Proximity-Labeling Reveals Novel Host and Parasite Proteins at the Toxoplasma Parasitophorous Vacuole Membrane

Abstract

Toxoplasma gondii is a ubiquitous, intracellular parasite that envelops its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell, such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the Toxoplasma PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three parasite proteins (TGGT1_269950 [GRA61], TGGT1_215360 [GRA62], and TGGT1_217530 [GRA63]) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the Toxoplasma PVM and, given that three of the validated host proteins are components of the ESCRT (endosomal sorting complexes required for transport) machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. IMPORTANCEToxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here, we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.

Keywords: Toxoplasma; proximity-labeling; quantitative proteomics.

FIG 1

The arginine-rich-amphipathic helix domain (RAH) of ROP17 is sufficient to localize miniTurbo to the Toxoplasma PVM in infected cells. (A) Schematic of the miniTurbo fusion proteins expressed in HFFs depicting the epitope tag (V5), miniTurbo ligase (miniTurbo), nuclear export signal (NES), flexible linker (linker [amino acids KGSGSTSGSG]), and RAH domain of ROP17 (ROP17 RAH, [amino acids 104 to 223]). (B) Representative immunofluorescence microscopy images of tachyzoite-infected HFF populations stably expressing miniTurbo fusion proteins depicted in panel A. RHΔhptΔku80 tachyzoites were allowed to infect the corresponding HFFs for 24 h before the infected monolayers were fixed with methanol. The miniTurbo fusion proteins were detected with mouse anti-V5 antibodies (green), tachyzoites were detected with rabbit anti-SAG2A antibodies (magenta), and host and parasite nuclei were visualized using DAPI (blue). Scale bar is 10 μm.

FIG 2

miniTurbo can biotinylate proteins in human foreskin fibroblasts. (A) Western blot of whole-cell lysates. The indicated lines of HFFs were either infected or left uninfected (+/–) with RHΔhptΔku80 tachyzoites for 24 h and then incubated with or without (+/–) 50 μg of biotin for 1 h. Biotin labeling was terminated by cooling the cells to 4°C and washing away excess biotin. Whole-cell lysates (2 μg) were subsequently prepared and blotted with streptavidin-HRP to visualize biotinylated proteins and mouse anti-V5 antibodies to visualize expression of the miniTurbo fusion proteins. Bands present in the non-miniTurbo-expressing HFFs and the HFFs not incubated with biotin represent endogenously biotinylated host and parasite proteins. * denotes the location on the streptavidin blot at the approximate size of miniTurbo-RAH, and ** denotes the location on the streptavidin blot at the approximate size of miniTurbo. Approximate migration of a ladder of size standards (sizes in kDa) is indicated. (B) Representative immunofluorescence microscopy images of HFF populations stably expressing miniTurbo fusion proteins. The indicated line of HFFs was either infected with RHΔhptΔku80 tachyzoites or left uninfected for 24 h and then subjected to a 1-h incubation with biotin as described in panel A. After washing, the monolayers were fixed with methanol. The miniTurbo fusion proteins were detected with mouse anti-V5 antibodies (magenta), biotinylated proteins were detected with streptavidin-HRP (green), and the entire monolayer was visualized with phase microscopy. White arrowheads indicate a representative PV. Scale bar is 10 μm.

FIG 3

The Toxoplasma PVM-localized protein ROP17 is enriched in miniTurbo-RAH cells. (A) Western blot of whole-cell lysates and subsequent elutions after streptavidin affinity purification. The indicated line of HFFs was either infected or left uninfected (+/–) with RHΔrop17::ROP17-3xHA tachyzoites for 24 h and then incubated with or without (+/–) 50 μg of biotin for 1 h. Whole-cell lysates were subsequently prepared, and 200 μg of each lysate was incubated with streptavidin-coated magnetic beads overnight. The beads were washed and proteins were eluted from the beads by boiling in the presence of 2 mM biotin and BME. Samples of the input lysate, unbound lysate (flowthrough), and eluates were separated on an SDS-PAGE gel and blotted with streptavidin-HRP to visualize all biotinylated proteins, mouse anti-HA-HRP antibody to visualize ROP17-3xHA, and mouse anti-GAPDH to visualize host protein as a loading control. 25× indicates that the lanes containing eluted material had 25-fold more material loaded compared to input and flowthrough lanes (1×). Approximate migration of a ladder of size standards (sizes in kDa) is indicated. (B) Cartoon depiction of expected regions of biotinylation (depicted by red cloud) near the Toxoplasma PVM and their proximity to the localization of ROP17 (blue dots) at the PVM based on miniTurbo fusion localization (yellow stars) and addition of biotin.

FIG 4

Quantitative mass spectrometry-based proteomic experimental setup and quality control checks. (A) Overview of proteomics experimental design and cartoon depiction of expected regions of biotinylation (depicted by red cloud) near the Toxoplasma PVM based on miniTurbo fusion localization (yellow star) and +/– biotin in the 11 samples submitted for LC/MS-MS. The number of replicates for each sample is indicated in parentheses below the description. The indicated samples were infected with RHΔrop17::ROP17-3xHA parasites for 22 h and then treated with 50 μM biotin for 1 h. After treatment and biotin-labeling, cells were lysed, and biotinylated proteins were enriched with streptavidin magnetic beads; 5% of the beads were saved for a quality control check, the remaining protein was digested on-bead, and subsequent peptides were conjugated to TMT labels. All 11 samples were then combined and analyzed by LC-MS/MS. (B, INPUT) Western blot of whole-cell lysates. A sample of each whole-cell lysate depicted in panel A was run on an SDS-PAGE gel and blotted with streptavidin-HRP to visualize all biotinylated proteins, mouse anti-HA-HRP antibodies to visualize endogenous ROP17, and mouse anti-V5 antibodies to visualize miniTurbo expression. (ELUTION) Silver stain and Western blots of eluates from streptavidin affinity purification (from the 5% of beads saved prior to on-bead digestion). The beads were washed and proteins eluted from the beads by boiling in the presence of 2 mM biotin and BME. Equivalent volumes of each elution were run on an SDS-PAGE gel and stained with silver to visualize total protein material eluted from the beads. Equivalent volumes of each elution were additionally run on a separate SDS-PAGE gel and blotted with mouse anti-HA-HRP antibodies to check for specific enrichment of endogenous ROP17 in the PVM-miniTurbo localized samples (miniTurbo-RAH + Toxo, lanes 1–4). Approximate migration of a ladder of size standards (sizes in kDa) is indicated. (C) Principal-component analysis of the 11 samples analyzed in the proteomics experiment. Numbers in square brackets indicate the number of replicates for each sample type analyzed, as described in panel A.

