Volume 26, Issue 9, Pages 1253-1262.e5 (September 2019) Discovery of Druggable Host Factors Critical to Plasmodium Liver-Stage Infection  Rene Raphemot, Maria Toro-Moreno, Kuan-Yi Lu, Dora Posfai, Emily Rose Derbyshire  Cell Chemical Biology  Volume 26, Issue 9, Pages 1253-1262.e5 (September 2019) DOI: 10.1016/j.chembiol.2019.05.011 Copyright © 2019 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2019 26, 1253-1262. e5DOI: (10. 1016/j. chembiol Copyright © 2019 Elsevier Ltd Terms and Conditions

Figure 1 A Druggable Genome RNAi Screen Identifies Host Factors of P. berghei Liver Stage (A) Schematic representation of the siRNA screen. HepG2 cells were reverse transfected with pooled siRNAs in a 384-well plate. After 48 h post-siRNA transfection (30 nM), P. berghei sporozoites expressing luciferase (Pb-Luc) were added to the cells (MOI = 0.3) and relative liver cell viability and parasite load were measured at 48 h post-infection. (B) In this screen, the positive control of pooled siRNAs targeting SCARB1 (siSCARB1) led to a decrease in parasite load (gray bars) of approximately 60% compared with the negative controls of mock and non-targeting scrambled siRNA (NT siRNA). No significant effects on HepG2 cell viability (>50%) were observed (red circles). Data are means ± SEM; n = 46 technical replicates. (C) Distribution of the robust SSMD scores of Plasmodium liver-stage infection for roughly 7,000 targeted genes in the druggable genome library. Anti-infection hits exhibit an increase in parasite load upon gene suppression and are defined by a robust SSMD score ≥2 (green). Pro-infection hits exhibit a decrease in parasite load upon gene suppression and are defined by a robust SSMD score ≤−2 (red). The number of genes implicated as pro-infection or anti-infection is shown. (D) Functional group network view for Reactome pathway (circles) and GO terms (hexagons) generated with ClueGO. Terms are functionally grouped based on shared genes (kappa score) and are shown with different colors. (E) Heatmap of percent P. berghei parasite load inhibition for 58 selected pro-infection genes tested in HepG2 and Huh7 cell lines. Two different siRNAs per gene (gene_1 and gene_2) were tested. (F) Venn diagram of unique and shared targeted genes that significantly decreased (≥50%) liver-stage parasite load within the two hepatoma cell lines. Cell Chemical Biology 2019 26, 1253-1262.e5DOI: (10.1016/j.chembiol.2019.05.011) Copyright © 2019 Elsevier Ltd Terms and Conditions

Figure 2 Evaluation of the Pro-infection Hits, COPB2, COPG1, and GGA1 (A and B) Rescue of P. berghei parasite load in HepG2 cells after COPB2, COPG1, and GGA1 gene knockdown. HepG2 were co-transfected with siRNA and a siRNA-resistant plasmid specific to each candidate gene (i.e., COPB2, COPG1, and GGA1). After incubation for 48 h, cells were infected with P. berghei-Luc (MOI = 0.3). Parasite load (A) and liver cell viability (B) were assessed at 48 h post-infection, respectively. Liver cell viability was not inhibited under the experimental conditions. The p values display one-way ANOVA with Dunnett's multiple comparison test for each condition compared with control siRNA (Ctrl siRNA). *p < 0.05, ***p < 0.001, ****p < 0.0001. Data are means ± SEM; n = 2–5 biological replicates. (C) Huh7 cells were reverse transfected with siRNAs targeting COPB2, COPG1, and GGA1 (30 nM). After 48 h, cells were infected with P. berghei-Luc (MOI = 0.3) and fixed at 48 h post-infection. Cells were stained with anti-PbUIS4 (red), a PVM-resident protein. Nuclei were stained with DAPI (blue). A non-targeting scrambled siRNA was used as a control. Scale bars, 30 μm. (D) P. berghei parasite size in Huh7 cells transfected with siRNAs targeting COPB2 (n = 20), COPG1 (n = 48), and GGA1 (n = 48) as measured by immunofluorescence microscopy. Cells were fixed at 48 h post-infection and treated as described in (C). PbUIS4 size was measured by microscopy to quantify parasite size. Non-targeting scrambled siRNAs were used as negative controls (n = 101). Three independent experiments were performed, and data are shown as means ± SEM (red line). Each symbol indicates one parasite. *p < 0.05, ***p < 0.001, ****p < 0.0001 (one-way ANOVA, Dunnett‘s multiple comparison test). (E) Rate of P. berghei infection of Huh7 cells transfected with siRNAs targeting COPB2, COPG1, and GGA1. Cells were fixed at 48 h post-infection and treated as described in (C). The total number of P. berghei schizonts with PbUIS4 staining in each treatment condition was determined by microscopy. One-way ANOVA, p = 0.2696. Data are shown as means ± SEM; n = 2–3 biological replicates. Cell Chemical Biology 2019 26, 1253-1262.e5DOI: (10.1016/j.chembiol.2019.05.011) Copyright © 2019 Elsevier Ltd Terms and Conditions

