Developmental Cell Article Genome-Wide RNAi Screens Identify Genes Required for Ricin and PE Intoxications Dimitri Moreau,1 Pankaj Kumar,1 Shyi Chyi Wang,1 Alexandre Chaumet,1 Shin Yi Chew,1 He´ le` ne Chevalley,1 and Fre´ de´ ric Bard1,* 1Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore *Correspondence: [email protected] DOI 10.1016/j.devcel.2011.06.014 SUMMARY In the lumen of the ER, these toxins are thought to interact with elements of the ER-associated degradation (ERAD) pathway, Protein toxins such as Ricin and Pseudomonas which targets misfolded proteins in the ER for degradation. exotoxin (PE) pose major public health challenges. This interaction is proposed to allow translocation to the cytosol Both toxins depend on host cell machinery for inter- without resulting in toxin degradation (Johannes and Ro¨ mer, nalization, retrograde trafficking from endosomes 2010). to the ER, and translocation to cytosol. Although Obviously, this complex set of membrane-trafficking and both toxins follow a similar intracellular route, it is membrane-translocation events involves many host proteins, some of which have already been described (Johannes and unknown how much they rely on the same genes. Ro¨ mer, 2010; Sandvig et al., 2010). Altering the function of these Here we conducted two genome-wide RNAi screens host proteins could in theory provide a toxin antidote. identifying genes required for intoxication and Consistently, inhibition of retrograde traffic by drugs such as demonstrating that requirements are strikingly Brefeldin A (Sandvig et al., 1991)(Yoshida et al., 1991) or Golgi- different between PE and Ricin, with only 13% over- cide A (Sa´ enz et al., 2009) and Retro-1 and 2 (Stechmann et al., lap. Yet factors required by both toxins are present 2010) can reduce intoxication of cultured cells by some toxins. In from the endosomes to the ER, and, at the morpho- addition, Retro-2 can rescue mice challenged by Ricin nasal logical level, the toxins colocalize in multiple exposure (Stechmann et al., 2010). However, this drug may structures. Interestingly, Ricin, but not PE, depends not be useful for other toxins because previous studies have re- on Golgi complex integrity and colocalizes signifi- vealed that different toxins can have different host gene require- cantly with a medial Golgi marker. Our data are con- ments. For example, diphtheria toxin (DT) is highly dependent on clathrin, while Ricin can be internalized by independent mecha- sistent with two intertwined pathways converging nism(s) (Moya et al., 1985). Similarly, PE traffics between the and diverging at multiple points and reveal the Golgi and the ER in a COPI-dependent fashion by binding to complexity of retrograde membrane trafficking in KDEL receptor (KDELR), while Shiga toxin is COPI independent mammalian cells. and Rab6 dependent (Wolf and Elsa¨ sser-Beile, 2009; Girod et al., 1999; White et al., 1999). The molecular description of the retrograde-trafficking path- INTRODUCTION ways remains limited with relatively few genes identified (Johannes and Popoff, 2008; Pavelka et al., 2008; Sandvig Ricin is a plant protein with high toxicity present in high concen- et al., 2010). As a consequence, it is not clear how much the tration in the beans of Ricinus communis. Because of its stability requirements for host proteins of different toxins overlap. Inter- and relative ease of preparation, it represents a potential biolog- estingly, Ricin and PE, like diphtheria and Shiga toxins, all target ical weapon. Pseudomonas exotoxin A (PE) is a protein secreted the mammalian translation machinery (Hartley and Lord, 2004; by Pseudomonas aeruginosa, an opportunistic pathogen that Falnes and Sandvig, 2000). affects immunocompromised patients, causing pulmonary and This allowed us to use the same assay and very similar exper- urinary tract infections as well as infections of burn injuries imental conditions to reliably compare the gene sets required for (Wolf and Elsa¨ sser-Beile, 2009). different toxins. We present two genome-wide RNAi screens Although from different origins, both PE and Ricin share identifying human genes required for Ricin and PE intoxication. a similar mode of action: hijacking cellular processes to cross Our results reveal a large number of genes required for maximal the cell membrane and targeting protein synthesis. Like many toxic effect, some of them previously known to be involved in other toxins, PE and Ricin bind cell surface receptors that are en- membrane traffic. Furthermore, we find that the requirements docytosed. After internalization, some toxins, such as diphtheria, for both toxins differ significantly and at multiple levels. Interest- are able to translocate from the endosomal lumen to the cytosol ingly, the toxins also share genetic requirements and similar (Ratts et al., 2003). Ricin, PE, Shiga, and other toxins instead subcellular localizations at various levels of their trafficking, hijack retrograde membrane transport to traffic from the endo- suggesting two intertwined pathways converging and diverging somes to the