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Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform
BISIO SABARIS, Hugo, et al.
BISIO SABARIS, Hugo, et al. Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform. Nature Microbiology, 2019
PMID : 30742070
DOI : 10.1038/s41564-018-0339-8
Available at:
http://archive-ouverte.unige.ch/unige:114242
Disclaimer: layout of this document may differ from the published version.
Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform
Hugo Bisio1, Matteo Lunghi1, Mathieu Brochet1 and Dominique Soldati-Favre1*
Affiliations
1Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland
*Correspondence to: dominique.soldati-favre@unige.ch
Keywords: Apicomplexa, Toxoplasma gondii, egress, microneme secretion, diacylglycerol kinase, phosphatidic acid, cGMP-dependent protein kinase G, guanylate cyclase, flippase, P4-ATPase, CDC50.
Toxoplasma gondii establishes a life-long chronic infection in humans and animals1. Host cell entry and egress are key steps in the lytic cycle of this obligate intracellular parasite, ensuring its survival and dissemination. Egress is temporally orchestrated, underpinned by the exocytosis of secretory organelles called micronemes. At any point during intracellular replication, deleterious environmental changes like loss of host cell integrity can trigger egress2 via the activation of the cGMP-dependent protein kinase G (PKG)3. Remarkably, even in the absence of extrinsic alarming signals, the parasites egress from infected cells in a coordinated manner after 5 to 6 cycles of endodyogeny multiplication. Here, we show that diacylglycerol kinase 2 (DGK2) is secreted into the parasitophorous vacuole where it produces phosphatidic acid (PA). PA acts as an intrinsic signal eliciting natural egress upstream of an atypical guanylate cyclase (GC) uniquely conserved in alveolates4 and ciliates5 and composed of a P4-ATPase and two GC catalytic domains. Assembly of GC at the plasma membrane depends on two associated cofactors, cell division control 50.1 (CDC50.1) and unique GC organizer (UGO). This study reveals the existence of a signaling platform responding to an intrinsic lipid mediator and extrinsic alarming signals to control programmed and induced egress.
Micronemes release numerous adhesins (MICs), perforins and proteases6 that are instrumental for egress, gliding motility and re-invasion. Intracellular parasites replicate inside a parasitophorous vacuole (PV) surrounded by a membrane resulting from the invagination of the host plasma membrane (PM) during invasion. Activation of PKG triggers a signaling cascade that involves a phosphoinositide phospholipase C (PI-PLC)7 and leads to a rise in intracellular calcium and the production of phosphatidic acid (PA), key mediators of microneme exocytosis. Intracellular PA, produced through the reversible reaction of diacylglycerol kinase 1 (DGK1) and phosphatidic acid phosphatases (PAPs/lipins) is sensed by the apical plextrin homology domain containing protein (APH) acylated at the surface of the micronemes8, which allows membrane fusion in a DOC2.1-dependent manner9. The type I RH parasites typically egress around 48 hours post-infection and this event is accompanied by a drop in vacuolar pH10 (Fig. 1a).
Although abscisic acid was previously reported to control calcium-dependent egress11, evidence for an abscisic acid biosynthetic pathway and a related receptor involved in egress are lacking. In consequence, the molecular mechanisms and intrinsic signals that govern such a programmed egress are not known.
Members of the coccidian subgroup of Apicomplexa encode for a putative secreted DGK212. RHΔku80 tachyzoites were modified to express DGK2-Ty, epitope-tagged in the endogenous locus8. DGK2-Ty migrates at the predicted size of 70 kDa by western blot (Fig. 1b), accumulates in the PV (Fig. 1c) and co-localizes with the vacuolar marker GRA1 (Fig. 1c). Constitutive secretion of DGK2 was observed by Excreted Secretory Antigens (ESA) assay (Supplementary Figure 1a). To investigate DGK2 function, conventional knockout (DGK2-KO) and tetracycline repressor-based inducible knockdown (DGK2-iKD) mutant parasites were generated (Fig. 1d, Supplementary Figure 1b-d). Plaque assays revealed the formation of smaller plaques, indicative of a defect in one or more steps of the lytic cycle (Fig. 1e, Supplementary Figure 1e). DGK2- KO and DGK2-iKD in presence of anhydrotetracycline (ATc) exhibit no defect in intracellular growth (Supplementary Figure 1f-g), invasion (Supplementary Figure 1h-i) or induced egress (Fig. 1f and Supplementary Figure 1j) but a severe natural egress phenotype (Fig. 1g-i, Supplementary Figure 1k). At 60 hours post-infection (hpi), the
parental or DGK2-iKD parasites in absence of ATc have initiated a new lytic cycle which will egress 96 hpi (Fig. 1a, g-i) whereas most of the vacuoles containing DGK2-iKD +ATc are still intact (Fig.1g-i and Supplementary Figure 1k). These parasites are trapped in enlarged vacuoles that eventually mechanically rupture in a host cell type independent manner (Supplementary Figure 1l).
