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. 2014 Apr 14;111(17):6437–6442. doi: 10.1073/pnas.1316841111

Fig. 2.

Fig. 2.

Phagocytosed PTG undergoes phagosome to vacuole invasion (PTVI) to establish parasitophorous vacuoles in resting macrophages. (A) Internalized PTG form moving junctions intracellularly. Arrow points to an extracellular PTG positively stained by surface antigen SAG1 antibody. Arrowhead points to an intracellular PTG (SAG1−) forming a moving junction as visualized by α-RON4 staining. (B) Intracellular PTG form moving junctions from within phagosome. Arrowhead points to a constriction indicating the localization of the moving junction. Nascent PVM was stained by α-GRA7 Ab, and phagosome was stained by α-LAMP-1 Ab. (C) Sensitivity of PVM formation by PTG strain to Baf A1 inhibition of phagosomal maturation. RAW264.7 cells were pretreated with 25 nM Baf A1 for 1 h followed by PTG infection for 40 min in absence of the drug. Frequency of GRA7+ vacuoles within internalized PTG is shown, and data are presented as mean ± SD in triplicates. ***P < 0.001, Student t test. (D) PVM formation by RH is insensitive to Baf A1 pretreatment of host cells but becomes sensitive after opsonization with the mouse α-SAG1 IgG2a. The data were analyzed as in C. (E) Stripping of opsonized antibody from phagocytosed RH surface as parasites transit from phagosome to vacuole. The number 1-, 2-, and 3-coded RH parasites show opsonized Ab at the three different stages of stripping during invasion, with opsonized Ab intact on surface of parasite 1 still residing in a phagosome (LAMP-1+), completely stripped from the surface of parasite 3, which has established a vacuole (ROP1+) and undergoing stripping from the surface of parasite 2 during passage through the MJ. Opsonized α-SAG1 Ab was visualized by donkey anti-mouse AF568 (blue), PVM was stained with α-ROP1 (white) and phagosome with α-LAMP-1(red). The yellow dotted line indicates the putative localization of the moving junction. Please note that magnification of the cropped image of parasite 2 is higher than that of parasite 1 and 3. (F) Schematic depiction of PTVI pathway, the two-step infection process, is initiated with phagocytosis of T. gondii followed by parasite active invasion from within phagosome. (Scale bars: 5 µm.)