A Toxoplasma palmitoyl acyl transferase and the palmitoylated armadillo repeat protein TgARO govern apical rhoptry tethering and reveal a critical role for the rhoptries in host cell invasion but not egress

Abstract

Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that inject a protein complex into the host cell to form a tight-junction for parasite entry. Little is known about the molecular factors that govern rhoptry biogenesis, their subcellular organization at the apical end of the parasite and subsequent release of this organelle during invasion. We have identified a Toxoplasma palmitoyl acyltransferase, TgDHHC7, which localizes to the rhoptries. Strikingly, conditional knockdown of TgDHHC7 results in dispersed rhoptries that fail to organize at the apical end of the parasite and are instead scattered throughout the cell. While the morphology and content of these rhoptries appears normal, failure to tether at the apex results in a complete block in host cell invasion. In contrast, attachment and egress are unaffected in the knockdown, demonstrating that the rhoptries are not required for these processes. We show that rhoptry targeting of TgDHHC7 requires a short, highly conserved C-terminal region while a large, divergent N-terminal domain is dispensable for both targeting and function. Additionally, a point mutant lacking a key residue predicted to be critical for enzyme activity fails to rescue apical rhoptry tethering, strongly suggesting that tethering of the organelle is dependent upon TgDHHC7 palmitoylation activity. We tie the importance of this activity to the palmitoylated Armadillo Repeats-Only (TgARO) rhoptry protein by showing that conditional knockdown of TgARO recapitulates the dispersed rhoptry phenotype of TgDHHC7 knockdown. The unexpected finding that apicomplexans have exploited protein palmitoylation for apical organelle tethering yields new insight into the biogenesis and function of rhoptries and may provide new avenues for therapeutic intervention against Toxoplasma and related apicomplexan parasites.

Figure 1. Identification of palmitoyl acyl transferases targeted to the Toxoplasma rhoptries or IMC.

(A) The expression profile of 18 candidate PATs (DHHC-CRD containing genes) was compared to known IMC and rhoptry genes across the Toxoplasma cell cycle (Figure S1). Five candidate genes were found to display an expression signature similar to the IMC/rhoptry pattern, peaking in a narrow window during daughter cell budding. (B–C) IFA of two candidate IMC/rhoptry PATs localized by introduction of a 3xHA epitope tag at the endogenous C-terminus. (B) TgDHHC7 was found to localize to the rhoptries, as assessed by colocalization with both the rhoptry body protein ROP13 and rhoptry neck protein RON11. Red: anti-HA antibody detected by Alexa594-anti-mouse IgG. Green: rabbit anti-ROP13 antibody detected by Alexa488-anti-rabbit IgG Blue: rat anti-RON11 antibody detected by Alexa350-anti-rat IgG. (C) TgDHHC14 was found to localize to the IMC, as assessed by colocalization with the IMC protein IMC1. Red: anti-HA antibody detected by Alexa594-anti-rabbit IgG. Green: mouse anti-IMC1 antibody detected by Alexa488-anti-mouse IgG. Blue: Hoechst stain. All scale bars = 5 µm.

Figure 2. A conserved region of the TgDHHC7 C-terminus is required for rhoptry targeting.

Analysis of determinants for rhoptry targeting of TgDHHC7. (A) Diagram showing different truncation mutants utilized in this study with an expanded view of the C-terminal PaCCT motif (red box) and downstream conserved region (CR, blue box). Homology between TgDHHC7 residues 474–510 and various orthologs is shown (see Figure S3 for full alignment details). (B) A second copy of the full length TgDHHC7 coding sequence with a C-terminal HA epitope tag was expressed in wild type parasites under the control of the RON5 promoter and found to localize to the rhoptries in the same fashion as the endogenous protein. The localization of several truncations mutants was then evaluated. Red: anti-HA antibody detected by Alexa594-anti-mouse IgG. Green: rabbit anti-ROP13 antibody detected by Alexa488-anti-rabbit IgG. (C) Removal of residues 1–162 had no apparent effect on rhoptry localization (Δ1–162). Red: anti-HA antibody detected by Alexa594-anti-rabbit IgG. Green: mouse anti-ROP2/3/4 antibody detected by Alexa488-anti-mouse IgG. (D) Truncation of residues 511–537 to remove a non-conserved extreme C-terminal region of the protein did not grossly impact targeting of TgDHHC7. Red: anti-HA antibody detected by Alexa594-anti-rabbit IgG. Green: mouse anti-ROP2/3/4 antibody detected by Alexa488-anti-mouse IgG. (E) However, in some parasites TgDHHC7_Δ511–537 signal was detected outside the rhoptries adjacent to the Golgi marker GRASP55. Red: anti-HA antibody detected by Alexa594-anti-mouse IgG. Green: GRASP55-YFP. (F–G) In contrast, further C-terminal truncations that remove the CR (residues 489–537) or the CR and PaCCT motif (residues 474–537) completely abrogate rhoptry targeting. These mutants were found to localize to the Golgi, as assessed by co-localization with the GRASP55, indicating the CR is necessary for TgDHHC7 transit from the Golgi to its final rhoptry destination. Red: anti-HA antibody detected by Alexa594-anti-mouse IgG. Green: GRASP55-YFP. Scale bars = 5 µm.

