Fig 1.
Disruption of the host ESCRT machinery impairs uptake of host cytosolic proteins by Toxoplasma gondii.
A. Schematic of the ESCRT machinery function in MVB formation. Ubiquitinated cargo targeted for degradation is recognized by ESCRT-0 and ECSRT-I, which further recruits ESCRT-II and the ESCRT accessory protein ALIX. At the last steps, ESCRT-III forms spirals at the membrane to mediate membrane constriction and the VPS4 complex facilitates scission and disassembly of the machinery by hydrolyzing ATP. B. Distribution of the ESCRT-III component CHMP4B at the host cytosol (i) and the PV (ii) in HeLa cells transiently transfected with VPS4AWT-mCherry and infected with RH parasites for 24 h. C. Distribution of the ESCRT-III component CHMP4B at the host cytosol (i) and the PV (ii) in HeLa cells transiently transfected with VPS4AEQ-mCherry and infected with RH parasites for 24 h. The PV was labeled with anti-TgGRA7. Images were analyzed by structural illuminated microscopy (SIM). Scale bar is 5 μm. D. Growth assay of RH parasites in HeLa cells transiently transfected with exogenous VPS4WT or VPS4AEQ compared to untransfected control. The percentage of PV with 1, 2, 4 or 8+ parasites was calculated for each cell subset. At least 20 PV were counted per blinded sample. Data represents means from 7 biological replicates. E. Mean number of parasites per PV from growth assay. Statistical analysis was by Student’s t-test. F. Quantification of ingestion of host cytosolic Venus at 30 mpi in RH or RΔcpl parasites harvest from cells were transiently co-transfected with a plasmid encoding cytosolic Venus fluorescent protein expression and exogenous expression of either VPS4AWT or VPS4ADN. Representative images for parasites with ingested host-derived cytosolic Venus (shown in magenta) at 30 mpi included at the top. Scale bar is 5 μm. G. Quantification of host cytosolic Venus ingestion at 24 hpi in RH or RΔcpl parasites. At least 200 parasites were analyzed per blinded sample. Representative images for parasites with ingested host-derived cytosolic Venus (magenta) at 24 hpi are included at the top. Scale bar is 5 μm. Data represents the mean from ≥ 3 biological replicates. Statistical analysis was by Student’s t-test. Only statistical differences are shown. *p<0.05, **p<0.01.
Fig 2.
The GRA14 C-terminal domain facilitates ESCRT-dependent release of VLPs.
A. Schematic of TgGRA14 topology at the PVM. The TgGRA14 C-terminus encoding the late domain motifs PTAP and YPX(n)L is exposed to the host cytosol whereas the N-terminus is exposed to the PV lumen. B. Schematic representation of the predicted recruitment of the ESCRT recruitment by TgGRA14 through the late domain motifs in comparison with their known function in HIV-1 budding. The PTAP and YPX(n)L motifs can mediate interactions with host ESCRT-I components TSG101 and the ESCRT accessory protein ALIX, respectively. C. Experimental design for the substitution of the HIV-1 Gag p6 domain for the TgGRA14 C-terminus portion encoding late domain motifs to generate the GagGRA14. aa, amino acid. Note that the aa numbering in the hybrid Gag/GRA14 is for GRA14. D. Analysis of VLP release by HIV-1 Gag, GagΔp6 and GagGRA14. Representative immunoblots of VLP release include top: VLP release sample (100%) and bottom: cellular lysate sample (4%). The role for the ESCRT machinery in GagGRA14 release was assessed by disruption of the ESCRT machinery using the VPS4A dominant negative form (VPS4AEQ). Data represents the mean from ≥ 3 biological replicates. Statistical analysis was by Student’s t-test. Only statistical differences are shown. *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
TgGRA14 is proximal to GFP-TSG101 and ALIX.
A. Recruitment of host ALIX and TSG101 with TgGRA14. GFP-TSG101 HeLa cells infected with TgGRA14-HA over expressing parasites (R:GRA14OE) were stained for the anti-ALIX and anti-HA. Representative images analyzed by structured illumination microscopy. 3D Projections. Scale bar is 5 μm. B. Proximity Ligation assay (PLA) reaction from samples infected with either WT or R:GRA14OE strains. PLA reactions were performed using antibodies for the host ALIX and the HA-tag to analyze the interaction of TgGRA14 with ALIX. PLA reaction using antibodies against host MAPK7 and HA-tag, was used a negative control. Representative images from at least 3 biological replicates. Scale bar is 5 μm. C. PLA reaction from GFP-TSG101 HeLa infected with either WT or TgGRA14-HA strains. PLA reactions were performed using antibodies for GFP and the HA-tag to analyze the interaction of TgGRA14 with TSG101. PLA reaction using antibodies against GFP and the HA-tag in WT HeLa cells was used a negative control. Representative images from at least 3 biological replicates. Scale bar is 5 μm.
Fig 4.
TgGRA14 influences recruitment of GFP-TSG101 to the PV but not association of ALIX with the PV.