Environmental acidification of the PV is known to occur shortly before or during natural egress10 with acidic pH acting as a potent trigger of microneme secretion in a process that depends on PKG and protein kinase A1 (PKAc1)13. Of relevance, disruption of DGK2 in PKAc1-iKD delays the acidification-dependent premature egress phenotype of PKAc1-iKD (Fig. 1j, Supplementary Figure 1m)13. Artificial acidification of the vacuole following permeabilization of the host PM with digitonin still induces egress of DGK2-iKD parasites in presence of ATc (Fig. 1k), suggesting that DGK2 acts in concert with pH to trigger egress.
DAG kinase activity of DGK2 was confirmed by expression of the recombinant protein in mammalian cells followed by cell lysate-based enzymatic assay (Fig. 1l-m). T.
gondii DGK1, DGK2 and mammalian DGKα catalyzed the conversion of DAG into PA by consumption of ATP, an activity which was ablated by the introduction of a point mutation in the ATP binding site14 (Fig. 1l-m). In the PV, DGK2 potentially produces extracellular PA (ePA) either in the outer leaflet of the parasite PM, the inner leaflet of the PV membrane (PVM) or the membranous nanotubular network (MNN)15. We hypothesized that ePA accumulates in the outer leaflet of the parasite PM to facilitate the signal transduction into the parasite cytoplasm. Concordantly, exposure of extracellular parasites to nanomolar concentrations of ePA is sufficient to trigger microneme exocytosis (Supplementary Figure 1n-o), concordant with low levels of PA present in cells, reaching a fast saturation. Under the same experimental conditions, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fail to trigger microneme secretion (Supplementary Figure 1p). Remarkably, ePA-dependent microneme exocytosis is blocked by a PKG inhibitor (Fig. 1n), confirming that ePA acts upstream of PKG in the intracellular signaling cascade leading to microneme exocytosis.
Transmembrane guanylyl cyclases (GCs) are known as receptors able to integrate directly extracellular signals to produce intracellular cGMP16. T. gondii possesses a single
gene coding for an atypical GC composed of two GC catalytic domains fused to a P4- ATPase domain (Supplementary Figure 2a). Of relevance here, a major group of P4- ATPases corresponds to phospholipid flippases that translocate phospholipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet of biological membranes17. Epitope- tagged GC localizes to the apical and basal poles of the parasite PM13. When fused to the auxin-inducible degron (mAID) at the endogenous locus, GC-mAID-HA (Fig. 2a and Supplementary Figure 2b) is rapidly degraded upon addition of 3-indole acetic acid (IAA)3 (Fig.2b). The plaque assay reveals a dramatic loss of fitness in absence of GC (Fig. 2c), associated with a severe impairment in invasion (Fig. 2d), egress (Fig. 2e-f, Supplementary Figure 2c) and microneme exocytosis (Fig. 2g) but without impact on intracellular growth (Supplementary Figure 2d). Depletion of GC-mAID-HA thus recapitulates the phenotype observed for PKG inhibition or degradation3. PKG triggers calcium mobilization from intracellular stores18 and the calcium ionophore, A23187 could bypass the requirement for GC, partially rescuing egress of GC-mAID-HA depleted parasites (Fig. 2e). Intriguingly, treatment with propranolol, a PAP inhibitor, also rescued egress however extracellular parasites are still connected at their posterior pole (Fig. 2e-f). This highlights a second role for GC in the fission of basal pole connected parasites, which occurs concomitantly to egress. The cell-cell communication between intravacuolar parasites was previously shown to critically dependent on myosin I (MyoI)19. Logically, the disruption of MyoI in GC-mAID-HA parasites (Supplementary Figure 2e) leads to a complete rescue of the clumping observed in propranolol-induced egress in presence of IAA (Fig. 2e).