Figure 3. Establishment of a TgDHHC7 conditional knockdown.

(A) Western blot comparing TgDHHC7 protein levels in parental and TgDHHC7cKO strains without Atc treatment. Exchange of the endogenous TgDHHC7 promoter with a Tet-repressible, conditional promoter results in lower levels of basal TgDHHC7 expression. ISP3 serves as a loading control. (B–C) Growth of TgDHHC7cKO parasites in the presence of Atc results a depletion of TgDHHC7. (B) Western blot showing TgDHHC7 levels after 72 hours −/+ Atc. (C) IFA showing TgDHHC7 signal after 48 hours −/+ Atc. Red: anti-HA antibody detected by Alexa594-anti-mouse IgG. Scale bars = 5 µm.

Figure 4. TgDHHC7 is required for apical rhoptry docking.

(A–C) IFA detecting the rhoptry body protein ROP2/3/4 and rhoptry neck protein RON11. (A) During normal rhoptry biogenesis, 6–14 rhoptries are generated, each with a polarized morphology consisting of a posterior, bulbous body and tapered anterior neck domain. These individual rhoptries are bundled together with the necks docked at the extreme apical end of the parasite (arrow). (B) TgDHHC7cKO parasites, which express lower levels of TgDHHC7 than the parental line, maintain an apical bundle of rhoptries (arrow) but also contain some individual rhoptries in posterior areas of the cell (arrowhead). (C) Upon depletion of TgDHHC7 by Atc treatment, apical rhoptry bundles are lost with individual rhoptries scattered throughout the cell cytosol. The rhoptry neck and body domains are clearly maintained in each scattered organelle. Red: rat anti-RON11 antibody detected by Alexa594-anti-rat IgG. Green: mouse anti-ROP2/3/4 antibody detected by Alexa488-anti-mouse IgG. All IFA scale bars = 5 µm. (D) Rhoptry organization defects incurred by loss of TgDHHC7 occur after pro-rhoptry formation. Mature rhoptries labeled by RON11 are scattered throughout the cell following Atc treatment of TgDHHC7cKO parasites. In contrast, the pro-rhoptry marker proROP4 is only observed within forming daughter buds labeled by IMC1, similar to what is observed in the parental line. Red: anti-IMC1 antibody detected by Alexa594-anti-mouse IgG. Green: rabbit anti-proROP4 antibody detected by Alexa488-anti-rabbit IgG Blue: rat anti-RON11 antibody detected by Alexa350-anti-rat IgG. (E–F) TEM analysis of parent and TgDHHC7cKO lines. (E) A normal apical bundle of rhoptries (R) are visible in wild-type parasites and in untreated TgDHHC7cKO cells. Following Atc treatment of TgDHHC7cKO parasites, bundles of rhoptries docked at the extreme apex of the cell are no longer observed. Rhoptries are instead scattered, with many no longer oriented in a longitudinal fashion (R). Importantly, a standard apical arrangement of micronemes (M) is still present in these conditions. (F) In TgDHHC7 depleted cells, individual rhoptries are observed scattered throughout the cell cytosol. These scattered rhoptries are morphologically unchanged with obvious body (B) and neck (N) domains. Note the lumen of these rhoptries have a normal, mottled appearance.

Figure 5. TgDHHC7 is critical for host invasion but not egress.

(A–B) Parasites depleted of TgDHHC7 encounter a complete block in invasion. (A) Parental or TgDHHC7cKO parasites were grown for 72 hours−/+Atc and then allowed to invade into fresh host cells for one hour. Following depletion of TgDHHC7, parasites show a nearly complete block in host penetration (asterisk, p-value<0.001). A corresponding increase in attached, uninvaded parasites is not seen (blue bars). A minor decrease in penetration is also seen for untreated TgDHHC7cKO, likely due to the lower levels of TgDHHC7 expressed in this strain relative to the parental line. (B) Parasites depleted of TgDHHC7 cannot form plaques in fibroblast monolayers. Parental or TgDHHC7cKO parasites were grown 48 hours−/+Atc and then infected into fresh fibroblast monolayers at an infective dose of 200 parasites per well and allowed to incubate for nine days. TgDHHC7cKO parasites are unable to form plaques in the presence of Atc, even at an infective dose of 20,000 parasites per well. (C) Initial attachment is not affected upon knockdown of TgDHHC7. Parasites were grown for 60 hours−/+Atc before treatment with cytochalasin D to block motility and arrest the invasion process just after attachment. (D) Loss of TgDHHC7 impairs secretion of evacuoles by rhoptries (asterisk, p-value<0.001). Parasites were grown for 60 hours−/+Atc before treatment with cytochalasin D to block invasion and allow evacuole formation. Evacuoles were detected by staining for ROP2/3/4. (E) Parasite egress is unaffected by knockdown of TgDHHC7. Parasites were grown 60 hours−/+Atc and then induced to egress by treatment with calcium ionophore A23187 before fixation and staining for detection with anti-SAG1. The egress efficiency of parasites with defective rhoptries (lacking TgDHHC7) was not significantly different from parental or untreated TgDHHC7cKO parasites.

