Article Figures & Data
Figures
- FIG 1
Initial cyst wall pulldowns with BioID reveal hypothetical proteins. (A) Schematic diagram of the exogenous expression construct containing the promoter and coding sequence of the cyst wall gene of interest fused C terminally with BirA* and 3×HA followed by the DHFR selectable marker. (B) Immunofluorescence micrographs of Pru and BirA*-tagged parasites (CST1, MAG1, MCP4, BPK1, and GRA6) stained with anti-HA antibody and Alexa Fluor 488-conjugated streptavidin under pH 8 conditions supplemented with 150 μM biotin. Endogenously biotinylated proteins within the apicoplast are observed within these parasite strains. (C) Network graph showing potential parasite-specific interacting proteins of each BirA*-tagged cyst wall protein (CST1, MAG1, MCP4, BPK1, and GRA6) identified during the bradyzoite stage. BirA* cyst wall bait proteins, known cyst wall proteins, dense granule proteins, and hypothetical proteins are represented by purple, red, green, and blue nodes, respectively. Darker arrows correspond to higher normalized spectral abundance factor (NSAF) values of prey proteins identified in each pulldown.
- FIG 2
Iterative BioID pulldowns of cyst wall proteins identify a novel cyst wall protein. (A) Immunofluorescence images of tagged hypothetical proteins stained with anti-HA and anti-ALD1 (cytosolic marker) or anti-CST1 (cyst wall marker) antibodies. Tachyzoites: HA in red and ALD1 in green; bradyzoites: HA in red and CST1 in green. (B) Immunofluorescence micrographs of endogenously BirA*-tagged parasites (CST2, CST3, CST4, CST7, CST8, CST9, and MCP3) stained with anti-HA antibody and Alexa Fluor 488-conjugated streptavidin under pH 8 conditions supplemented with 150 μM biotin. (C) Network graph showing potential parasite-specific interacting proteins of each BirA*-tagged cyst wall protein (CST2, CST4, CST9, and MCP3). BirA* cyst wall bait proteins, known cyst wall proteins, novel cyst wall proteins, dense granule proteins, and hypothetical proteins are represented by purple, red, orange, green, and blue nodes, respectively. Darker arrows correspond to higher normalized spectral abundance factor (NSAF) values of prey proteins identified in each pulldown. (D) Immunofluorescence images of HA-tagged hypothetical proteins (HP) identified from the group 2 BirA* pulldowns stained with anti-HA and anti-ALD1 (cytosolic marker) or anti-CST1 (cyst wall marker) antibodies. Tachyzoites: HA in red and ALD1 in green; bradyzoites: HA in red and CST1 in green.
- FIG 3
Cyst wall interactome model analysis reveals distinct clusters. Venn diagrams showing the proteins that are common between the group 1 (A) or group 2 (B) cyst wall BirA* pulldowns. Each BirA* pulldown is represented by a different colored circle. (C) Network of the proteins identified in the group 1 and group 2 BirA* pulldowns. BirA* bait protein nodes are represented by a blue outline. Known cyst wall proteins, dense granule proteins, and novel cyst wall/matrix proteins are represented by red, green, and orange nodes, respectively. Darkness of arrows represents the normalized spectral abundance factor (NSAF) of the prey proteins identified in each pulldown. Size of each node represents the in-degrees or the amount of times a protein was identified within each pulldown. Protein clustering was performed using the Louvain method of community detection. (D) Graph of each node’s betweenness, which signifies the extent to which nodes stand between each other, versus their in-degrees, the frequency of each node appearing in a pulldown. (E) Heat map showing the percentage of identity between alpha tubulin and each T. gondii cyst wall and cyst matrix protein to their orthologs in other cyst-forming coccidians.
- FIG 4
Characterization of cyst wall proteins reveals MCP3 affects cyst size. (A) Schematic showing the endogenous editing of genomic loci using a targetable stop PAM sequence (TSPS) to introduce a Cas9-targetable sequence of premature stop codons. Complementation of the edited loci was performed by targeting the premature stop codons with a TSPS-specific sgRNA and replacing the TSPS with synonymous mutations of the coding region. The red region indicates the TSPS sequence, while the green region indicates synonymous mutations. (B) IFA showing CST4, CST8, CST9, and MCP3 knockout (top) and complement (bottom) bradyzoites stained with anti-HA (red) and anti-CST1 (green) antibodies. (C) Brain cyst counts of mice infected with Pru, CST4, CST8, CST9, and MCP3 knockout and complement parasite strains at 30 days postinfection. Numbers of brains counted are listed above each violin plot. No significant differences in cyst numbers between BirA*, knockout, and complement parasites in each strain group were observed (P > 0.05). (D) Size analysis of in vivo cysts obtained from Pru, CST4, CST8, CST9, and MCP3 knockout and complement parasite strains. Numbers of cysts imaged and analyzed are listed above each violin plot. NS, not significant; *, P < 0.05; ***, P < 0.001.
