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. 2017 Sep 12;6:e27480. doi: 10.7554/eLife.27480

Figure 6. 49c recapitulates the phenotype of ASP3 depletion in T. gondii.

(A) Chemical structure of compounds 49b and 49c. (B) IC50 on T. gondii RH-pTub-CBG99-luciferase parasites of 49b (13.8 μM), 49c (676 nM) and Pyrimethamine (295 nM), used here as a positive control. (C) 49c treatment impaired RH parasites invasion and (D) egress. Data are presented as mean ±standard deviation (SD) from 3 independent experiments. (E, F) Parasites, treated for 36 hr with DMSO or 49c, showed normal secretion of microneme proteins (MIC2, M2AP, MIC4), but altered processing in this fraction (MIC2, M2AP, MIC4). Arrowheads show full length and secreted fragments. Catalase was used as cytosolic control and GRA1 for constitutive secretion. (G) 49c treated during 48 hr of Δku80 parasites displayed a complete absence of rhoptry content secretion as assessed by probing against the rhoptry protein ROP1. (H) Processing of RON4, ROP18 and MIC6 was affected by 48 hr of 49c treatment while DMSO treated samples were not affected. AMA1 serves a control for an unprocessed microneme and Catalase as loading control. ROP18 was Ty-tagged at endogenous locus. Pepstatin was used as a negative control. (I) Invasion was blocked by 49c when parasites were treated for 6 hr or more prior to egress, but not less than 3 hr or when extracellular. (J) Parasites were blocked in egress when treated with 49c at least 3 hr before induced egress, but not later. (K) Schematic showing the various treatments for the induced egress assay in J.

Figure 6—source data 1. Source data of the triplicate experiments done on RHΔku80 parasites (DMSO or 49c treated) Intracellular growth assay (Figure 6—figure supplement 1A), Invasion assay (C), Egress assay (D), Invasion assay with washes (I) and Egress assay with washed (J).
elife-27480-fig6-data1.zip (13.1KB, zip)
DOI: 10.7554/eLife.27480.029
Figure 6—source data 2. Source data of the in vitro measurement of IC50 done on luciferase expressing T. gondii tachyzoites (Figure 6B).
elife-27480-fig6-data2.zip (66KB, zip)
DOI: 10.7554/eLife.27480.030

Figure 6.

Figure 6—figure supplement 1. 49c has no impact on parasite intracellular replication, gliding motility, and on the localization of rhoptry and microneme proteins.

Figure 6—figure supplement 1.

(A) DMSO or 49c treated RHΔku80 parasites revealed no defect in intracellular replication. Data is presented as mean ±SD from 3 independent experiments. (B) Gliding motility was not affected by 49c treatment. DMSO and cytoD were used as positive and negative controls respectively. (C) Processing of MIC5 was unaffected by 48 hr of 49c treatment. DMSO/49b/Pepstatin treatment has no impact on processing of these proteins. ROP2-4 serves a control for an ASP3 dependent processing, which is indeed inhibited by 49c, and Catalase as loading control. MIC5 was Ty-tagged at endogenous locus. (D) Processing of PLP1 was analyzed by 48 hr of treatment with 49c/49b/DMSO/Pepstatin. ROP2-4 serves a control for a ASP3 dependent processing and Catalase as loading control. PLP1 was Ty-tagged at endogenous locus. (E) Localization of RON4, ROP18, MIC6 and AMA1 remained unchanged upon 49c treatment. ROP18 was Ty-tagged at the endogenous locus.
Figure 6—figure supplement 2. 49c has no impact on ASP5 maturation, intravacuolar membranous nanotubular network (MNN) formation or on the maturation and export of ASP5 substrate, GRA16.

Figure 6—figure supplement 2.

(A) Formation of intravacuolar membranous nanotubular network was not affected by either 49c treatment or ASP3 depletion. DMSO treated wild type parasites were used as negative control. (B) Endogenously Ty tagged ASP5 strain, when treated with DMSO or 49c show no defect in ASP5 maturation. (C) Immunoblot evaluating the processing of a second copy expression of myc tagged GRA16 showed that 49c has no impact on its ASP5 dependent processing. (D) 49c has no impact on the ASP5 mediated export of GRA16 to the host cell nucleus. DMSO treated wild type parasites and ΔASP5 parasites were used as negative and positive controls respectively.