FIG 5

The newly identified Toxoplasma proteins 269950 (GRA61), 215360 (GRA62), and 217530 (GRA63) localize to the Toxoplasma parasitophorous vacuole in infected cells and derive from the dense granules. (A) Two-dimensional plot showing log₂ fold changes between miniTurbo-RAH and miniTurbo-infected samples (x axis) and log₂ fold changes between +biotin and –biotin miniTurbo-RAH samples (y axis). Toxoplasma proteins known to be exported or PV/PVM-localized or putatively nonsecreted (see Materials and Methods) are indicated by orange and purple symbols, respectively. Dotted lines indicate the optimal threshold for separation of known PV/PVM/exported versus nonsecreted Toxoplasma proteins. Yellow shading indicates the position on the plot of the proteins of interest described in Table 1. (B) Schematic representations of the indicated proteins showing predicted N-terminal signal peptides (SP), predicted transmembrane domains (TM), and predicted homology to conserved domains from ToxoDB.org (version 53). (C) Representative immunofluorescence microscopy images of endogenously tagged HA-protein (269950-3xHA and 217530-3xHA) or protein ectopically expressed under its native promoter (215360-HA and 216180-HA). The tachyzoite single clones expressing HA-tagged proteins of interest were allowed to infect HFFs for 20 h before the infected monolayers were fixed with methanol. The corresponding tagged proteins were detected with rat anti-HA antibodies (green), while all tachyzoites were detected with rabbit anti-SAG2A antibodies (magenta). Host and parasite nuclei were stained with DAPI (blue). Arrowheads indicate vacuoles with HA-staining outside the parasites. Scale bar is 10 μm. (D) Representative immunofluorescence microscopy images of free tachyzoites expressing endogenously HA-tagged protein (269950-3xHA and 217530-3xHA) or protein ectopically expressed under its native promoter (215360-HA). Staining was with rabbit anti-GRA7 antibodies (magenta) and rat anti-HA antibodies (green) with the data superimposed on phase contrast images of the parasites themselves in the right-most panels. Colocalization is apparent as white coloring in the merged images. Scale bar is 5 μm. (E) Western blotting of HA-tagged proteins. Lysates of HFFs infected with parasite single clones expressing GRA61/62/63 were resolved by SDS-PAGE, blotted, and probed with rat anti-HA antibodies to detect the HA-tagged proteins. Rabbit anti-SAG2A staining was used as a loading control for total parasite protein. Approximate migration of a ladder of size standards (sizes in kDa) is indicated.

FIG 6

Host ESCRT-associated proteins PDCD6, ALIX, and CC2D1A, and the host ER-organelle tethering protein MOSPD2, localize to the Toxoplasma parasitophorous vacuole membrane. (A) Two-dimensional plot showing log₂ fold changes between miniTurbo-RAH and miniTurbo-infected samples (x axis) and log₂ fold changes between infected and mock-infected miniTurbo-RAH samples (y axis). Toxoplasma proteins known to be exported or PV/PVM-localized or putatively nonsecreted are labeled using the same color scheme as in Fig. 5A. The dotted line indicates the optimal threshold for separation of known PV/PVM/exported-localized proteins and nonsecreted Toxoplasma proteins. Yellow shading indicates the position on the plot of the proteins of interest described in Table 2. (B) Significant terms from functional enrichment analysis performed on the candidate host PVM proteins from Table 2. Terms are shown for the Gene Ontology biological process functional database. (C) Result of STRING version 11 (61) protein-protein interaction analysis of the candidate host PVM proteins from Table 2 with singletons (proteins with no association to another protein) removed. The thickness of the lines between proteins indicates the degree of confidence of the interaction. (D) Representative immunofluorescence microscopy images of the localization of the host proteins CC2D1A and PDCD6IP/ALIX during Toxoplasma infection. HFFs were infected with RH parasites for 20 h before the infected monolayers were fixed with methanol. Rabbit anti-CC2D1A antibodies and rabbit anti-ALIX antibodies were used to detect the corresponding host proteins (green). Tachyzoites were detected with rabbit anti-SAG2A antibodies (magenta), and the infected monolayer was visualized with phase microscopy. Scale bar is 10 μm. (E) Representative immunofluorescence microscopy images of the localization of host proteins upon transient overexpression in HFFs. HFFs were transiently transfected with plasmids expressing the indicated tagged proteins and then infected with RH tachyzoites 24 h posttransfection. The parasites were allowed to infect for 16 h before the monolayers were fixed with methanol. Mouse anti-V5 and rat anti-HA antibodies (green) were used to detect the corresponding host proteins. Tachyzoites were detected with rabbit anti-SAG2A antibodies (magenta), and the infected monolayer was visualized with phase microscopy. Dashed white boxes indicate the PVs expanded in the insets (left-most panels). Scale bars are 10 μm.