Fig 1.
A VPS15 orthologue in P. falciparum and Toxoplasma gondii.
A. Domain organization of Pf/TgVPS15 revealed that TgVPS15 contains a kinase like domain (KD) at the N-terminus followed by helical HEAT repeats, which are connected by long linkers. WD40 could not be predicted with certainty as only weak sequence similarity was found to WD40 repeats in the case of TgVPS15 whereas PfVPS15 does not contain the C-terminal WD40 domain. B. Schematic illustrating the subdomains of canonical protein kinases and key putative residues involved in catalysis are indicated on the top and corresponding residues in Tg/PfVPS15 are indicated below. N.P.—not present. C. CLUSTALW alignment of the kinase domains of ScVPS15 with TgVPS15. Some of the key motifs and subdomains are indicated and putative residues possibly involved in kinase function are highlighted. D216 and E268, which were mutated in subsequent studies, are indicated by an arrow.
Fig 2.
TgVPS15 regulates PI3P formation and is critical for parasite replication.
A. Depletion of TgVPS15 in Toxoplasma gondii. TgVPS15-iKD parasites were treated with ATc for indicated duration followed by Western blotting of parasite lysate, which revealed that Myc-TgVPS15 migrates at an expected size and the addition of ATc resulted in its depletion. ΔKu80 was used as a negative control. B. IFA was performed on TgVPS15-iKD parasites that were left untreated or treated with ATc for 72h or 3d using anti-myc and anti-GAP45 antibodies. C. Plaque assays were carried out by infecting HFF monolayer with ΔKu80 or TgVPS15-iKD parasites in the presence or absence of ATc for 7 days (for quantitation please see Supp. S3B Fig). D. TgVPS15-iKD or ΔKu80 parasites were preincubated in culture medium for 48h or 2d with (+) or without (-) ATc and were subsequently allowed to invade fresh HFFs in the presence or absence of ATc and the number of parasites per vacuole was determined after 24h or 1d. Data represent Mean ± SE, n = 3 and at least 200 vacuoles were counted for each condition (n = 3, *** P<0.001 4 p/v, ** P<0.01 8 p/v, * P<0.05 16 p/v ANOVA, ns-not significant). E. TgVPS15-iKD or ΔKu80 parasites were treated with ATc for 1d, 3d or 5d and replication assay was performed as described in panel E and average intracellular parasites/vacuole present in each condition was determined. Fold change in average parasites upon ATc addition was determined (Mean±SE, n = 3, ANOVA, **** P<0.0001; ns-not significant). F. Invasion assays were performed on TgVPS15-iKD parasites that were left untreated or treated with ATc. No significant change in invasion was observed (P> 0.05, t test, n = 2, ns-not significant). G. ΔKu80/DD-GFP2xFYVE or TgVPS15-iKD/DD-GFP2xFYVE parasites that expressed GFP-2xFYVE domain infected HFF were left untreated (-) or treated (+) with ATc for 72h or 3d. Prior to fixation, Shield-1 was added for 30 minutes to stabilize the expression of GFP-2xFYVE, which was localized mainly to the apicoplast as revealed by IFA for Cpn60, an apicoplast protein. GFP-2xFYVE was found mainly in the cytoplasm of ATc-treated TgVPS15-iKD parasites. H. % vacuoles in which GFP-2xFYVE was found at the apicoplast was determined in IFAs for experiments reported in panel G (Mean±SE, n = 3, *** p<0.001, t-test).
Fig 3.
TgVPS15 regulates apicoplast biogenesis.
A. IFA was performed on ΔKu80 or TgVPS15-iKD parasites treated with ATc for 1-5d to detect apicoplast using an antibody against Cpn60 and the pellicle was stained using anti-SAG1. ATc treatment of TgVPS15-iKD parasites resulted in a loss of apicoplast. Representative images shown here are for experiments done after 4 and 5 days of ATc treatment. B. TgVPS15-iKD (red) or ΔKu80 (blue) parasites were treated with ATc for indicated duration as described in panel A and IFA was performed using anti-Cpn60 antibody. % parasites with an apicoplast were counted (Mean±SE, n = 3, ** P<0.01, ANOVA). C. ΔKu80 or TgVPS15-iKD parasites were treated with ATc for 96h or 4d. Western blotting was performed on parasite lysates using anti-Cpn60 antibody. Both mature (m) and precursor (pre) Cpn60 was detected in all cases except in ATc-treated TgVPS15-iKD parasites, which exhibited mainly the precursor Cpn60 form. D. Quantitative PCR was performed using total DNA from ΔKu80 or TgVPS15-iKD parasites treated with ATc for 96h or 4d and primers specific to nuclear and apicoplast coded genes. Fold change upon ATc treatment in genomic equivalents for apicoplast, which was normalized with respect to the nuclear genome, is provided (Mean±SE, n = 3, * P<0.05, ANOVA).