- FIG 5
Novel cyst wall proteins do not affect morphology of cyst ultrastructure. Transmission electron micrographs of in vitro cysts from the Pru, ΔCST4, ΔCST8, ΔCST9, and ΔMCP3 parasite strains.
Supplemental Material
FIG S1
BirA*-tagged proteins correctly localize to the cyst wall. (A) Immunofluorescence images showing that BirA*-tagged parasites (CST1, MAG1, MCP4, BPK1, and GRA6) localize correctly to the cyst wall as seen with colocalization with CST1. (B) Immunofluorescence assay of Pru and BirA*-tagged parasites stained with anti-HA antibody and Alexa Fluor 488-conjugated streptavidin under pH 8 conditions without exogenous biotin added to the medium. Endogenously biotinylated proteins within the apicoplast are observed within these parasite strains. (C) Western blot of lysates corresponding to Pru and BirA*-tagged parasites differentiated in pH 8 supplemented with and without 150 μM biotin was probed with streptavidin-horseradish peroxidase (HRP). ALD1 was used as the loading control. Download FIG S1, TIF file, 2.8 MB.
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FIG S2
Intracellular and extracellular immunofluorescence of BirA*- and HA-tagged cyst wall proteins. (A) Immunofluorescence assay of BirA*-tagged parasites differentiated to bradyzoites without exogenous biotin added to the induction medium. Fixed cells containing cyst vacuoles were stained with anti-HA antibody and Alexa Fluor 488-conjugated streptavidin, which labels the endogenously biotinylated proteins within the apicoplast. (B) Immunofluorescence micrographs of extracellular BirA*- (CST9) or HA-tagged (CST10) tachyzoites stained with anti-HA (red), anti-GRA1 (green), and DAPI (4′,6-diamidino-2-phenylindole; blue). Download FIG S2, TIF file, 2.8 MB.
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FIG S3
Multiple-sequence alignment tree diagram of cyst wall and cyst matrix proteins. Protein sequences of Toxoplasma gondii cyst wall and cyst matrix proteins and alpha tubulin were aligned to their orthologous proteins within Hammondia hammondi, Neospora caninum, Cystoisospora suis, and Sarcocystis neurona, based on protein identity. The similarity between each protein was visualized using a phylogenetic tree. Download FIG S3, TIF file, 0.9 MB.
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FIG S4
Sanger sequencing results of the loci targeted by Cas9 of ΔCST4, ΔCST8, ΔCST9, and ΔMCP3 parasites. Chromatograms showing the sequencing results of the N termini of ΔCST8 and ΔCST9 parasites demonstrating the inserted premature stop codons. ΔCST4 and ΔMCP3 parasites had the targetable stop PAM sequence (TSPS) inserted further downstream from the start codon. The red boxes depict the DNA sequences where stop codons or TSPSs were introduced to generate the knockout strains. Download FIG S4, JPG file, 2.0 MB.
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TABLE S2
Oligonucleotides used as donor DNA to generate HA-tagged, knockout, and complement strains. Mutations are designated in red, with stop codons designated as lowercase letters and the HA sequence in green. Download Table S2, DOCX file, 0.1 MB.
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FIG S5
Additional characterization of cyst wall proteins. (A) Quantification of the plaque size from each parasite strain. The number of plaques analyzed per strain is provided above each violin plot. No significant differences in plaque size between the parental, knockout, and complement strains were observed for each cyst wall protein. Immunofluorescence images of cyst wall protein knockout parasites stained for CST1 and MAG1 (B) or CST, DBA, and MCP4 (C) under bradyzoite conditions in vitro. (D) Survival curve of C57BL/6 mice challenged with 100,000 Pru, ΔCST4, ΔCST8, ΔCST9, and ΔMCP3 parasites (n = 10). Download FIG S5, TIF file, 1.8 MB.
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FIG S6
Ultrastructural localization of cyst wall proteins. Immunoelectron micrographs of in vitro cysts from Pru and BirA*-tagged parasites (CST4, CST8, CST10, and MCP3) showing labeling with the anti-HA antibody. White arrows point to particles from the gold-conjugated anti-rat antibody. Download FIG S6, TIF file, 2.6 MB.
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DATA SET S1
LC-MS/MS results obtained from the group 1 and group 2 BirA* pulldowns. Numbers indicate the NSAF values obtained from Scaffold4 software after all the pulldown data were merged together. Nonspecific proteins from the Pru strain were filtered out from each pulldown. Proteins highlighted in red are the hypothetical proteins identified in this study. Group 1 pulldowns were duplicated and pooled before analysis. Group 2 pulldowns were performed once. Download Data Set S1, XLS file, 0.5 MB.
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TABLE S1
Primers used in this study. Overhanging regions for Gibson assembly or KLD reaction are designated in green. Download Table S1, DOCX file, 0.1 MB.
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