Supporting Information Zhao et al. 10.1073/pnas.1316841111 SI Materials and Methods Immunofluorescence and Ultrastructural Microscopy Assay. To infect RAW264.7 cells, 0.3 × 106 cells were suspended into invasion medium [RPMI-1640 + 5% (vol/vol) FBS] and plated onto each coverslip in a well of 24-well plate in the absence or presence of 20 U/mL IFNγ (PeproTech) as stated in figure legends. Twentyfour hours later, the cells were infected with T. gondii. To synchronize infection, the plates carrying cells were precooled at 4 °C for 10 min. Then, 2 × 106 parasites per well were added onto plates and rested for 15 min at 4 °C to allow the parasites to settle down. To initiate infection, the cold medium was gently replaced with 37 °C prewarmed invasion medium. The infections were carried out in a 37 °C incubator supplied with 5% CO2. For observation of F-actin induction by attached T. gondii, the infected cells were fixed in 4% (wt/vol) paraformaldehyde (Electron Microscopy Science) 8 min after temperature switch. The cells were surface stained without permeabilization by phalloidin-Alexa Fluor350 conjugates (Invitrogen) and mouse α-SAG1 primary mAb combined with the donkey α-mouse AF568. In this case, only extracellular parasites were positively stained with anti-SAG1 Ab. For quantification, 150–200 cellcontacting parasites per coverslip were counted from 10 random selected fields to calculate F-actin induction rate. For enumeration of PVMs formation, the infected cells were fixed in 4% (wt/vol) PFA 40 or 60 min after infection followed by acetone permeabilization at −20 °C for 3 min. Fixed cells were subjected to staining with phalloidin-AF350, rat α-LAMP-1, rabbit α-GRA7 and mouse α-SAG1 (omitted when using fluorescently tagged Toxoplasma strains). In a case when two different rabbit primary antibodies had to be used, a Zenon Tricolor labeling kit (Invitrogen) was used to prelabel the two antibodies with AF568 and AF647-conjugated donkey anti-rabbit IgG. Two hundred to 300 internalized parasites per coverslip were scored from 12 randomly selected fields and the rate of PVM formation was calculated as the percent of GRA7+ vacuoles among intracellular parasites. For Bafilomycin A1 pretreatment (2), the host cells were incubated in culture medium supplemented with 25 nM Baf A1 or equal volume of DMSO diluent at 37 °C for 1 h. The treated cells were extensively washed five times with invasion medium to remove Baf A1 before infection. For infection of naïve peritoneal macrophages and CPS-primed peritoneal macrophages, 2 × 106 PECs were loaded into each well and incubated at 37 °C for 4 h. Nonadherent cells were washed away and adherent cells were infected by 2 × 106 Toxoplasma tachyzoites. The procedure for electron microscope imaging of phagocytosed PTG was reported (1). Fluorescent images were acquired by using Eclipse Ti-E epifluorescent microscope equipped with Plan Apo 100×/1.4 N.A. and Plan Fluor 60×/ 1.25 N.A. Iris objectives (Nikon), Clara interline CCD camera (Andor) and DAPI, FITC, TRITC, and Cy5 filter sets (Chroma). The images were processed with NIS-Elements AR3.2 (Nikon). 1. Zhao Y, et al. (2009) Virulent Toxoplasma gondii evade immunity-related GTPasemediated parasite vacuole disruption within primed macrophages. J Immunol 182(6): 3775–3781. 