Fig 1.
Vicia villosa lectin staining in Toxoplasma.
(A-D) VVL-FITC was used to stain and visualize intracellular parasites using (A-C) formaldehyde fixation, or (D) methanol fixation. (A) Formaldehyde fixation results in both apical and posterior staining in the parasites, (B) some vacuoles also show staining in the vacuole (arrow). (C) Co-staining of VVL with IMC3 shows some co-localization at the periphery (arrowheads) and in daughter buds (arrow). Co-staining was also seen at the apical periphery with micronemes (MIC2, double arrowheads), and in the vacuole with GRA14 (asterisk), but not in the golgi (GRASP55). (D) Methanol fixation results in specific staining in the rhoptries as shown by co-localization with ROP7. Scale bar is 20μm.
Fig 2.
Identification of VVL-binding proteins.
(A) Schematic for identifying VVL-binding proteins. (B, C) VVL Western blot (B) and commassie staining (C) of the eluted materials from VVL affinity chromatography. (D) Proteins identified from the VVL pull down. The bolded proteins are the ones we either discuss or study in the paper.
Fig 3.
SRS44 is a vacuolar protein with 13 SAG repeats and a C-terminal mucin-like domain.
(A-C) IFA labeling with SRS44 polyclonal antibody in either WT tachyzoites (A,B) or Δsrs44 parasites (C). (A) Staining shows different levels of SRS44 expression between two different vacuoles (B) Staining shows SRS44 detection in some vacuoles but not all (arrow). (C) Disruption of SRS44 was verified by IFA. (D) Mouse brain cysts of the pruΔku80 strain have a GFP under a bradyzoites (cysts) promoter and show staining of SRS44 on the cyst wall. Scale bar is 20μm.
Fig 4.
TgME49_283540 is a novel VVL-binding micronemal protein.
(A) Heterologous expression of TgME49_283540-HA is detected in the micronemes, as shown by co-localization with MIC2. (B) VVL Western blotting of eluted materials from MIC20-HA pull down confirms that MIC20 is a VVL-binding protein.
Fig 5.
Δron11 parasites show a minor fitness defect compared to the WT.
(A) IFA demonstrating RON11 localization to the rhoptry necks in WT parasites, using previously described rat anti-serum raised against RON11[24]. (B, C) Δron11 parasites were generated via CRISPR/Cas9-mediated disruption of RON11 as shown by IFA (B) and Western blot (C). (D) Confluent HFF monolayers were infected with equal numbers of WT and Δron11 tachyzoites. After seven days of incubation, parasites were fixed and stained with crystal violet as previously described [24]. For each experiment, 30 plaques were counted and measured from both the WT and Δron11. The bar graph represents the average mean for three experimental replicates. The error bars represent ±SD, and a paired two-tails t-test was performed comparing the mean plaque area of each Δron11 experimental replicates to the WT to assess statistical significance (p = 0.033; *p<0.05).
Fig 6.
TgME49_218240 is an IMC membrane protein that is proteolytically processed.
(A) TgME49_218240-HA shows IMC staining in both the maternal IMC and in daughter buds in parasites undergoing endodyogeny. (B, C) The IMC25 gene was disrupted via CRISPR/Cas9 and Δimc25 parasites were confirmed by IFA (B) and Western blot (C). (D) VVL pull down Western blot probed with anti-HA antibody showing total (T), precolumn (S), flow through (FT) and elution (E). The eluted material detects the less abundant IMC25 precursors, indicating that the glycosylation is upstream of the IMC25 cleavage site (~20–25 kDa). This also suggests that the C-terminal fragments of IMC25 are not linked to the larger upstream glycosylated portion of IMC25. (E) Detergent extraction demonstrates that IMC25-HA is detergent-labile (T = total, S = detergent soluble, P = detergent insoluble IMC cytoskeleton), demonstrating that this portion of IMC25 is part of the membrane portion of the IMC. ISP3 and IMC1 are used as controls for the membrane and cytoskeletal portions of the IMC, respectively.
Table 1.
Oligonucleotide primers used in this study.