Fig 4.
Regulation of TgATG8 and TgATG18 by TgVPS15.
A. TgVPS15-iKD parasites expressing GFP-TgATG8 were treated with ATc for 5d. IFA was performed to detect apicoplast protein Cpn60 and GFP fluorescence was used to localize TgATG8. TgATG8 co-localized with the apicoplast and was also observed in the cytoplasm of untreated parasites. It was predominantly present in the cytoplasm of parasites and was largely absent from the apicoplast upon ATc treatment. Right Panel, % parasites in which TgATG8 was localized to the apicoplast (Mean±SE, n = 3, t-test, **, P<0.01). Please note that only those parasites that possessed an apicoplast as indicated by Cpn60 staining were used for quantitation. B. TgVPS15-iKD parasites expressing GFP-TgATG8 were treated with ATc. Subsequently, parasite lysates were electrophoresed using urea-SDS PAGE followed by Western blotting using anti-GFP antibody to detect unmodified or PE conjugated form of TgATG8 and anti-Cpn60 antibody which detected precursor or mature Cpn60. Actin was used as a loading control. C. TgVPS15-iKD/TgATG18-HA parasites in which TgVPS15 and TgATG18 are myc and HA-tagged respectively were treated with ATc for 5d. IFA was performed using anti-myc and anti-HA antibodies followed by confocal microscopy. ATc-treatment impaired the punctate and vesicular structures of TgATG18 (-ATc) as it was less punctate and more diffuse in the cytoplasm (+ATc). The brightness of red channel in images in +ATc condition is increased for better visualization. D. TgVPS15-iKD/TgATG18-HA/GFP-TgATG8 parasites expressing GFP-TgATG8 and in which TgATG18 was HA-tagged were treated with ATc for 5d. IFA was performed using anti-HA antibody and anti-GFP antibody. Maximum Intensity Projection (MIP) is provided which revealed that TgATG18 was present in punctate vesicular structures but ATc treatment retained it mainly in the parasite cytoplasm.
Fig 5.
TgVPS15 regulates autophagy in Toxoplasma.
A. TgVPS15/GFP-TgATG8 tachyzoites were left untreated or treated with ATc for 2d. Subsequently, extracellular tachyzoites were incubated in complete medium (0h) or HBSS (8h). Parasites were fixed and analyzed by fluorescence microscopy. GFP-ATG8 was mainly cytoplasmic but upon starvation in HBSS it labelled autophagosomes. Upon ATc addition to parasites in HBSS medium these punctii/autophagosomes were lost in a significant number of parasites, which were quantitated by counting at least 50 parasites (lower panel). Data represent mean±SE, n = 3, ANOVA, ****, P<0.0001, **, P<0.01. B. Autophagy was induced in TgVPS15/GFP-TgATG8 tachyzoites as described in A. Subsequently, parasite lysates were electrophoresed by using urea-SDS PAGE followed by Western blotting using anti-GFP antibody to detect unmodified or PE conjugated form of TgATG8. Actin was used as a loading control. C-D. TgVPS15-iKD/TgATG18-HA/GFP-TgATG8 parasites were left untreated or treated with ATc for 2d. Subsequently, extracellular tachyzoites were incubated complete medium (0h) or HBSS (8h). Parasites were fixed to perform IFA using anti-HA antibody, which revealed TgATG18-HA localization to larger puncta observed in HBSS was lost upon addition of ATc. TgATG18-HA puncta were quantitated by counting at least 50 parasites (Panel D). Data represents mean±SE, n = 3, ANOVA, **, P<0.01. E. Myc-TgVPS15-iKD/TgATG18-HA extracellular tachyzoites were incubated in complete medium (0h) or HBSS (8h). Subsequently, parasites were fixed to perform IFA using anti-HA and anti-myc antibody, which revealed that TgATG18-HA as well as TgVPS15 localization changed to a larger puncta in HBSS. In some cases, they co-localized to these larger puncta (arrows). F. Quantitation of parasites in which TgATG18 and TgVPS15 co-localized by counting at least 50 parasites (panel E). Data represents mean±SE, n = 3, ANOVA, ****, P<0.0001.