Importantly, ePA does not trigger microneme secretion nor raise intracellular cGMP levels in GC-mAID-HA depleted parasites (Fig. 2g, Supplementary Figure 2f) suggesting that the P4-ATPase domain could sense ePA and activate GC. To determine if the P4-ATPase domain of GC exhibits a flippase activity, fluorescent analogues of PA, phosphatidylserine (PS) and PC were incubated with intact extracellular parasites using the membrane impermeable quencher DPX20. Substantial flipping of PS is detected whereas flipping of PA and PC is marginal (Supplementary Figure 2g). Bulk, time- dependent increase of non-quenchable fluorescent analogues of all three phospholipids remains unchanged upon depletion of GC (Supplementary Figure 2h). To address the role
of the P4-ATPase domain in GC activation, we attempted to introduce a point mutation in the conserved aspartate residue (Asp782) known to be auto-phosphorylated during the flipping event21. CRISPR/Cas9-mediated locus replacement of a DNA fragment encompassing the Asp782 was successful when the wild type amino acid sequence was maintained but failed with the fragment harboring the Asp782Glu mutation. Failure to recover mutant parasites indirectly suggested that the P4-ATPase activity of GC is critical for parasite fitness.
Flippases are polytoptic proteins that typically depend on a CDC50 co-factor protein functioning as a chaperone to facilitate activity and/or trafficking to their final destination22. We engaged in crosslinking experiments coupled to immunoprecipitation (IP) and mass spectrometry to identify interacting partners of GC that might regulate its activity (Supplementary Table 1). Among the 55 proteins immunoprecipitated with GC, four prominent hits were retained for further analysis. Two of these putative partners were deemed dispensable for the lytic cycle of the parasite and were not analyzed further (Supplementary Figure 3a-c). On the other hand, a CDC50-related protein (CDC50.1;
TGGT1_230820) that we C-terminally tagged at the endogenous locus with mAID-HA (CDC50.1-mAID-HA) partially co-localizes with GC (Fig. 3a). The interaction between CDC50.1 and GC was confirmed by co-IP coupled to western blot analysis (Fig. 3b, Supplementary Figure 3d). Depletion of CDC50.1-mAID-HA with IAA (Fig. 3c, Supplementary Figure 3e) leads to a strong plaque assay phenotype (Fig. 3d) associated to a natural egress defect (Fig. 3e, Supplementary Figure 3f). A second interacting partner of GC, named Unique Guanylate cyclase Organizer (UGO; TGGT1_238390) is found in all Apicomplexa and is composed of stretches of conserved transmembrane spanning domains at both the N- and C-terminal regions whereas the predicted central ectodomain shows amino acid sequences composition specific to parasite species (Supplementary Figures 3g-h and 4). UGO-mAID-HA partially co-localizes with GC-Ty (Fig. 3f) and is critical for parasite survival (Fig. 3g-h). Remarkably, GC-Ty is unstable and remains trapped along the secretory pathway in CDC50.1-mAID-HA or UGO mAID-HA lines in presence of IAA (Fig. 3c and 3i-k). Depletion of either CDC50.1 or UGO does not affect the trafficking of other proteins through the secretory pathway (Supplementary Figure 5a), indicating that these partners are instrumental to selectively bring GC to its final
destination. The localization of UGO is affected by depletion of CDC50.1, confirming the existence of a membrane macro-complex formed together with the GC (Supplementary Figure 5b-c).
