Protein kinase TgCDPK7 regulates vesicular trafficking and phospholipid synthesis in Toxoplasma gondii

Abstract

Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for intracellular development of both Apicomplexa Plasmodium falciparum and Toxoplasma gondii. However, the mechanisms via which it regulates parasite replication have remained unknown. We performed quantitative phosphoproteomics of T. gondii lacking TgCDPK7 to identify its parasitic targets. Our analysis lead to the identification of several putative TgCDPK7 substrates implicated in critical processes like phospholipid (PL) synthesis and vesicular trafficking. Strikingly, phosphorylation of TgRab11a via TgCDPK7 was critical for parasite intracellular development and protein trafficking. Lipidomic analysis combined with biochemical and cellular studies confirmed that TgCDPK7 regulates phosphatidylethanolamine (PE) levels in T. gondii. These studies provide novel insights into the regulation of these processes that are critical for parasite development by TgCDPK7.

Fig 1. TgCDPK7 is critical for parasite division and localization of SAG1/3.

A. Schematic representation of TgCDPK7. It contains a PH domain adjacent to the kinase domain at the C-terminus and two EF hand motifs near the N-terminus. In addition, the TgCDPK7 contains a putative myristoylation motif (red) near its N-terminus. B. TgCDPK7-iKD parasites were pre-incubated for 48h with ATc and were subsequently allowed to invade fresh HFFs in the presence or absence of ATc. The number of parasites per vacuole was determined after 24h. Data represent mean ± SE, n = 3 and at least 200 vacuoles were counted for each condition (n = 3*, p<0.001 for 8/16 parasites/vacuole, ANOVA). C and D. TgCDPK7-iKD or ΔKu80 parasites were treated with 1μg/ml ATc for 2 days. After parasites egressed, fresh HFF were infected along with additional 24h ATc treatment and IFA was performed on untreated or ATc treated parasites using SAG1 (C) or SAG3 (D) and ROM4 antibodies. E. Quantification of vacuoles containing normal SAG1 staining, which was at parasite periphery, from experiments described in panel C. Data are mean ± SE of three independent experiments and at least 200 vacuoles were counted for each condition (*, n = 4, p<0.001, ANOVA).

Fig 2. Comparative phosphoproteomics of TgCDPK7-iKD parasites.

A. Schematic representation of the overall workflow employed for comparative proteomic and phosphoproteomic analysis. A TMT-labeling approach was used for TgCDPK7-iKD parasites (for details see Materials and Methods S1 Data 1.1). B. The phosphorylation fold-change ratios of phosphopeptides identified from TgCDPK7-iKD tachyzoites cultured in the presence or absence of ATc was normalized with respect to total protein fold-change. The ratios for all phosphopeptides from various replicates are provided in S1 Data, 1.1. The S-curve for normalized data is provided and some of the significantly altered phosphorylation sites belonging to key proteins (S1 Data, 1.2) are indicated. Representation of phosphosites and their corresponding abundance fold change in TgCDPK7 depleted parasites. Some of the differentially phosphorylated sites are highlighted in red and blue color, respectively and key target proteins that are relevant to the reported studies are indicated in green. C. Pathway analysis of proteins that exhibited reduced phosphorylation upon TgCDPK7 depletion and the possible metabolic pathways these proteins may regulate (S1 Data, 1.4). D and E. Protein-protein interactions were predicted between differentially phosphorylated proteins using STRING resource. The analysis exhibited high confidence protein-protein interactions between the candidate genes (S10 Fig and S1 Data, 1.6). Two major protein-protein interaction clusters involving TgRab11A (D) and GPAT protein (E) are illustrated.

Fig 3. TgCDPK7 phosphorylates TgRab11a and regulates its cellular localization.

