Figure 1.
Monoclonal antibody SalmonE binds to the cyst wall of cysts isolated in vitro and in vivo.
(A) Human foreskin fibroblasts (HFF) were infected with ME49 strain of T. gondii under alkaline conditions (pH 8.1) for 3 days. Anti-BAG1 antibody (red) staining demonstrates that the smaller parasitophorous vacuole on the right is an in vitro cyst; mAb SalmonE (green) binds to the parasitophorous vacuole of in vitro cyst, but not to the larger parasitophorous vacuole containing tachyzoites on the left. Bar, 10 µm. (B) Immuno-electron microscopic labeling of the cyst wall with mAb SalmonE. A ME49 brain tissue cyst isolated from an infected mouse was processed for immuno-electron microscopy and probed with mAb SalmonE. The gold particles demonstrate that mAb SalmonE localizes to the granular layer (arrow) under the limiting membrane of cyst wall. Bar, 200 nm.
Figure 2.
Monoclonal antibody SalmonE recognizes cyst wall protein CST1 (TGME49_064660).
(A) The two peptides (red and blue bars) identified by mass spectrometry were mapped to the TGME49_064660 (CST1) protein. The recombinant protein rTGME49_064660 is the first 200 amino acids of the predicted gene (green bar). (B) CST1 (TGME49_064660) antiserum stains in vitro cyst walls. HFF cells were infected with ME49 strain of T. gondii under alkaline conditions for 3 days. Anti-rTGME49_064660 antiserum (mice immunized with TGME49_064660 recombinant protein green) stains the parasitophorous vacuole containing BAG1-positive parasites (red). Bar, 10 µm.
Figure 3.
The mucin domain of CST1 is highly O-glycosylated.
(A) The glycosylation potential of CST1 was predicted by the NetOGlyc 3.1. Red line indicates the threshold and blue line indicates the potential at each S/T amino acid position with domain-scheme overlay. This method predicts that the mucin domain is likely to be highly O-glycosylated. (B) mAb SalmonE immunoprecipitates are bound by DBA lectin. The left lane is 10 µl of whole parasite lysate (ME49 at pH 8.1) and the right lane is the sample (equivalent to 200 µl lysate) that was immunoprecipitated with mAb SalmonE, separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was probed with AP-conjugated DBA lectin.
Figure 4.
Characterization of CST1 knock out (Δcst1) and CST1 complemented T. gondii strains.
(A) Schematic representation of disruption of CST1 and complementation of Δcst1 mutant. Upper half of the diagram represents the deletion of whole CST1 gene with Δcst1 vector. Lower half represents the complementation of CST1 genes at UPRT locus. (B) Immunoblot of T. gondii parasite cultures grown in normal and alkaline conditions probed with mAb SalmonE. SalmonE reactive antigen (CST1) is induced in alkaline conditions. Δcst1 knockout parasites are not recognized by mAb Salmon E, but reactivity is restored in full-length complement (Δcst1::cst1) but CST1Δmuc protein lacks the reactivity due to the loss of mucin-like domain. The parasite specific dense granule protein GRA1 is used as a loading control. (C) CST1 and DBA co-localize on in vitro cyst wall. HFF cells infected with WT, Δcst1, Δcst1::cst1, or Δcst1::cst1Δmuc T. gondii strains under alkaline conditions were probed with SalmonE (red) or DBA lectin (green). Monoclonal antibody SalmonE and DBA staining co-localized. The presence of the full-length CST1 gene is necessary for mAb SalmonE and DBA staining on cyst wall. Bar, 10 µm.
Figure 5.
SalmonE and mAb 73.18 recognize CST1 (TGME49_064660).