Depletion of CDC50.1 blocks natural egress but does not affect parasite replication, motility and invasion, as observed for DGK2, suggesting a role in ePA sensing (Fig. 3m and Supplementary Figure 5d). In contrast, UGO is necessary for both natural and induced egress (Supplementary Figure 3f), motility and invasion (Fig. 3l-m and Supplementary Figure 5e) as observed for GC (Fig. 2d-e and Supplementary Figure 3f). Consistently, both mutants fail to trigger microneme secretion in response to ePA (Fig. 3n-o). However, BIPPO induces microneme secretion in the absence of CDC50.1 (Fig. 3o) suggesting that GC-dependent cGMP synthesis is still functional and responsible for tonic signaling despite its change in localization. Conversely, in the absence of UGO, BIPPO is unable to trigger microneme secretion indicating that cGMP production by GC is abolished (Fig 3n-o). In agreement with these results, BIPPO- or ePA-induced cGMP production was shown to be dependent of the GC and UGO expression while only ePA-mediated raise in cGMP was affected in CDC50.1 depleted parasites (Fig. 3p). A23187 or propranolol fully rescued the egress defect in the absence of CDC50.1 but only partially in absence of UGO (Fig. 3l) further confirming the important role of UGO for effective cGMP synthesis. Changes in pH act as putative modulator of GC activity10. Depletion of both CDC50.1 and UGO leads to a drastic reduction in the response to changes in vacuolar pH (Fig. 3q and Supplementary Figure 5f), indicating that the PM localization of GC is important for proton sensing.
Parasites depleted in CDC50.1 failed to egress in response to BIPPO (Fig 3l) despite the fact that extracellular parasites were able to secrete micronemes normally in response to BIPPO (Fig. 3o). The variable effect of BIPPO suggests substantial differences in basal level of cGMP production in intracellular and extracellular parasites. Altogether, CDC50.1 and UGO appear to act as chaperone cofactors on the P4-ATPase and the GC catalytic domains of GC, respectively.
Our study sheds light on the neglected mechanism by which parasites program their natural egress from infected cells23. T. gondii divides by endodyogeny and remains invasive at any point during its intracellular growth phase. This adaptation allows a quick
response to insults of the infected host cells and host immune system. Here, we demonstrate that parasites not only respond to extrinsic alarming signals but also produce their own intrinsic signal that calibrates their multiplication and spreading in a time- dependent manner (Fig. 4). We propose that timely controlled local raise in ePA concentrations produced by DGK2 likely in the parasite PM and possibly also in the MNN or PVM acts as an intrinsic signal to regulate egress. The impact of a programmed egress in vivo remains to be assessed. Previous studies indicate that static cells (such as mesothelial cells) contain populated PV24, whereas migratory cells are mostly infected with PV containing two to four parasites in vivo. This suggests that egress is likely triggered by the hostile environment of immune cells and bypasses DGK224. Cell death- inducing signals such as Fas and perforins have also been shown to induce egress of T.
gondii and could provide an alternative route to balance egress rates in inflammatory conditions25. The levels of cGMP have been proposed to be regulated via cAMP levels and PKAc1 activity, whose inhibition leads to the phosphorylation of phosphodiesterase 2 and concomitantly controlling PKG activity13. Here, we show that premature egress of PKAc1-iKD depends on the presence of DGK2 with PKAc1 potentially acting as a negative regulator of egress. Moreover, two dense granule proteins have been previously associated to egress in T. gondii (GRA22 and GRA41)26,27, and given their localization in the PV, these proteins may impact on DGK2 substrate or activity. Natural egress might serve as an adaptation of this generalist pathogen to respond to differential pressure of the immune system in diverse host organisms and environments.
The essential signaling platform composed of GC-CD50.1-UGO described here integrates intrinsic (PA, pH drop10) and extrinsic signals (K+2, pH drop10 and seralbumin28) governing egress (Fig. 3q and Supplementary Figure 5g-h). GC coordinates simultaneously the exocytosis of microneme at the apical pole and the disconnection of parasites at the basal pole to free motile tachyzoites, ready to reinvade. DGK2 and CDC50.1 do not impact on the parasites capacity to respond to potent triggers of induced egress and to glide and invade host cells whereas GC and UGO are strictly essential for cGMP production to initiate and ensure parasite egress, motility and invasion. Taken together, we propose a model by which CDC50.1 assists the folding of P4-ATPase domain on GC to sense and possibly directly bind to PA, whereas UGO is an essential
co-factor to produce a catalytically active GC. In Plasmodium yoelii a related complex composed of GCb and CDC50a has been shown to be important to control parasite gliding during mosquito transmission29. The conservation of this signaling platform across the Apicomplexa and the molecular mechanistic details on how GC integrates intrinsic and extrinsic signals awaits further investigation.
