1. Volume 11, Issue 5, Pages 515-527 (May 2012)
Toxoplasma Sortilin-like Receptor Regulates Protein Transport and Is Essential for Apical Secretory Organelle Biogenesis and Host Infection 
Pierre-Julien Sloves, Stephane Delhaye, Thomas Mouveaux, Elisabeth Werkmeister, Christian Slomianny, Agnes Hovasse, Tchilabalo Dilezitoko Alayi, Isabelle Callebaut, Rajshekhar Y. Gaji, Christine Schaeffer-Reiss, Alain Van Dorsselear, Vern B. Carruthers, Stanislas Tomavo 
Cell Host & Microbe 
Volume 11, Issue 5, Pages (May 2012)
DOI: /j.chom
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1
T. gondii Sortilin-like Receptor Resides in the Golgi and Endosome-Related Structures
(A) Schematic representations of key domains of human sortilin, TgSORTLR, the lumenal domain of TgSORTLR, and its truncated versions fused to GST. PS, peptide signal; TM, transmembrane; m, membrane-adjacent motif; ac-LL, acidic cluster-dileucine motif. See also Figure S1.
(B) Immunoblot probed with mouse polyclonal anti-TgSORTLR antibodies reveals a single protein of ∼105 kDa in T. gondii whereas no protein was detected in human foreskin fibroblast (HFF) cell extract.
(C) Localization of TgSORTLR by confocal images using the mouse polyclonal anti-TgSORTLR antibodies. Bars, 5 μm.
(D) TgSORTLR colocalized with Golgi-endosomal and trafficking markers (see also Movie S1). Intracellular T. gondii tachyzoites stably expressing GRASP-RFP (cis-Golgi and medial Golgi), HDEL-RFP (endoplasmic reticulum), and DrpB-YFP (Golgi proximal cytoplasmic inclusion) or transiently expressing GalNAcT-GFP (trans-Golgi network), Rab5a (early endosome), and Rab7 (late endosome), or rabbit polyclonal anti-VP1 (late endosome and lysosome-related compartment) antibodies were used. Bars, 5 μm.
(E) Quantification of TgSORTLR colocalization Golgi-endosomal markers. Bars represent mean values of n = 3 independent experiments ± standard deviation.
(F) Schematic representation of T. gondii showing its different compartments and organelles. Cryo-IEM of TgSORTLR localized in the Golgi apparatus of transgenic TgSORTLR-cMyc parasite (left panel), wild-type parasite (middle panel), and Golgi-proximal vesicles of transgenic TgSORTLR-cMyc strain (right panel). Black arrowheads indicate the nuclear envelope. Mn, microneme; Rh, rhoptry; DG, dense granule; N, nuclei. Bars, 200 nm.
Cell Host & Microbe  , DOI: ( /j.chom )
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3. Figure 2
The Cytoplasmic Tail of TgSORTLR Is Essential for Correct Subcellular Localization of Endogenous TgSORTLR, Rhoptry, and Microneme Proteins
(A) Expression of TgSORTLR protein lacking its C-terminal tail (TgSORTLRΔCt-GFP, green) resulted in the mislocalization of endogenous TgSORTLR stained with the anti-TgSORTLR antibodies (middle panel, red). TgSORTLRΔNt-GFP (right panel) and the full-length TgSORTLR-GFP (left panel) mediate the correct localization in the Golgi-endosomal structures. The endogenous TgSORTLR was labeled with the mouse anti-TgSORTLR and goat secondary antibodies.
(B) Expression of TgSORTLRΔCt-HA (rat polyclonal anti-HA9 and 488 nm-Alexa goat secondary antibodies, green) resulted in the mislocalization of MIC5 (rabbit anti-MIC5 and 594 nm-Alexa goat secondary antibodies, red). Endogenous TgSORTLR was stained with the mouse polyclonal anti-TgSORTLR and 633 nm-Alexa goat secondary antibodies.