A. Representative images from three biological replicates comparing the recruitment of ALIX and GFP-TSG101 between WT and RΔgra14. Images were analyzed by confocal microscopy. Scale bar is 5 μm. B. Quantification of ALIX recruitment to the PV between RH, RΔgra14, R:GRA14OE. Data represent the mean ALIX intensity at the PV relative to the cytosol (PV/cytosol). Each point represents the well average. (~18 wells per biological replicates (n = 3)). C. Quantification of TSG101 recruitment to the PV between RH, RΔgra14, R:GRA14OE parasites. Data represent the mean TSG101 intensity in the PV relative to the cytosol (PV/cytosol). Each point represents the well average. (~18 wells per biological replicates (n = 3)). Statistical analysis was done using Kruskal-Wallis test. Only statistical differences are shown. ****p<0.0001.
Fig 5.
Host ESCRT components immunoprecipitated with TgGRA14.
A. Volcano plot of the host proteins immunoprecipitated with TgGRA14 under tachyzoite infection conditions showing the ESCRT-accessory proteins (orange), ESCRT-I components (blue), ESCRT-III components (green) and other non-ESCRT associated proteins (purple). Colored dots represent proteins with >1 log2 fold change in tagged vs control lysates and a negative log2 p-value >3.32 (equivalent to p<0.1). B. Immunoblots for the analysis of immunoprecipitation samples confirming the TgGRA14 interaction with host ESCRT components. C. Volcano plot representative of the T. gondii proteins immunoprecipitated with TgGRA14. Some of the enriched dense granule proteins are highlighted in purple. Colored dots represent proteins with >1 log2 fold change in tagged vs control lysates and a negative log2 p-value >3.32 (equivalent to p<0.1). D. Immunoblot analysis of immunoprecipitation samples confirming the TgGRA14 interaction with some of the enriched dense granule proteins.
Fig 6.
Replicating parasites deficient of TgGRA14 do not internalize host cytosolic proteins as efficiently as wildtype.
A. Experimental design for the analysis of the internalization of host cytosolic proteins of TgGRA14-deficient parasites. (1) Inducible mCherry HeLa cells were infected with parasites for 4 hours, (2) at 4 hpi, extracellular parasites were removed, and the infected monolayer was treated with LHVS for 20 h, (3) parasites were harvested at 24 hpi and analyzed by microscopy. B. Quantification of host cytosolic mCherry uptake at 24 hpi by RH or RΔgra14 type I strains treated with DMSO or LHVS for 20 h. C. Quantification of host cytosolic mCherry uptake at 24 hpi by ME49 or MΔgra14 type II strains treated with DMSO or LHVS for 20 h. At least 200 parasites were analyzed per blinded sample. Data represents the mean from ≥ 3 biological replicates. Statistical analysis was by Student’s t-test. *p<0.05, **p<0.01, ***p<0.001.
Fig 7.
Late domain motifs encoded in TgGRA14 C-terminus can mediate ESCRT-dependent HIV virus-like particle release.
A. Schematic of the generation of mutants in the late domain motifs PTAP and YPX(n)L encoded by TgGRA14. B. Analysis of virus-like particle release by GagGRA14 and GagGRA14 mutants. Representative immunoblots of VLP release include top: VLP release sample (100%) and bottom: cellular lysate sample (4%). Data represents the mean from ≥ 6 biological replicates. Statistical analysis was by Student’s t-test. Only statistical differences are shown. *p<0.05, **p<0.01, ***p<0.001.
Fig 8.
TgGRA14 PTAP late domain motif is necessary for GFP-TSG101 recruitment but not for uptake of host cytosolic proteins.
A. Representative images from 3 biological replicates for the GFP-TSG101 recruitment in RΔgra14GRA14 complemented strains. PVM was labeled using an antibody against TgGRA8. Images were analyzed by confocal microscopy. Scale bar is 5 μm. B. Quantification of TSG101 recruitment to the PV between WT, RΔgra14 and RΔgra14 complementation mutants. Data represent the mean TSG101 intensity in the PV relative to the cytosol (PV/cytosol). Each point represents the well average. (~18 wells per biological replicates (n = 3)). Statistical analysis was done using Kruskal-Wallis test. Statistical analysis between complement strains, RH with Δgra14 and Δgra14 with Δgra14:GRA14WT are shown. ****p<0.0001. C. Quantification of host cytosolic mCherry uptake by RΔgra14 and RΔgra14GRA14 mutants treated with LHVS for 20 h. At least 200 parasites were analyzed per blinded sample. Data represents the mean from ≥ 3 biological replicates. Statistical analysis was by Student’s t-test.
Fig 9.
Model of TgGRA14-ESCRT interactions for the uptake of host cytosolic proteins.
Our data supports a model for the interaction of TgGRA14 with the host ESCRT machinery at the PVM for the uptake of host cytosolic proteins. We hypothesize that through this interaction, vesicles packaging host cytosolic proteins are formed at the PVM and are further endocytosed by the parasites by an unknown mechanism. ESCRT components like TSG101 and ALIX could potentially be packaged within these vesicles resembling what occurs in ESCRT-dependent exosome formation. Furthermore, other transmembrane or soluble dense granule proteins are likely contributing to this pathway.