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Full Methods
Cell culture and establishment of stable parasite lines
Cell culture were performed in Human foreskin fibroblasts (HFFs) (American Type Culture Collection-CRL 1634), HeLa cells (ATCC CCL-2) and Vero cells (American Type culture Collection CCL 81). Cell lines were not tested for mycoplasma contamination.
T. gondii tachyzoites (RH-ΔHGPRT, ΔKU80, and TATi-11 were grown in human foreskin fibroblast (HFF) monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO) supplemented with 5% fetal calf serum (FCS), 2 mM glutamine and 25 µg/ml
gentamicin (37°C, 5% CO2). Parasite transfections were performed by electroporation as described previously 2. The hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) 3, chloramphenicol selection 4 and dihydrofolate reductase thymidylate synthase (DHFR-TS) genes 5 were used as positively selectable markers as described previously.
Parasite cloning strategies
All primers used in this study are listed in Table S2. Genomic DNA was isolated with the Wizard SV genomic DNA purification system (Promega). RNA was purified wing Trizol (Invitrogen) extraction. Total cDNA was generated by RT-PCR using the Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's protocol.
Tet-repressive knockdown of DGK2 (DGK2-iKD) mutant was generated using a PCR fragment encoding the TATi-1 trans-activator, the HXGPRT cassette and the TetO7S1 promoter produced using the KOD DNA polymerase (Novagen, Merk) with the vector 5′MyoF-TATi1-HX-tetO7S1MycNtMyoF10 as template and the primers 5021/5022 that also carry 30 bp homology with the 5′ end of DGK2. A specific sgRNA was generated to introduce a double stranded break at the 5’ of DGK2 (primers used to generate the guide (5020/4883)).
To generate DGK2-KO and MyoI-KO, the CRISPR-Cas9 system was used to disrupt the genes by insertion of a HXGPRT or DHFR-TS selection cassette into the coding sequence. To do so, a PCR product was generated using the KOD DNA polymerase (Novagen, Merk) with the vector 5′MyoF-TATi1-HX-tetO7S1MycNtMyoF10 or p2854- DHRF as template and the primers 6753/6754, 6022/6023 that also carry 30 bp homology
with the respective locus. A specific sgRNA was generated to introduce a double stranded break in the DGK2 locus (primers used to generate the guide (6752/4883)).
Auxin-inducible degradation of GC, CDC50.1 and UGO were generated using a PCR fragment encoding the mAID-HA and the HXGPRT cassette produced using the KOD DNA polymerase (Novagen, Merk) with the vector pTUB1:YFP-mAID-3HA as template and the primers 7427/7428, 7905/7906, AIDfo_208420/AIDrev_208420, AIDfo_233220/AIDrev_233220, AIDfo_238390/AIDrev_238390 that also carry 30 bp homology with the 3′ end of GC. A specific sgRNA was generated to introduce a double stranded break at the 3’ of GC (primers used to generate the guide (7426, gRNA_208420, gRNA_233220, gRNA_238390 /4883)).
To replace 5’ genomic locus of GC by a cDNA derived sequence two pcr fragments were generated: PCR1: 450bp of the GC promoter were amplified using the primers 7384/7385 and cloned by Gibson assembly to generate the pl. PCR2: 2.7Kb of the 5’ coding sequence of the GC was amplified using the primers 7386/7387 from cDNA and introduced by Gibson assembly into the previous generated vector. A 3xmyc tag was introduced in the N-terminal of the protein. Point mutation was generated in one step using the primers 7390/7391. Linearization was performed using ApaI/PacI before transfection. Two guides were used to introduce two double breaks in the genome locus of the GC. Guides were generated using the primers (7388/4883) and (6845/4883).