Figure 6. A key cysteine residue predicted to be required for TgDHHC7 catalytic activity is necessary for rhoptry function.

(A) IFA showing localization of C371S mutant version of TgDHHC7. Targeting to the rhoptries is unaffected by the C371S mutation as assessed by co-localization with the rhoptry body protein ROP2/3/4. Red: anti-HA antibody detected by Alexa594-anti-rabbit IgG. Green: mouse anti-ROP2/3/4 antibody detected by Alexa488-anti-mouse. Scale bar = 5 µm. (B) Complementation of TgDHHC7cKO assessed by plaque assay. TgDHHC7_FL-WT rescues the defect incurred by the knockdown of TgDHHC7 while TgDHHC7_FL-C371S does not, failing to form plaques even with an infectious dose of 20,000 parasites per well. TgDHHC7 N-terminal truncations removing the first 162 or 206 residues also rescue the defect while C371S mutant versions of these truncation mutants do not.

Figure 7. TgARO is required for apical rhoptry tethering and host cell invasion.

(A) Expression of TgARO is delayed about one hour after other rhoptry proteins. Expression of representative genes from several classes of rhoptry proteins are shown including ROP1 (rhoptry body proteins), RON2 (rhoptry neck proteins) and the Na+/H+ exchanger TgNHE2 and TgDHHC7 (multipass integral membrane rhoptry proteins). (B) IFA showing endogenously tagged TgARO-3xHA is distributed along the surface of the entire rhoptry and concentrated at the apex of the organelle. Red: rabbit anti-HA antibody detected by Alexa594-anti-rabbit IgG. Green: mouse anti-ROP2/3/4 antibody detected by Alexa488-anti-mouse IgG. Blue: rat anti-RON11 antibody detected by Alexa350-anti-rat IgG. All scale bars = 5 µm. (C) Western blot showing lower levels of TgARO following promoter replacement. ISP3 serves as a loading control. (D) Untreated TgAROcKO parasites maintain an apical rhoptry bundle (arrow) but also display some dispersed rhoptries (arrowhead). TgARO is visible not only in the apical bundle but also at the apex of each of the individual dispersed rhoptries. (E) Western blot showing knockdown of TgARO after 48 hours of growth with Atc. ISP3 serves as a loading control. (F) Following Atc treatment, cells are depleted of TgARO and the apical bundle of rhoptries is completely lost with the rhoptries scattered throughout the cytosol. (G) Parasites depleted of TgARO encounter a complete block in invasion. Parental or TgAROcKO parasites were grown for 48 hours−/+Atc and then allowed to invade into fresh host cells for one hour. Following depletion of TgARO, parasites show a nearly complete block in host penetration (asterisk, p-value<0.001). A corresponding increase in attached, uninvaded parasites is not seen (blue bars). A minor decrease in penetration is also seen for untreated TgAROcKO, likely due to the lower levels of TgARO expressed in this strain relative to the parental line. (H) Parasites depleted of TgARO cannot form plaques in fibroblast monolayers. Parental or TgAROcKO parasites were grown 48 hours−/+Atc and then infected into fresh fibroblast monolayers at an infective dose of 200 parasites per well and allowed to incubate for nine days. TgAROcKO parasites are unable to form plaques in the presence of Atc, even at an infective dose of 20,000 parasites per well.

Figure 8. Model of TgDHHC7 and TgARO function in rhoptry biogenesis.

TgDHHC7 is positioned in the rhoptry membrane with the catalytic DHHC-CRD domain in the cytosol where it recruits TgARO to the cytosolic face of the rhoptries by palmitoylation. Rhoptry biogenesis occurs de novo during each round of parasite replication and recruitment of TgARO by TgDHHC7 during this process facilitates apical rhoptry docking. The failure to recruit TgARO to the rhoptries following knockdown of TgDHHC7 or direct knockdown of TgARO results in the synthesis of rhoptries that are morphologically intact with respect to ultrastructure and cargo, but scattered throughout the parasite cytosol. Without proper tethering at the cell apex, these rhoptries are unable to secrete their contents. While egress from one host cell and attachment to the next occurs normally, the loss of functional rhoptries results in a block in penetration (likely due to a failure to inject rhoptry neck proteins and form a moving junction) and subsequent detachment from the host cell.