Fig 6.
TgPI3K and TgATG18 regulate autophagy.
A. TgATG18-iKD/GFP-ATG8 parasites were treated with ATc and extracellular tachyzoites were either cultured in complete medium (0h) or HBSS (8h) medium. Subsequently, parasites were fixed and analyzed by fluorescence microscopy. Upon ATc addition to parasites in HBSS medium, GFP-ATG8 positive autophagosomes were lost in a significant number of parasites, which were quantitated by counting at least 50 parasites (bottom panel). Data represents mean±SE (n = 3, ANOVA, *, P<0.05). B. TgATG18-iKD/GFP-ATG8 extracellular tachyzoites were treated with DMSO (-) or 100 μM LY294002 and cultured in complete medium (0h) or HBSS (8h). Subsequently, parasites were fixed and analyzed by fluorescence microscopy. The number of parasites with TgATG18 or TgATG8 positive puncta was counted under each condition (bottom panel). Data represents mean±SE (n = 3, ANOVA, **, P<0.01, *, P<0.05).
Fig 7.
Role of catalytic site residues in the TgVPS15 function.
A. DD-GFP-2xFYVE was expressed in TgVPS15-iKD parasites or these parasites complemented with a copy of Ty-tagged WT TgVPS15, or its D216A or E268A mutants. Subsequently, parasites were treated with ATc for 72h (3d) or left untreated. Prior to fixation, Shield-1 was added for 30 minutes to stabilize the expression of GFP-2xFYVE. IFA was performed using anti-Cpn60 antibody. The image of only those parasites in which apicoplast was present is shown. While WT TgVPS15 complementation restored PI3P formation indicated by 2xFYVE staining at the apicoplast, majority of parasites complemented with D216A and E268A exhibited diffuse 2xFYVE in the cytoplasm. B. TgVPS15-iKD parasites were complemented with a copy of Ty-tagged WT TgVPS15 or its D216A and E268A mutants. The indicated parasite lines were preincubated for 96h or 4d in culture medium in the presence (+) or absence (-) of ATc and were subsequently allowed to invade fresh HFFs in the presence or absence of ATc. The number of parasites per vacuole was determined after 24h. Data represent mean ± SE, n = 3 and at least 200 vacuoles were counted for each condition (n = 3, ****, P<0.0001, ANOVA, 8p/v, ns-not significant). C. The indicated parasite lines were pre-treated with ATc for 4d and IFA was performed using anti-Cpn60 antibody after additional 24h (S6 Fig). % parasites containing the apicoplast were determined by counting parasites in IFA images (S6 Fig). Data represent mean±SE, n = 3, ** P<0.01, ANOVA, ns-not significant. D. GFP-ATG8 was ectopically expressed in indicated parasite lines. Parasites were treated with ATc for 2d and extracellular tachyzoites were either cultured in complete (0h) or HBSS medium (8h). Subsequently, parasites were fixed and analyzed by fluorescence microscopy (S7 Fig). GFP-ATG8 positive autophagosomes were counted from at least 50 parasites and % parasites possessing autophagosomes was determined (Mean±SE, n = 3,* P<0.05, **P<0.01, ANOVA, ns-not significant).
Fig 8.
A signaling pathway involving TgVPS15 regulates apicoplast inheritance and autophagy.
TgVPS15 may regulate TgPI3K and facilitate the formation of PI3P (Fig 2B). Under steady state conditions, PI3P regulates the localization of TgATG18 to vesicular compartments (Fig 4C and 4D), which in turn promotes the trafficking of TgATG8 to the apicoplast membrane [10] and is critical for the inheritance of the apicoplast during parasite division. Under nutrient limiting conditions, TgVPS15 may facilitate the generation of PI3P via TgPI3K at pre-autophagosome-like vesicles (Fig 5E) resulting in the localization of TgATG18 to this compartment (Fig 5C and 5E), which sets the stage for the formation of autophagosome to which TgATG8 is trafficked and where it is conjugated to autophagosomal membrane (Figs 5A and 6A) and contributes to the process of autophagy.