Unconventional endosome-like compartment and retromer complex in Toxoplasma gondii govern parasite integrity and host infection

Abstract

Membrane trafficking pathways play critical roles in Apicomplexa, a phylum of protozoan parasites that cause life-threatening diseases worldwide. Here we report the first retromer-trafficking interactome in Toxoplasma gondii. This retromer complex includes a trimer Vps35-Vps26-Vps29 core complex that serves as a hub for the endosome-like compartment and parasite-specific proteins. Conditional ablation of TgVps35 reveals that the retromer complex is crucial for the biogenesis of secretory organelles and for maintaining parasite morphology. We identify TgHP12 as a parasite-specific and retromer-associated protein with functions unrelated to secretory organelle formation. Furthermore, the major facilitator superfamily homologue named TgHP03, which is a multiple spanning and ligand transmembrane transporter, is maintained at the parasite membrane by retromer-mediated endocytic recycling. Thus, our findings highlight that both evolutionarily conserved and unconventional proteins act in concert in T. gondii by controlling retrograde transport that is essential for parasite integrity and host infection.

Figure 1. Interactome reveals cargo-selective complex and other interactors with the retromer of T. gondii.

(a) Immunoblots of co-immunoprecipitates of TgVps35-HA and TgVps26-HA (designated IP retromer) probed with rabbit polyclonal specific anti-HA antibodies (lanes 2 and 3) using total detergent protein extracts from the knopck-in TgVps35-HA and TgVs26-HA parasites, respectively, and anti-HA beads. Immunoblot of co-immunoprecipitate of TgVps29-cMyc (IP retromer) probed with rabbit polyclonal anti-cMyc antibodies (lane 4) using total detergent protein extract from the knock-in TgVps29-cMyc parasites and anti-cMyc beads. Negative controls (Neg control) using total detergent protein extracts from untagged parental RH TaTi parasites incubated with anti-HA (lane 5) and anti-cMyc (lane 6) beads. Lane 1 (designated input) corresponds to equally mixed sample of all three detergent extracts containing TgVps35-HA, TgVps26-HA and TgVps29-cMyc proteins and revealed by a mixed probe containing both anti-HA and anti-cMyc antibodies. Molecular weights (kDa) of protein markers are shown on left. IgG_h means heavy chain of IgG. (b) Immunoblots of TgVps35-HA, TgVps26-HA and TgVsp29-cMyc as described in a probed with rat specific anti-TgSORTLR antibodies. (I, input) corresponds to total detergent protein extracts from TgVps35-HA, TgVps26-HA and TgVps29-cMyc knock-in parasites, respectively; (U) unbound lysates to the anti-HA or anti-cMyc beads and (E) eluates corresponding to co-immunoprecipitates. The blots were simultaneously incubated with rat anti-TgSORTLR and rabbit anti-HA or rat anti-TgSORTLR and rabbit anti-cMyc antibodies. Protein markers (kDa) are also shown on left. IgG_h means heavy chain of IgG, IgG_L means light chain of IgG. (c) Retromer interactome was constructed by analysing the co-immunoprecipitates of TgVsp35-HA, TgVps26-HA and TgVps29-cMyc validated by immunoblotting in a and mass spectrometry (Supplementary Data 1). The interactome identified TgVps35, TgVps29 and TgVps26 (red) and TgSORTLR (blue) in addition to the putative phosphatidylinositol synthase (PPS), transporter major facilitator family protein (TMFFP), putative N-ethylmaleimide sensitive fusion protein (NSF), multi-pass transmembrane protein (MTP), Rab5, Rab11B, Rab7-GTPase-activating protein (GAP) regulator TBC1D5A homologue and nine parasite-specific HP. (d) Confocal imaging of TgVps35, TgVps26 and TgVps29 that co-localize with TgSORTLR, proM2AP and vacuolar protein 1 (VP1) using intracellular tachyzoites of the respective knock-in parasites stained with anti-HA or anti-cMyc antibodies followed by probing with anti-TgSORTLR, anti-proM2AP and anti-VP1 antibodies, respectively. Bar, 2 μm.

