A Toxoplasma gondii patatin-like phospholipase contributes to host cell invasion
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.