(C) Expression of TgSORTLRΔCt-HA resulted in the mislocalization of ROP1. TgSORTLRΔCt-HA, endogenous TgSORTLR, and ROP1 were labeled as above.
(D) Expression of TgSORTLRΔCt-HA has no effect in the Golgi proximal localization of DrpB-YFP. These data indicate that rhoptry and microneme proteins are present in the Golgi-endosomal associated structures in which TgSORTLR resides (see also Figure S2). Bars, 5 μm for all panels.
Cell Host & Microbe  , DOI: ( /j.chom )
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4. Figure 3 Conditional Ablation of TgSORTLR Gene and Phenotypic Studies
(A) Schematic of the approach used for the conditional ablation of TgSORTLR gene.
(B) PCR analysis using the primers indicated in (A) confirms the conditional ablation TgSORTLR. Superoxide dismutase (SOD), positive control.
(C) Immunoblots of wild-type and TgSORTLR-deficient lines grown in the presence or absence of ATc for 48 hr and probed with anti-TgSORTLR antibodies. Actin was detected as a loading control.
(D) Confirmation of TgSORTLR conditional depletion by confocal imaging using anti-TgSORTLR antibodies after 48 hr ATc treatment.
(E) Immunoblots of wild-type TgSORTLR or ectopic TgSORTLR-cMyc coimmunoprecipitated with monoclonal anti-ROP2–4 or anti-MIC1 antibodies. See also Table S1.
(F) TgSORTLR does not coimmunoprecipitate with ROP2–4 or MIC1 after conditional depletion with ATc for 48 hr.
(G) Coomassie blue staining of the purified recombinant lumenal domain of TgSORTLR, its different truncations or GST alone.
(H) Expression of recombinant rMIC5-HA (lane 2) and rROP1-HA (lane 4) proteins by wheat germ in vitro transcription/translation. Immunoblots were probed with rabbit polyclonal anti-HA9 antibodies. Lanes 1 and 3, total protein extracts stained with Ponceau red. Immunoblots of TgSORTLR-deficient mutants grown without (lane 5) or with ATc for 48 hr (lane 6) incubated with anti-TgSORTLR, rabbit polyclonal anti-MIC5 (lane 7), or monoclonal anti-ROP1 (lane 8) antibodies.
(I) Pull-down assays using the protein extracts from TgSORTLR-deficient mutant and rSORT GST, rΔ1SORT GST, rΔ2SORT GST, or rΔ3SORT GST (see schematic representations in Figure 1A). Blots were incubated with rabbit polyclonal anti-MIC5 or anti-ROP1 antibodies.
(J) Pull-down assays using protein extracts containing rMIC5-HA or rROP1-HA protein incubated with rSORT GST or rΔ1SORT GST beads (upper panel). Blots were stained with rabbit anti-HA antibodies.
Cell Host & Microbe  , DOI: ( /j.chom )
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5. Figure 4
The Conditional Depletion of TgSORTLR Mislocalizes Microneme and Rhoptry Proteins
(A) In the presence of ATc for 48 hr, several rhoptry ROP1, R0P2–4, ROP5, ROP7, and RON1 proteins were mislocalized in TgSORTLR-deficient mutant, whereas the apical localization was observed in the absence of ATc, as expected. Only one confocal image of wild-type parasite corresponding to the first anti-ROP antibody is shown when several proteins have an identical pattern.
(B) Several microneme proteins including MIC2, MIC3, MIC4, and MA2P were mislocalized in TgSORTLR-deficient mutant after 48 hr ATc treatment. Only one confocal image of wild-type parasite was shown as above.
(C) Subcellular localizations of the inner membrane complex (GAP45), dense granules (GRA3), endoplasmic reticulum (SERCA), apicoplast (Atrx2; Api), or mitochondrial (HSP60; Mito) markers were unchanged in the TgSORTLR deficient mutant. These data indicate that TgSORTLR regulates ROP and MIC protein transport during rhoptry and microneme biogenesis (see also Figure S3).