Figure 2. Conditional ablation of TgVps35 gene.

(a) Schematic of the vector and experimental approach used for the conditional ablation of the TgVps35 gene. (b) PCR analysis of three clones with conditional disruption of TgVps35 and the parental line. Superoxide dismutase (SOD) served as the positive control. Also see the primers used for these PCR in Supplementary Table 3. (c) Immunoblots of the three conditional iKoTgVps35 mutants and RH TaTi parasites, which were grown in the presence or absence of ATc for 48 h, harvested and purified. Each lane refers to a total SDS-protein extract corresponding to the equivalent of 2 × 10⁶ parasites. Immunoblots were probed with anti-HA antibodies. Actin probed with specific monoclonal antibodies served as a loading control. Molecular weights (kDa) of protein markers are indicated on left. (d) Intracellular vacuole containing 16-daughter iKoTgVps35 mutants corresponding to one of the three clones analysed by PCR and western blots and PCR confirmed the conditional depletion TgVps35 protein (right panel) by confocal imaging after 48 h post-infection in the presence of ATc. The left panel showed the same mutant in which TgVps35 protein was detected in the endosome-like comportment (ELC) closely located to the nuclei in the absence of ATc, as expected. 4′, 6-diamidino-2-phenylindole (DAPI) was used to stain nuclei. Rabbit specific anti-HA antibodies was also used. Bar, 2 μm.

Figure 3. Resultant phenotypic traits of conditional disruption of TgVps35 gene.

(a) Host cell invasion was assayed in iKoTgVps35 and complemented mutants (Comp-iKoTgVsp35) in the presence and absence of ATc. Bars indicate mean±s.d. (n=3, P<0.001 by Student's test). (b) Host cell lytic plaques were examined in TgVsp35-deficient mutants, Comp-iKoTgVsp35 and parental RH TaTi parasites in the presence or absence of ATc. (c) Survival of mice infected with lethal doses of TgVsp35-deficient mutants, Comp-iKoTgVsp35 and parental RH TaTi parasites in the presence and absence of ATc in the drinking water. The ATc was removed after 12 days and mice survival was monitored for 30 days post-infection. (d) Avirulent TgVps35-depleted parasites do not confer protective immunity to reinfection with lethal doses of RH wild-type parasites. ATc treatment and mice survival were monitored as above, except that sub-lethal doses of parasites were used during the primo-infection.

Figure 4. The TgVsp35 gene is essential for secretory organelle biogenesis and parasite morphology.

(a) Transmission electron microscopy showing normal banana-shape morphology of iKoTgVps35 parasites in the absence of ATc. (b) An iKoTgVps35 mutant showing an aberrant morphology. (c) Four replicated daughter iKoTgVsp35 mutants with disorganized body shapes without rhoptries, micronemes and dense granules. (d) Complementation of iKoTgVps35 mutants restored normal parasite morphology with the presence of secretory organelles. C, conoid; DG, dense granules; G, Golgi; M, mitochondria; Mn, micronemes; N, nucleus; Rh, rhoptries. Bar, 200 nm.

Figure 5. Conditional ablation of TgVps35 results in mis-sorting of ROP, MIC and GRA proteins.

(a) Confocal immunofluorescence microscopy of ROP 2-3, MIC5 and GRA1 proteins in iKoTgVps35 mutants in the presence (lower panels) or absence of ATc (upper panels) for 48 h using specific antibodies to ROP2-3, MIC5 and GRA1 proteins (see the complete list of antibodies in Supplementary Table 5). Bar, 2 μm. (b) Confocal immunofluorescence microscopy of MIC5, ROP1 and GRA3 proteins in complemented iKoTgVps35 mutants in the presence (lower panels) or absence of ATc (upper panels) as above. Bar, 2 μm.

Figure 6. Disruption of TgVsp35 accumulates unprocessed and immature ROP and MIC proteins.

