FIG 6.

Host ESCRT-associated proteins PDCD6, ALIX, and CC2D1A, and the host ER-organelle tethering protein MOSPD2, localize to the Toxoplasma parasitophorous vacuole membrane. (A) Two-dimensional plot showing log₂ fold changes between miniTurbo-RAH and miniTurbo-infected samples (x axis) and log₂ fold changes between infected and mock-infected miniTurbo-RAH samples (y axis). Toxoplasma proteins known to be exported or PV/PVM-localized or putatively nonsecreted are labeled using the same color scheme as in Fig. 5A. The dotted line indicates the optimal threshold for separation of known PV/PVM/exported-localized proteins and nonsecreted Toxoplasma proteins. Yellow shading indicates the position on the plot of the proteins of interest described in Table 2. (B) Significant terms from functional enrichment analysis performed on the candidate host PVM proteins from Table 2. Terms are shown for the Gene Ontology biological process functional database. (C) Result of STRING version 11 (61) protein-protein interaction analysis of the candidate host PVM proteins from Table 2 with singletons (proteins with no association to another protein) removed. The thickness of the lines between proteins indicates the degree of confidence of the interaction. (D) Representative immunofluorescence microscopy images of the localization of the host proteins CC2D1A and PDCD6IP/ALIX during Toxoplasma infection. HFFs were infected with RH parasites for 20 h before the infected monolayers were fixed with methanol. Rabbit anti-CC2D1A antibodies and rabbit anti-ALIX antibodies were used to detect the corresponding host proteins (green). Tachyzoites were detected with rabbit anti-SAG2A antibodies (magenta), and the infected monolayer was visualized with phase microscopy. Scale bar is 10 μm. (E) Representative immunofluorescence microscopy images of the localization of host proteins upon transient overexpression in HFFs. HFFs were transiently transfected with plasmids expressing the indicated tagged proteins and then infected with RH tachyzoites 24 h posttransfection. The parasites were allowed to infect for 16 h before the monolayers were fixed with methanol. Mouse anti-V5 and rat anti-HA antibodies (green) were used to detect the corresponding host proteins. Tachyzoites were detected with rabbit anti-SAG2A antibodies (magenta), and the infected monolayer was visualized with phase microscopy. Dashed white boxes indicate the PVs expanded in the insets (left-most panels). Scale bars are 10 μm.