(A) Immunoblot of mAb SalmonE immunoprecipitated material (ME49), WT lysate (Pru) and Δcst1 lysate probed with mAb SalmonE (green) and mAb 73.18 (red). This immunoblot demonstrated that mAb SalmonE and mAb 73.18 bind to the same major species which is not detectable in Δcst1 parasites. (B) IFA of in vitro cysts probed with CST1 specific monoclonal antibody 73.18 (red) and DBA lectin (green). The presence of full-length CST1 is required for both mAb 73.18 and DBA cyst wall staining. Bar, 10 µm.
Figure 6.
BAG1 expression in WT, Δcst1, Δcst1::cst and Δcst1::cst1Δ
muc parasites. HFF cells were infected with either WT, Δcst1, Δcst1::cst and Δcst1::cst1Δmuc parasites and probed with anti-CST1 antiserum (red) and rabbit anti-BAG1 (green). This demonstrates that differentiation occurs in the Δcst1 and Δcst1::cst1Δmuc parasites.
Figure 7.
Brain cyst burden and survival rate of infected mice at 4 weeks post infection.
(A) Survival curve of the mice challenged with the WT and mutant parasites (n = 20). (B) Brain cyst count of C57/BL6 mice infected with either WT, Δcst1, Δcst1:cst1 or Δcst1:cst1Δmuc parasites sacrificed at 4 weeks post infection. The bars represent mean and standard deviation (n = 12). *p<0.05 (Mann-Whitney U test). (C) Immunohistochemistry of brain sections from infected mice probed with anti-GFP antibody demonstrates the formation of brain cysts in vivo. Pru parasites are stably transfected with LDH2-GFP enabling identification of bradyzoites within cysts. Bar, 10 µm.
Figure 8.
The mucin domain of CST1 is required to form mechanical stress-resistant cyst wall structures.
(A) Brain cysts of WT and mutants after gentle brain homogenization, observed under epifluorescence microscopy. Bar, 10 µm. (B) Electron micrograph of T. gondii brain cysts demonstrates an alteration in the cyst wall with disruption and loss of the granular layer found underneath the cyst wall membrane. This disruption is visible in the Δcst1 and Δcst1::cst1Δmuc cysts, but no disruption is seen in the WT and Δcst1::cst1 cysts. Red arrows indicate the cyst wall membrane and the blue arrows indicate the cyst wall granular layer. Measurements of the cyst wall: WT 153±28 nm, Δcst1 24±10 nm, Δcst1::cst1 284±65 nm, and Δ cst1::cst1Δmuc 34±14 nm (p<0.05 WT vs Δcst1, WT vs Δ cst1::cst1Δmuc, and WT vs Δ cst1::cst1). The original image magnification was 20,000× on all of these EM images. Bar, 200 nm.
Figure 9.
Growth of Δcst1 parasite is impaired at pH 8.1 but not at pH 7.1.
(A) Growth of WT (blue) or Δcst1 parasites (red) in HFF cells at pH 8.1 were measured as 3H-uracil incorporation into parasites DNA. Mean and standard deviation are shown. n = 3, * p<0.005. This experiment was repeated 3 times with similar results. (B) Number of parasites inside vacuoles in HFF cells at pH 8.1 per 600 host cells. 3H-uracil incorporation was not done due to the lack of UPRT gene in complement strains.
Figure 10.
Lack of CST1 reduces bradyzoite gene expression.
(A) Heat map of fold change in gene expression (pH 8/pH 7) of wild type and cst1 mutants. The top 50 upregulated genes in wild type T. gondii are displayed in order. The number is the gene name in TGME49 (ToxoDB ver 6.1). Gene upregulation is reduced in the Δcst1 strain. The full length complement strain Δcst1:cst1 has a restoration of gene upregulation to control (WT) levels, but the mucin-null complement strain Δcst1:cst1Δmuc did not have restoration of these gene levels. (B) Upregulation of known bradyzoite specific gene expressions shows the same pattern. Note that the housekeeping and tachyzoite specific genes did not follow the same expression pattern. Both (A) and (B) graphs were generated from the same RNA-seq data set.