A. Schematic illustrating TgRab11a domain architecture. The catalytic domain is in green, and the two cysteines (CC) that may be critical for lipid modification like prenylation are indicated. The phosphorylation of S207 was reduced upon TgCDPK7 depletion. MS/MS spectra is provided for the corresponding phosphopeptide. TMT channels highlighted in green in the inset represents independent biological replicates of TgCDPK7 depleted parasites and wild type samples are highlighted in black. B. A deletion mutant of TgCDPK7 (ΔTgCDPK7) that possesses the PH and kinase domain was expressed as a GST tagged protein and was used to phosphorylate recombinant 6xHis-TgRab11a or its S207A mutant in vitro using γ³²P-labelled ATP. The reaction mixture was separated by SDS-PAGE and gel was used for phosphorimaging. C. DD-Myc-TgRab11a was ectopically expressed in the presence of Shield-1 in T. gondii parasites in which TgCDPK7 was tagged with HA (TgCDPK7-HA) at the C-terminus. TgCDPK7 was immunoprecipitated using anti-HA antibody. Western blotting was performed on TgCDPK7-IP (lane 3) from these parasites or ΔKu80 (negative control, lane 1) or whole cell lysate (lane 2) using anti-myc antibody. A band corresponding to DD-myc-TgRab11a was observed in TgCDPK7-IP. *, a possible non specific or break down product. D. DD-Myc-TgRab11a was ectopically expressed in T. gondii in TgCDPK7-iKD parasites. Shld-1 was added for 4h to stabilize the expression of TgRab11a and in addition ATc was added for 72h to deplete TgCDPK7. IFA was performed using anti-myc and anti-GAP45 antibodies, which revealed that punctate localization of TgRab11a was lost upon TgCDPK7 depletion. Right Panel, Quantification of vacuoles containing normal punctate TgRab11a staining, from experiments described in the left panel. Data are mean ± SE of three independent experiments and at least 200 vacuoles were counted for each condition (*, n = 3, p<0.001, t-test). E. DD-Myc-TgRab11a was ectopically expressed in T. gondii in TgCDPK7-iKD-HA parasites, ATc treatment was given for 72h and parasites were incubated with Shld-1 for 4h. Western blotting was performed using anti-myc antibody or anti-actin, which was used as loading control. F. IFA was performed using anti-TgRab11a antibody on untreated or ATC-treated TgCDPK7-iKD parasites. ATc treatment resulted in loss of punctate TgRab11a localization and was diffuse.

Fig 4. TgCDPK7-mediated phosphorylation of TgRab11a at S207 is critical for its function.

A. DD-Myc-TgRab11a or its S207A mutant was ectopically expressed in T. gondii in ΔKu80 parasites. Shld-1 was added for 4h or parasites were left untreated. Western blotting was performed using anti-myc antibody and Actin was used as a loading control. B. Shld-1 was added for 4h to parasites ectopically expressing DD-Myc-TgRab11a or its S207A and S207D mutants in ΔKu80 parasites. IFA was performed using anti-myc and anti-GAP45 antibody. While WT TgRab11a and S207D exhibited punctate localization, S207A mutant was diffused in parasite cytoplasm in most cases. Right Panel, Quantification of vacuoles containing normal punctate TgRab11a or S207A/D staining, from experiments described in the left panel [Mean ±SE, *, n = 3 (WT and S207A) and n = 2 (S207D), p<0.0001, t-test]. C. DD-Myc-TgRab11a or its S207A, T205A, S207D mutants were ectopically expressed in T. gondii. Shld-1 was added for 16h and the number of parasites per vacuole was determined after additional 8h. Data represent Mean ± SE, n = 3 and at least 200 vacuoles were counted for each condition (ANOVA; WT, S207A, S207D: n = 3, T205A: n = 2. *, p<0.05, ns-not significant). D. IFA was performed on parasites expressing WT TgRab11a or its S207A mutant in the absence or presence of Shld-1 as described in panel B using antibodies against SAG1 and GAP45. Right Panel, Quantification of vacuoles containing normal SAG1 staining, which was at the parasite periphery, from experiments described in left panel. Data are mean ± SE of three independent experiments and at least 200 vacuoles were counted for each condition (*, n = 4, p<0.001, ANOVA).

Fig 5. TgCDPK7 is involved in phospholipid metabolism.