Figure 3 Pharmacological Evaluation of Protein Trafficking Inhibition on Liver-Stage Parasite Load (A) Dose-response curves for brefeldin A and golgicide A in P. berghei-infected HepG2 (red) and Huh7 (black) cells. Data are means ± SEM; n = 3–4 biological replicates. (B) Schematic illustration of the experimental setup (top panel). P. berghei-Luc sporozoites were pretreated with DMSO (vehicle control), 0.5 μM of brefeldin A (BFA), or 3.5 μM of golgicide A (GCA) for 20 min at room temperature. Following pretreatment, sporozoites were used to infect HepG2 cells (pretreated SPZ, bottom panel). In parallel, BFA and GCA were added to cells immediately before infection (liver stage, bottom panel). Parasite load was measured at 48 hpi. Data were normalized to the DMSO control and shown as means ± SEM; n = 4 biological replicates. *p < 0.05, **p < 0.01; ns, non-significant (Student's t test). (C) Effects of golgicide A and brefeldin A on PbUIS4 trafficking to the PVM during early liver-stage schizogony. Representative confocal images of DMSO- (vehicle control), golgicide A-, and brefeldin A-treated Huh7 cells infected with P. berghei sporozoites and fixed at 8 hpi. Cells were stained with anti-PbUIS4 (red), anti-PbHSP70 (green), and DAPI (blue) (left). Schematic illustrating the experimental setup (top right). Quantification of the proportion of PbUIS4-positive pixels not overlapping with PbHSP70 pixels in each condition (n = 11–12) (bottom right). ****p < 0.0001 (one-way ANOVA, Dunnett’s multiple comparison test). Scale bars, 10 μm. (D) Schematic illustration of the experimental setup (top panel). Time-course analysis of golgicide A treatment (3.5 μM) on liver-stage Plasmodium. Golgicide A was added to HepG2 cells infected with P. berghei-Luc sporozoites at 0, 4, 8, 24, or 36 hpi. Parasite load was measured at 48 hpi. Data are means ± SEM normalized to corresponding control condition (DMSO, vehicle) for each time point; n = 3 biological replicates. **p < 0.01; ns, not significant (Student's t test). (E) P. berghei-infected HepG2 cells were treated with DMSO (vehicle control) or 3.5 μM GCA from 0 to 48 hpi (DMSO, n = 176; GCA, n = 82) or 8–24 hpi (DMSO, n = 119; GCA, n = 49) and fixed at 48 hpi. Cells were stained with anti-PbUIS4, a PVM-resident protein, to enable quantification of parasite size by microscopy. Data are means ± SEM (red line). ****p < 0.0001; ns, not significant (Kruskal-Wallis test). Cell Chemical Biology 2019 26, 1253-1262.e5DOI: (10.1016/j.chembiol.2019.05.011) Copyright © 2019 Elsevier Ltd Terms and Conditions

Figure 4 Localization of β′-COP and GGA1 with Exo-Erythrocytic Forms during Liver-Stage Plasmodium Infection (A and B) Huh7 cells were infected with P. berghei-Luc sporozoites and fixed at 3, 24, and 48 hpi. Cells were stained with anti-β′ COP (A) (red) or anti-GGA1 (B) (red), and anti-PbHSP70 (green) to visualize Plasmodium. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. See Figure S4A for GGA1-stained uninfected cells at 24 and 48 hpi. (C) Representative confocal images of P. berghei-infected Huh7 cells at 48 hpi. β′-COP (red, top panel) and GGA1 (red, bottom panel) colocalize with PbUIS4 (green, both panels), a PVM-resident protein. Nuclei were stained with DAPI. Scale bars, 10 μm. (D) Hepa1-6 cells were infected with P. yoelii sporozoites and fixed at 48 hpi. Cells were stained with anti-β′ COP (red, top panel) or anti-GGA1 (red, bottom panel), and anti-PbHSP70 (green, both panels). Nuclei were stained with DAPI (blue). Scale bars, 20 μm. Cell Chemical Biology 2019 26, 1253-1262.e5DOI: (10.1016/j.chembiol.2019.05.011) Copyright © 2019 Elsevier Ltd Terms and Conditions