(a) Immunoblots of iKoTgVps35 mutants probed with specific anti-ROP1, ROP2, proROP4, ROP4, M2AP, proMIC5 and MIC5 antibodies after 48 h post-infection in the presence or absence of ATc. TgSORTLR and ENO2 protein levels were identical in both ATc-treated and untreated mutants. (b) Immunoblots of complemented iKoTgVps35 mutants probed with specific anti-ROP1, ROP2, proROP4, ROP4, M2AP, proMIC5 and MIC5 antibodies after 48 h post-infection in the presence or absence of ATc. ENO2 protein levels were identical in both ATc-treated and untreated mutants.

Figure 7. Recycling of TgSORTLR is blocked in Rab5A- and Rab7-decorated ELCs.

(a) SIM imaging of the co-distribution of TgSORTLR with Golgi marker GRASP in iKoTgVps35 mutants in the absence or presence of ATc. Bar, 2 μm. (b) Quantification of the co-distribution of TgSORTLR with Golgi marker GRASP in iKoTgVps35 mutants by SIM in the absence or presence of ATc. Bars indicated the mean n=90 parasites from 7 to 8 vacuoles±s.d., P<0.0001. (c) Co-distribution of TgSORTLR with the marker of early endosome Rab5A in iKoTgVps35 mutants grown in the presence or absence of ATc and treated for SIM imaging as above. Bar, 2 μm. (d) Quantification of TgSORTLR co-distributing with the marker of early endosome Rab5A in iKoTgVps35 mutants by SIM in the absence or presence of ATc. Bars indicated the mean n=90 parasites from 7 to 8 vacuoles±s.d., P<0.0001. (e) Co-distribution of TgSORTLR with the marker of late endosome marker Rab7 in iKoTgVps35 mutants by SIM imaging as above. Bar, 2 μm. (f) Quantification of TgSORTLR co-distributing with the marker of late endosome marker Rab7 in iKoTgVps35 mutants by SIM in the absence or presence of ATc. Bars indicated the mean n=90 parasites±s.d., P<0.0001. The Pearson correlation coefficient (PCC) and the Mander's overlap coefficient (MOC) used to quantify the degree of colocalization between the red and green fluorophores were shown on the right.

Figure 8. TgHP12 is a novel parasite-specific retromer-associating partner.

(a) Alignment of the helical region in TgHP12 protein and in orthologues from apicomplexan parasites with human syntaxin (pdb 1n7s_B, syntaxin), rabenosyn-5 (1z0j_B and 1z0k_B), and FIP2 (pdb 3tso_C). Secondary structures are reported above and below the alignment. Stars indicate amino acids involved in Rab binding (rabenosyn-5 and FIP2) and the conserved glutamine of syntaxin that participates in the ionic central layer of SNARE complexes. Genbank identifier (gi) and N- and C-terminal limits are as follows: Theileria annulata (85000999, aa 239-297), Neospora caninum (401395596, aa 42-100), Eimeria maxima (557188226, aa 283-342), Babesia equi (510902511, aa 253-311) and Plasmodium falciparum (583212139, aa 375-433). Also see Supplementary Fig. 4. (b) Confocal immunofluorescences showing co-distribution of TgHP12 protein with TgVps35 protein, and TgSORTLR protein (upper panel) while no colocalization was detected with cathepsin L (CPL) or proM2AP protein. Mouse or rabbit polyclonal anti-cMyc antibodies were used in addition to rat anti-TgSORTLR, rabbit anti-HA, anti-CPL and proM2AP antibodies. Bar, 2 μm. (c) Reverse immunoprecipitation using total detergent extract proteins from the knock-in TgVps35-HA, TgVps29-cMyc or TgVps26-HA parasites demonstrated that all three components of the core retromer complex can be pulled down by TgHP12. IgG_h, IgG heavy chain; IP, immunoprecipitations; Neg control, negative control using naïve sera; molecular weights (kDa) were shown on left. (d) TgHP12-cMyc and TgVps35-HA proteins were concomitantly co-immunoprecipitated from total detergent protein extracts from iKoTgVps35 parasites in which TgHP12-cMyc protein was expressed by knock-in strategy. The blots were probed with rabbit anti-cMyc and anti-HA. IgG_L, IgG light chain; IP, immunoprecipitations; Neg control, negative control using naïve sera; molecular weights (kDa) were shown on left. (e) TgHP12-cMyc and TgSORTLR proteins were concomitantly co-immunoprecipitated from parasites total detergent protein extracts from iKoTgVps35 parasites in which TgHP12-cMyc protein was expressed by knock-in strategy. The blots were stained with rabbit anti-cMyc and rat anti-TgSORTLR. IgG_L, IgG light chain; IP, immunoprecipitations; Neg control, negative control using naïve sera; molecular weights (kDa) were shown on left.

