Fig 1.
Predicted domains of TgPL3 based on sequence and structural alignments.
(A) In orange, two serine-rich domains (S) flank the microtubule binding domain (MtBD) in green. The first serine-rich domain contains 34 consecutive serines and the second contains 37 serines interrupted by 2 prolines and 2 alanines. The putative PLP domain is shown in black. (B) The PLP domain contains all the necessary conserved amino acids for activity based on sequence alignment. The known conserved patatin motifs are underlined and catalytic S/A dyad are colored red. The serine to alanine mutation (S1409A) is indicated by an arrow. (C) Structural alignment of the TgPL3 PLP domain (orange) to the patatin PLA2 crystal structure (blue) predicts correct folding to form the active site. The secondary and tertiary structures of the TgPL3 PLP domain were predicted using I-TASSER and the resulting model was aligned to the PDB model of patatin (1OWX) in PyMOL. The catalytic serine and aspartate dyad are shown in green in each model.
Fig 2.
TgPL3 localizes to the apical end of tachyzoites.
Top three panels show immunofluorescence of TgPL3 (red) in intracellular tachyzoites co labeled with (A) MIC2, (B) RNG1 or (C) CAP1 (green). Bottom two panels show TgPL3 localization in extracellular tachyzoites after (D) induced conoid extrusion or (E) deoxycholate extraction (DOC). (F) Schematic showing costain localization in intracellular parasites (created with BioRender.com). Images for panels A, B, C and D were taken under the same magnification and the scale bars in the lower right corners equal 5 μm.
Fig 3.
Deletion of TgPL3 from RHΔKu80 parasites using FACS sorting.
(A) Schematic for the targeted knockout of the TgPL3 gene in T. gondii by double homologous crossover. Parental strain RHΔku80ΔHXGPRT parasites were electroporated with the CRISPR/Cas9 plasmid containing gRNA targeting the 5’ end of TgPL3 and the knockout plasmid containing TgPL3 5’ and 3’ flanking regions surrounding mCherry which allows for sorting mCherry positive parasites using FACS. Sorting GFP negative clones in tandem ensures a double crossover event. (B) Genomic DNA of clones were screened by southern blot using a probe targeting the 5’ upstream (US) flanking region of TgPL3. Parental RHΔku80ΔHXGPRT (WT), complement (C1) and S1409A (SA) parasites show a 12kb band, ΔTgPL3 (KO) strains show a 3kb band. (C) Parasite strains were further analyzed by immunofluorescence of TgPL3 (red) co-labeled with MIC2 (green). Images were taken under the same magnification and the scale bar in the lower right corner equals 5 μm.
Fig 4.
TgPL3 is critical for invasion but not attachment.
(A) Triplicate monolayers of HFFs were infected with 3.4 x 104 T. gondii parasites. At 18 or 26 hours post infection, tachyzoites per vacuole were scored in at least 100 randomly encountered vacuoles per replicate. Average tachyzoites per vacuole is shown at each time point. P value was nonsignificant in a two tailed t-test compared to WT. (B) 9 days post infection, HFF monolayers were stained with crystal violet to reveal T. gondii plaque formation. White arrows indicate the small plaques created by the KO and SA parasites. (C) The percentage of parasites that were successfully able to invade the host cell was determined using the red/green invasion assay [21]. Four independent experiments were performed in triplet, and the combined results are shown here. *** p<0.0001. (D) The percentage of parasites that were attached to glutaraldehyde fixed host cells was examined but no significant differences were seen. Shown here the combined results from two independent experiments performed in triplet.
Fig 5.
TgPL3 enhances rhoptry but not microneme secretion.
Excreted/secreted antigen (ESA) was induced in 1 x 108 parasites by the addition of 3% fetal bovine serum and 1% ethanol (FBS/EtOH). Supernatant (A) and pellet (B) were run on a 12% SDS-page gel, transferred to PVDF membranes and simultaneously probed with rabbit anti-MIC2 and mouse anti-SAG (monoclonal DG52). (A) Western blotting showing processed MIC-2 (95–100 kDa) and no SAG1. Size markers are indicated on the right side in kDa. (B) Pellet or loading control shows the full-size MIC-2 band (115 kDa) and a lower band corresponding to SAG1 (30 kDa). (C) Quantification of rhoptry discharge during e-vacuole formation. Triplicate monolayers of HFFs were infected with 5x106 parasites that were pre-treated with cytochalasin D. After a 15-minute incubation, the parasites were stained with a mouse α-SAG1 antibody and a rabbit α-ROP1 antibody. After counting, percent rhoptry secretion was normalized to the WT strain. Three independent experiments were performed, and the combined results are shown here. *** p<0.001 (D) Rhoptry secretion by the SeCreEt assay [28] and the number of GFP++ host cells was quantified by FACS analysis, as shown on the y-axis. After 1 hour of invasion, ΔTgPL3 parasites complemented with S1409A showed a significant difference in rhoptry secretion compared to parasites complemented with the WT gene (** p<0.01). After 6 hours, this difference was less significant (* p<0.05). Shown here the results from one of two independent experiments performed in triplet.
Fig 6.
ΔTgPL3 and S1409A parasites are avirulent.
(A) Shown is a combination of two independent experiments of 3–4 NMRI mice each, either all male or all female, with a total of 7 mice per strain. Mice were infected with 1000 T. gondii parasites and survival was monitored for 28 days post infection. No brain cysts were seen in surviving mice. *** p<0.001. (B) Serum collected 28 days post infection in surviving mice tested for seropositivity with individual strip blots for each mouse (labeled 1, 2, 3 or 4). Seroconversion was seen in all ΔTgPL3 and S1409A infected mice but not in uninfected mice (UN) or mice infected with heat-killed parasites (HK). Shown is a representative of two independent experiments.
Fig 7.
ΔTgPL3 and S1409 parasites are unable to disseminate in vivo.
In 2 independent experiments, 4 male NMRI mice were infected with 1000 T. gondii parasites. 3 days post infection (A) spleens and (B) lungs were removed and homogenized. Homogenate was serially diluted, added to HFF monolayers in duplicate, and plaqued for 7 days. The monolayers were stained with crystal violet and PFU/ml calculated. (C-F) Serum was collected from the same mice at 3 days post infection and cytokine levels of (C) IFNγ, (D) MCP1, (E) TNF and (F) IL6 were determined using a cytokine bead array kit (Biosciences). Cytokine analysis was repeated with female mice and shown is one representative experiment. Uninfected mice and mice infected with 1000 heat-killed parasites were used as negative controls * p<0.05, **p <0.01 and ***p<0.001.
Fig 8.
ΔTgPL3 and S1409 parasites induce a protective immunity against subsequent infections.
3 male NMRI mice were infected with 1000 ΔTgPL3 or S1409 parasites, or uninfected as a negative control. 30 days post infection, mice were tested for seropositivity and challenged with 1000 RHΔKu80ΔHPT parasites. Survival was recorded for 14 days post challenge.