Figure 2. The role of TgTRPPL-2 in T. gondii growth.

(A) Schematic representation of the generation of ΔTgTRPPL-2 in the T. gondii RH strain. (B) qPCR of total RNA from ΔTgTRPPL-2 (Δ), ΔTgTRPPL-2-trppl2 (C), and parental strains (P) using primers upstream and downstream of the insertion site of the dihydrofolate reductase-thymidylate synthase cassette. (C) Immunofluorescence analysis of extracellular parasites showing plasma membrane labeling with αTgTRPPL-2 (1:1000) and co-localization with αSERCA (1:1000). (D) Plaque assays of parental (P), ΔTgTRPPL-2 (Δ), and ΔTgTRPPL-2-trppl2 (C) parasites. Quantification of plaque sizes from three independent biological experiments using Student’s t-test. Values are means ± SEM. ****p<0.0001. (E) Red green assays of parental, and ΔTgTRPPL-2 cells quantifying invaded and attached intracellular parasites. Assays were done at two concentrations of extracellular Ca²⁺: 0.5 and 1.8 mM. Values are means ± SEM. **p<0.001, ****p<0.0001. (F) Time to egress stimulated by saponin/Ca²⁺ at 1.8 mM extracellular Ca²⁺ of both parental and the ΔTgTRPPL-2 mutant. (G) Statistical analysis of average egress time stimulated by saponin or Zaprinast in the presence or absence of extracellular Ca²⁺. Analysis was performed from three independent biological replicates using Student’s t-test. Values are means ± SEM, **p<0.003, ****p<0.0001. Black bars represent parental strain, blue bars represent ΔTgTRPPL-2. Scale bars for C represent 5 µm.

Figure 2—figure supplement 1. Validation of the anti-TgTRPPL-2 antibody.

(A) Genomic validation of the insertion of the dihydrofolate reductase-thymidylate synthase cassette in the ΔTgTRPPL-2 locus. Primers used are shown in Figure 2A. (B) Western blot analysis of total lysates from different cells lines were run in an SDS-PAGE and developed with αTgTRPPL2 (1:1000) and αTubulin (1:10,000) (1: ΔKu80Tati; 2: TgTRPPL2-smHA; 3: RH; 4: ΔTgTRPPL-2). Note that all cell lines show a band at ~115–120 kDa and lane 2 (tagged line) shows two bands one at ~115–120 kDa and an additional band of ~150 kDa due to the presence of the smHA tag, which adds approximately 39 kDa to the protein. The sum of these two bands (120 + 150 kDa) represents the size predicted for TgTRPPL2 tagged with the smHA tag in ToxoDB. (C) Western blots of lysates from parental, ΔTgTRPPL-2 (Δ) and ΔTgTRPPL-2-trppl2 (C) cells were run in an SDS-PAGE cell and developed with the anti-TgTRPPL-2 antibody at 1:1000. αTubulin (1:10,000) was used as loading control. (D) Immunofluorescence analysis of extracellular tachyzoites with αTgTRPPL-2 (1:1000) co-localized with αSERCA (1:1000) in ΔTgTRPPL-2 and ΔTgTRPPL-2-trppl2. Scale bars represent 5 μm. Images were taken with equivalent time and laser power.