2. Conte MP, et al. (1996) The effects of inhibitors of vacuolar acidification on the release of Listeria monocytogenes from phagosomes of Caco-2 cells. J Med Microbiol 44(6): 418–424. Antibodies and Chemical Reagents. Bafilomycin A1 was purchased from Sigma. Alexa Fluor 350 or Alexa Fluor 555-conjugated phalloidin (Invitrogen) were used for filamentous actin staining. The α-granule protein 7 (GRA7) Y1 antiserum was made in laboratory with three immunizations of rabbit by using the full length of purified GRA7 protein. Rabbit α-Rab5a (S-19) was obtained from Santa Cruz Biotechnology. Rat α-FcγRIII/II (2.4G2), rat α-LAMP-1 (1D4B), rat α-CD19 (1D4), B220 (RA36B2), α-TCRβ (H57-597), and rat α-CD11b (M1/70) were purchased from BD Biosciences. Affinity-purified rabbit α-toxoplasma and mouse α-SAG1 (P30/3) were procured from GenWay. Rabbit α-RON4 was a gift of John Boothroyd (Stanford University, Stanford, CA). Rabbit α-ROP1 and mouse α-MIC2 Abs were from the laboratory of David Sibley (Washington University in St. Louis, St. Louis). All secondary antibodies were donkey antimouse, rabbit, rat, or goat IgG and were directly conjugated with Alexa Fluor (AF) 350, AF488, AF568, or AF647, respectively (Invitrogen). Preparation of Naïve and Carbamoyl Phosphate Synthase Mutant -Primed Mouse Peritoneal Macrophages. To obtain mouse naïve macrophages, peritoneal exudate cells (PECs) from C57BL/6 mice were harvested by peritoneum lavage with 10 mL of RPMI 1640 medium plus 1% FBS. PECs were plated onto 24-well plate and settled for 4 h before being extensively washed to remove nonadherent cells. For obtaining primed macrophages, mouse was injected i.p. with 2 × 106 irradiated carbamoyl phosphate synthase mutant (CPS) at day 0 and 1 × 106 CPS at day 4. Total PECs were harvested on day 7 by following the same procedure mentioned above (1). Zhao et al. www.pnas.org/cgi/content/short/1316841111 1 of 3 Fig. S1. Avirulent T. gondii strain PTG induced membrane ruffling and actin polymerization once attached to primary mononuclear cells derived from mouse and human. Mononuclear cells harvested from naïve mouse peritoneum or human PBMC were seeded on coverslips and infected by GFP-PTG or GFP-RH as in Fig. 1 A and B. The host F-actin was stained by Phalloidin-Alexa Fluor 555 and the PVMs were stained with α-GRA7 antisera. Representative images are shown on Left to illustrate the induction of host actin polymerization by PTG but not by RH parasites. The frequency of phagocytic cup induction by attached parasites is shown on Right. (Scale bars: 5 μm.) Fig. S2. Phagocytosed PTG parasites form moving junctions within highly activated macrophages. Three internalized PTG tachyzoites at the three putatively sequential stages of PTVI were captured in a single fluorescent image. Day 7 CPS-primed mouse peritoneal macrophages were infected by GFP-PTG for 15 min. Infected cells were stained with phalloidin-AF350, α-RON4 and α-MIC2 Abs. Arrows pinpoint moving junctions (RON4). Parasite 1 (initiation) is intracellular and has started to form a moving junction. Parasite 2 (progression) is also intracellular and is in the process of transitioning from a phagosomal to a vacuolar niche. Parasite 3 (completion) has nearly completed PTVI showing that MIC2 is found behind the MJ and is shed from the posterior end of the parasite. Zhao et al. www.pnas.org/cgi/content/short/1316841111 2 of 3 Fig. S3. Avirulent T. gondii strain PTG does not exhibit infection preference for T cells in vitro and in vivo. (A) PECs were harvested from the peritoneum of naïve C57BL/6 mice and infected ex vivo with GFP-PTG or mCherry-RH alone (single infection, s.i) or in combination (dual infection, d.i) at moi = 0.5 per strain for 50 min at 37 °C. Infection rate in T cells (TCRβ+CD11b− B220−) was determined by flow cytometry. Data are presented as mean± SD (n = 3). This experiment was repeated four times with similar results. (B) Naïve C57BL/6 mice were i.p infected by either 1 million GFP-PTG or 1 million mCherry-RH per mouse during single infection (Single, s.i, n = 2), or by 1 million GFP-PTG and 1 million mCherry-RH per mouse during dual infection (Dual, d.i, n = 4) for 60 min. In vivo infected PECs were harvested and analyzed using FACSAria. T cells (TCRβ+CD19−) infection rates in individual mice are shown. This experiment was repeated three times with similar results. Zhao et al. www.pnas.org/cgi/content/short/1316841111 3 of 3