Cell Host & Microbe  , DOI: ( /j.chom )
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6. Figure 5
TgSORTLR Is Crucial for the Biogenesis of Micronemes and Rhoptries
(A) Transmission electron micrograph showing rhoptries (Rh) and micronemes (Mn) in the wild-type parasite.
(B) Ultrathin section of a TgSORTLR-deficient parasite lacking typical micronemes and rhoptries after ATc treatment for 72 hr.
(C) Longitudinal ultrathin section of TgSORTLR-deficient parasite lacking typical micronemes and rhoptries but accumulating unknown vesicles (asterisks) after ATc treatment for 72 hr. See also Figure S4.
(D) Cryo-IEM of rhoptries in the wild-type parasite using anti-ROP1 antibodies.
(E) In the TgSORTLR-deficient parasites, gold-labeled ROP1 was only detected in the vacuole after ATc treatment for 48 hr.
(F) Cross-sections through the anterior end of wild-type parasite showing numerous gold-labeled micronemes using anti-MIC5 antibodies.
(G) TgSORTLR-deficient parasites showed gold-labeled MIC5 mainly in the PV after ATc treatment for 48 hr as a result of mistargeting from the microneme pathway. The remaining gold-labeled MIC5 was detected in the dense granules (DG, see the inset (H)). N, nucleus; PV, parasitophorous vacuole; M, mitochondrion. Bars, 500 nm for all panels.
Cell Host & Microbe  , DOI: ( /j.chom )
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7. Figure 6
TgSORTLR Colocalizes with TgVsp26 and Dictates Its Membrane Association
(A) Extensive colocalization between TgSORTLR (mouse polyclonal antibody, red) and endogenously tagged TgVps26-HA (rabbit polyclonal antibody, green).
(B) Z stack acquisitions by confocal microscopy of transiently transfected iKO tachyzoites expressing TgVSP26-HA under the control of the GRA1 and grown in the absence (left panels) or presence (right panels) of ATc for 48 hr. Antibodies used are the same as in (A).
(C) Z stack confocal acquisitions of tachyzoites stably expressing endogenously tagged TgVps26-HA in RHΔku80 strain before (left panels) or after (right panels) transient transfection with TgSORTLR lacking its C-terminal tail. These data indicate that the C-terminal tail of TgSORTLR recruits cytoplasmic sorting proteins (see also Table S2). The confocal images were captured in increments of 0.37 or 0.43 μm for (B) and (C), respectively. Bars, 5 μm for all panels.
Cell Host & Microbe  , DOI: ( /j.chom )
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8. Figure 7
TgSORTLR Is Essential for Gliding Motility, Host Infection, and Parasite Virulence
(A) The wild-type parasites grown in the presence or absence of ATc after 48 hr.
(B) The conditional TgSORTLR depletion dramatically prevents the lytic and invasion cycles, thus affecting plaque formation in the presence of ATc for 48 hr. Bars, 200 μm.
(C) Motility was impaired in the conditional TgSORTLR-deficient line after it was grown in ATc for 48 hr (see also Movie S2). Bars, 50 μm.
(D) Host cell invasion was severely impaired in TgSORTLR deficient parasites after ATc treatment for 48 hr. Bars represent means ± standard deviations (n = 3, p < by Student's t test).
(E) TgSORTLR depletion abrogates virulence in mice. A group of mice infected with TgSORTLR-deficient mutants for 5 days (green) prior to the addition of ATc to drinking water.
(F) A group of six mice inoculated with doses ranging from 5 × 103 up to 2.5 × 106 iKO parasites survived with ATc treatment.
(G) TgSORTLR-depleted parasites are avirulent in mice but do not confer protective immunity to reinfection with 1 × 103 of wild-type RH tachyzoites.
(H) Model of TgSORTLR function in protein sorting and the biogenesis of apical secretory organelles.
Cell Host & Microbe  , DOI: ( /j.chom )
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