoles were scored on the same fields/images (C). Three independent experiments with triplicates were performed. Data are shown as a mean ± SEM with one-way ANOVA with Tukey’s multiple comparison. ***, p<0.0001, scale = 5 μm. (D-F) Fatty acid synthesis and TCA metabolites were significantly reduced in IAA induced AID parasites. Parasites were grown in IAA for 18 hours and harvested for untargeted metabolomics with GCMS and LCMS (N=3), and metabolites detected were analyzed by Dunnette’s multiple comparison by comparing the AID parasites to the TIR1. Three AID lines shared 13 differential metabolites (D). These shared metabolites included fatty acids and lipids, such as myristic acid, palmitoleic acid, oleic acid, and phosphatidylcholine (PC) (E), and metabolites related to the TCA cycle (F). *, p<0.05; **, p<0.001; ***, p<0.0001.

Depletion of AMT2 resulted in ACC1 instability.

(A-B) No reciprocal effect on protein levels by depletion of AMT1 or AMT2. AMT1 and AMT2 were reciprocally tagged at C-terminus with 6HA in the AID lines, and the HA fusions were detected by Western blots in parasites in ±IAA for 36 hours (A), and by IFA in parasites in ±IAA for 24 hours (B). IFA was performed with IMC1 (green) and Ty/HA (red).

(C-D) ACC1 was lost upon depletion of AMT2-AID. ACC1 was endogenously tagged with 6HA at its C-terminus in the AMT1-AID and AMT2-AID lines, and parasites were grown in IAA for 36 hours for Western blots (C) and IFA (D). IFA analyses were performed with anti-ACP and anti-HA antibodies, while Western blots were done with anti-HA and streptavidin Li-COR 800CW. Actin served as the control.

(E-F) Representative images of ACC1 on IFA in parasites of AMT2-AID induced in IAA for 24 hours. ACC1 (red) was completely lost in parasites of a vacuole (type 1), remained only in one parasite of a single vacuole (type 2), or in two parasites of a single vacuole (type 3) (E). The ACC1 types were scored in AMT2-AID induced in IAA for 24 hours, and expressed as percentages of different types (F). At least 150 parasites were scored in each replicate. Three experiments with triplicates were performed, and data are shown as a mean ± SEM and analyzed by two-way ANOVA with multiple comparison, ***, p<0.0001.

(G) The remaining ACC1 was still biotinylated. Parasites were grown in ±IAA for 24 hours for IFA analysis with antibodies against HA and streptavidin Alexa Fluor 488. Parasites containing both ACC1-6HA (red) and biotinylated ACC1 (green) was observed.

Three independent experiments were performed with similar outcomes, and representative images were shown. Scale = 5 μm.

Examination of the apicoplast ultra-structures, and mouse virulence of the parasites upon depletion of the AID fusions.

(A) Transmission electron microscopy (TEM) observed less electron dense or electron lucent lumen and loosened membranes in the apicoplast in the AID parasites induced in IAA for 24 hours. Representative images were shown. Scale = 200 nm.

(B) Quantification of the apicoplast with the lumen less dense or lucent in parasites induced for 12, 24 and 36 hours and TIR in IAA for 36 hours. The TEM examined 300 parasites for three lines (TIR1, AMT1-AID and AMT2-AID) at each time point, and numbers of abnormal apicoplast (red numbers) and apicoplast identified (green numbers) were shown by the columns. The columns express the percentages of abnormal apicoplast in all the identified apicoplasts.

(C) Survival curve of BALB/C mice infected with 100 parasites intraperitoneally and treated with IAA for 20 days. The Gehan-Breslow-Wilcoxon test was used to compare differences between the survival curves (the AID line versus TIR1), ***, p<0.0001. The experiment of mouse infection was performed with 5 mice in each group, and results from three independent experiments (1st, 2nd and 3rd) were shown together with symbols for the mouse groups.