Figure 9. Retromer is required to maintain TgHP03 in the parasite membrane.

(a) Schematic representation of TgHP03 topology inside the parasite membrane. (b) Confocal imaging of extracellular parasites with knock-in TgHP03-cMyc in the iKoTgVps35 mutants. Immunofluorescence assay (IFA) was performed in the absence of ATc and detergent permeabilization. Nuclei of the parasites as stained with 4′, 6-diamidino-2-phenylindole (DAPI; blue). Bar, 2 μm. (c) Magnified image of an extracellular parasite expressing TgHP03-cMyc protein in the iKoTgVps35 mutants grown in the absence of ATc and detergent permeabilization (upper, left panel); intracellular parasites expressing TgHP03-cMyc protein in the iKoTgVps35 genetic background. IFA was performed in the absence of ATc and in the presence detergent permeabilization (upper, right panel); magnified image of an intracellular parasites expressing TgHP03-cMyc protein in the iKoTgVps35 genetic background in the presence of ATc and detergent permeabilization (lower, left panel) and intracellular parasites expressing TgHP03-cMyc protein in the iKoTgVps35 genetic background in the presence of ATc and detergent permeabilization (lower, right panel). Bar, 2 μm. (d) Immunoblots of parasites expressing TgHP03-cMyc protein in the knock-in iKoTgVps35 mutants grown in the presence or absence of ATc. ENO2 was used as a loading control. Molecular weights (kDa) were shown on left. (e) Quantification of TgHP03 levels in these parasites expressing TgHP03 protein in the knock-in iKoTgVps35 mutants that were grown in the absence or presence of ATc. (f) The surface localization of glycosyl–phosphatidyl inositol (GPI)-anchored SAG1 and SAG3 were determined in iKoTgVps35 mutants in the presence or absence of ATc using monoclonal antibodies specific to SAG1 and SAG3. Bar, 2 μm.

Figure 10. Model of retromer-mediated recycling of diverse sorting cargoes in T. gondii versus mammalian cells.

No recognizable sorting nexins, Bin/Amphyphysin/Rvs (BAR)-containing domain proteins or lysosomal-like protein degradation have been identified in T. gondii (a), in contrast to the situation in mammalian cells (b). Instead, the retromer-dependent recycling is essential for secretory organelle formation and parasite shape. We propose a model suggesting that Rab7 is the key small GTPase, which is involved in the endocytic recycling of TgSORTLR from endosomes to TGN for another round of ROP and MIC transport and secretory organelle biogenesis. We further raised the possibility that in contrast to mammalian cells, T. gondii lysosomal-like organelles only promote proteolytic maturation of proteins destined to secretion and that the endosomal system is adapted for organellar discharge of virulence-like factors required for the intracellular lifestyle of the parasite. Moreover, we provide the first evidence that a multiple ligand transmembrane transporter TgHP03 is maintained at the surface of T. gondii through endocytic recycling from endosomes to the plasma membrane. BAR, Bin/Amphyphysin/Rvs; MIC, microneme; ROP, rhoptry; TgHP03, T. gondii hypothetical protein 03.