Golgi and from there to the ER (Sandvig et al., 2010). at multiple levels. Developmental Cell 21, 231–244, August 16, 2011 ª2011 Elsevier Inc. 231 Developmental Cell Mapping Ricin and PE Host Gene Requirements RicinRicin PE Figure 1. High-Throughput siRNA Screening AB of Retrograde Transport of PE and Ricin Toxin Endocytosis CLTC 800 PE (A) Intoxication assay using HeLa cells stably ex- 700 Ricin pressing a luciferase with short half-life (Luc2CP). Cycloheximide Endosome 600 After binding at the cell surface, both toxins are 500 STX16 internalized into endosomes and transported to the Golgi 400 Golgi and then the ER, where they translocate to Luciferase 300 KDELR1 the cytosol. Both toxins then inhibit translation and Ribosome 200 luciferase production. Luciferase activity rapidly Stop translation and 100 drops because of the short half-life of the protein luciferase synthesis Luciferase signal (fold from baseline) due to the hCL1 and hPEST destabilization Translocation 0 0 2 4 6 8 sequences fused at the C-terminal end. The three ER Time of treatment (h) CMV Luc2 hCL1hPEST highlighted genes, CLTC1, STX16, and KDELR1, Control siRNA act on endocytosis, transfer to the Golgi, and Golgi C D CLTC to ER traffic, respectively. Membrane traffic pilot library KDELR1 (B) Time course of luciferase signal from cells 14 STX16 STX16 Luciferase expression exposed to PE (0.25 ng/ml), Ricin (250 ng/ml), or 12 No target cycloheximide (20 mg/ml). PLK1 72 hours 10 Knock down (C) Membrane-traffic pilot library (201 genes) STX3A screens: luciferase values are expressed in fold 8 CLTC RAB1A from the negative control and plotted on x axis for 6 USO1 STX12 Toxin treatment SAR1A Ricin or PE 8hours PE treatment and y axis for Ricin treatment. 4 STX11 STX17 Luciferase signal lost (D) Work flow of the genome-wide screen (run in KDELR1 2 SEC22L2 Luciferase signal rescued duplicate): cells are seeded in 384-well plates preprinted with siRNA for reverse transfection and 0 incubated for 72 hr, then challenged with Ricin or Ricin luciferase signal (fold from neg. control) 0 5 10 15 PE luciferase signal (fold from neg. control) PE for 8 hr before luciferase signals are measured. RESULTS membrane (Low et al., 2006); therefore, its likely role in regard to toxin trafficking is less obvious. A Pilot Screen Identifies STX16 as an Important Because STX16 showed a strong inhibition of both PE and Regulator of Both Ricin and PE Traffic Ricin trafficking, we chose it as one of our positive controls in Ricin and PE intoxicate cells by inhibiting protein synthesis. To the screen (Figure 1D). We also selected CLTC, which affected measure this effect, we used a destabilized luciferase with a short both toxins but more significantly PE. This is consistent with half-life as previously described (Zhao and Haslam, 2005)(Fig- previous reports where PE has been shown to be dependent ure 1A). In this assay, cells challenged by 25 ng/ml PE or on clathrin-mediated endocytosis, whereas Ricin also uses 250 ng/ml Ricin almost completely lose their luciferase signal clathrin-independent mechanisms (Moya et al., 1985; Sandvig after about 8 hr (Figure 1B), which was therefore used as the et al., 1987). Finally, we also selected KDELR1 because its endpoint for the screens. knockdown provides a modest inhibitory effect specifically for Knockdown of a gene important for intoxication will rescue PE intoxication. the luciferase signal in toxin-treated cells. To identify positive controls to use for screen quality control and data normalization, Whole-Genome Screens for PE and Ricin Reveal a Large we performed a pilot screen on a library of siRNA pools targeting Number of Potent Regulators of Toxin Trafficking 201 known membrane-trafficking regulators (Figure 1C). This Whole-genome screens were performed in duplicate using the library was designed to target all identified human SNARES, library siGENOME SMARTpool siRNAs that targets 21,121 which are essential regulators of membrane traffic, and previ- human genes with one pool of four siRNAs per gene. Following ously described regulators of toxin trafficking such as KDELR1, reverse transfection, cells were incubated for 72 hr and then Rab6a, and the clathrin heavy chain (CLTC). Knockdown of challenged with PE or Ricin for 8 hr before luminescence reading most SNARES did not significantly rescue intoxication by either (Figure 1C). toxins; however, loss of Syntaxins 3, 12, and 16 provided some Reproducibility between duplicates was high for both screens of the strongest rescue observed (Figure 1C). with average Spearman rank correlations between the replicates Interestingly, whereas it was reported that Syntaxin 16 (STX16) of 0.85 for PE and 0.84 for Ricin (see Figure S1 available online). is important for endosome to trans-Golgi network (TGN) retro- For normalization, we realized that the usual plate-based z-score grade traffic (Amessou et al., 2007; Pe´ rez-Victoria and Bonifa- normalization method was inadequate because of the non- cino, 2009; Ganley et al., 2008), Syntaxins 3 and 12 have not random organization of the library.