A. Total lipid isolated from TgCDPK7-iKD tachyzoites grown in the presence or absence of ATc and separated by HPTLC. After quantification by GC-MS and normalization with respect to cell number content relative to total fatty acids was determined. Phospholipid profiling showed the abundance of major phospholipid classes and points at the significant reduction of PE upon TgCDPK7 depletion (mean ± SE, *, n = 4, p<0.05, t-test). B. Metabolic labeling to monitor the synthesis of PC and PE. Extracellular TgCDPK7-iKD tachyzoites were incubated with ¹⁴C-Eth or ¹⁴C-Cho in the presence or absence of ATc. Subsequently, lipids were extracted and radiolabeled lipids were detected by phosphoimaging of TLC (Left panel). Right Panel, Radiolabeled spots corresponding to PE and PC (left panel) were quantitated by densitometry (right panel) and % change in PC and PE formation in ATc-treated parasites with respect to untreated parasites was calculated (mean ± SE, *n = 3, p<0.001, ANOVA). C. Metabolic pathways depicting the synthesis of PC and PE in P. falciparum and T. gondii. The enzymes conserved in two parasites are indicated in black. PMT is solely encoded by P. falciparum, is indicated in red and PTS/PSS involved in conversion of PE to PT or PS in T. gondii is shown in green [29]. D. TgCDPK7-iKD parasites were pre-incubated for 48h with ATc and were subsequently allowed to invade fresh HFFs in the presence or absence of ATc. In one case, 200 μM Eth was added to cultures prior to the addition of ATc. The number of parasites per vacuole was determined after 24h. Data represent mean ± SE, n = 3 and at least 200 vacuoles were counted for each condition (ANOVA, *, p<0.001 for 8/16 parasites/vacuole).

Fig 6. TgCDPK7 interacts with TgGPAT and regulates its localization.

A. Domain architecture of TgGPAT (ToxoDB ID: TGGT1_256980) indicating N-terminal signal peptide and three transmembrane domains. The domain analysis suggested that its GPAT domain is split due to the presence of an insert. Multiple sites (S244, S278) on GPAT protein were identified to be hypophosphorylated upon TgCDPK7 depletion that were located in the insert. The location of the myc/Ty tag introduced in TgGPAT between aa 256 and 257 is indicated in red. Bottom Panel: MS/MS spectra for S278, one of the sites which was hypophosphorylated upon TgCDPK7 depletion (green bars). B. A deletion mutant of TgCDPK7 (ΔTgCDPK7) that has the PH and kinase domain was used to phosphorylate recombinant fragment of GPAT as a GST tagged protein in vitro using γ³²P-labelled ATP. The reaction mixture was separated by SDS-PAGE and gel was used for phosphorimaging. Autophosphorylation of the kinase was also observed. C. A myc-tag was introduced in TgGPAT gene after a.a 256 using CRISPR-Cas9 in C-terminally HA tagged TgCDPK7 parasite line. Parasite lysates were prepared from these or parental (ΔKu80) parasites and were used for IP with anti-myc antibody. Subsequently, Western blotting was performed on total lysate and GPAT-myc IP using anti-HA antibody to detect TgCDPK7-HA. A band corresponding to TgCDPK7-HA was observed only in GPAT-myc IP from transgenic parasites, which was the same size as the band in the whole cell lysates. No band was observed in the case of ΔKu80 parasites (negative control). D. GPAT was Ty-tagged at endogenous locus as described in panel A in TgCDPK7-iKD parasites (TgCDPK7-iKD/GPAT-Ty). ATc was added for 72h to deplete TgCDPK7 followed by IFA for GPAT-Ty. There was a significant change in the localization of GPAT from perinuclear ER like compartment (-ATc) to predominantly cytoplasm upon TgCDPK7 depletion (+ATc). Right Panel, Quantification of vacuoles containing normal perinuclear GPAT localization, from experiments described in the left panel. Data are mean ± SE of three independent experiments and at least 200 vacuoles were counted for each condition (*, n = 3, p<0.001, t-test). E. TgCDPK7-iKD/GPAT-Ty parasites were treated with ATc as described in panel D. Subsequently, parasite pellets were isolated and used for extracting proteins in PBS, sodium carbonate pH 11.0 or 1% Triton X-100. The supernatant (sup) or pellet fraction was electrophoresed and subjected to Western blotting using anti-Ty antibody to detect GPAT. F. C-terminal Ty-tagged full length TgGPAT or its S244A/S278A mutant were transiently overexpressed in ΔKu80 parasites. After 48h, parasites were fixed and IFA was performed using anti-Ty and anti-GAP45 antibodies. Bottom Panel, Quantification of vacuoles containing normal ER-like perinuclear TgGPAT or S244A/S278A staining, from experiments described in the right panel. Data are mean ± SE of three independent experiments and at least 200 vacuoles were counted for each condition (*, n = 3, p<0.001, t-test).

Fig 7. A model for TgCDPK7 signaling.

TgCDPK7, which interacts with PIPs [8], targets trafficking of key proteins like GPI linked proteins like SAG1/3 via its ability to facilitate the phosphorylation of proteins like TgRab11a. It regulates PE metabolism in the parasite possibly via its ability to regulate enzymes involved in PL metabolism like GPAT and PAP and these events are critical for the division of T. gondii.