Expression plasmids for DGKs, p3-FLAG-CMV-DGK1 and p3-FLAG-CMV-DGK2 were generated by PCR amplification from T. gondii cDNA using the primers 6243/6244 and 6264/6246 and digested with XmaI/NotI. Point mutations were generated using the primers 6698/6699 and 6700/6701.
Plaque assay
A confluent monolayer of HFFs was infected with freshly egressed parasites for 7 days before the cells were fixed with PFA/Glu. Plaques were visualized by staining with Crystal Violet (0.1%).
Intracellular growth assay
HFF monolayers were infected with freshly egressed parasites and incubated for 24hrs before fixation with PAF/Glu. IFAs were performed using α-GAP45 to count individual parasites. Results represent mean +/- standard deviation of 100 parasites from three biological replicates.
Egress assay
Freshly egressed parasites were inoculated on HFF monolayers and incubated for 30 hrs.
Egress was stimulated by serum free media containing 50 µM BIPPO, 3 µM A23187, 250 µM propranolol or 0.06% DMSO as a control at 37°C/7-12 mins prior to fixation.
Parasites and PVM were labeled with GAP45 and GRA3, respectively. The proportion of egressed versus non-egressed vacuoles was calculated by counting 100 vacuoles for three independent experiments.
Low pH induced egress was performed as described before 6. Briefly, host cells were infected with parasites for 30-36 hs. Before assay, wells were washed with intracellular buffer in order to avoid low K+ induced egress. Finally, the buffer was replaced with intracellular buffer varying pH with or without 15µM digitonin.
Invasion assay
Syringe-released or freshly egressed parasites were allowed to invade a new host cell layer for 30 min before fixing with PAF/Glu for 7min. Non-permeabilized cells were incubated with α-SAG1 antibodies diluted in 2% BSA/PBS for 20 min and washed 3 times with PBS. Cell were then fixed with 1% formaldehyde/PBS for 7min, washed with PBS and permeabilized using 0.2% Triton X-100/PBS. Parasites were labeled using α- GAP45 antibodies. At least 100 parasites were counted for each strain and the percent of intracellular parasites is represented. The data shown are mean values from three independent experiments.
Natural egress
Potassium buffer shift was used to synchronize T. gondii invasion in the designated host cell type as previously described 7. In brief, freshly egress parasites were let to settle in a monolayer of cells at low multiplicity of infection in intracellular buffer for 20 minutes.
Buffer were subsequently replaced for a low-potassium permissive medium and parasite were allowed to invade for 2-5 minutes at 37°C, followed by extensive washing.
Percentage of egressed vacuoles were analyzed after 36, 44 or 60 hours post infection. At least 100 vacuoles were counted per time point.
Microneme secretion
Freshly egressed parasites were washed and resuspended in equal volume of intracellular (IC) buffer (5 mM NaCL, 142 mM KCl, 1 mM MgCl2, 2mM EGTA, 5.6 mM glucose, 25 mM HEPES, pH 7,5). Treatment with EtOH, PA or DMSO was performed for 30 min at 37ºC. Parasites were pelleted (1,000 x g/10 min/4ºC), and supernatant was transferred to new Eppendorf tubes and re-pelleted (2,000 x g/5 min/4ºC). Final supernatant (excreted
secreted antigens [ESAs]) and pellet fractions were resuspended in sample buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 2 mM EDTA, 2% SDS, 0.05% bromophenol blue, 100 mM DTT) and boiled prior to analysis by immunoblotting.
DGK enzymatic activity
293T cells (ATCC® CRL-3216™) expressing the 3xFLAG-tagged DGKs were harvested in lysis buffer (50 mM HEPES (pH 7.2), 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol and the Complete Protease Inhibitor mixture (Roche)). After sonication and centrifugation at 15000g for 30 min, the resultant supernatant was used for the DGK activity assays. The octyl glucoside mixed micellar DGK activity assay was performed as previously described 8. Briefly, the assay mixture contained 50 mM MOPS (pH 7.4), 50 mM n-octyl-β-D-glucoside (Sigma), 1 mM dithiothreitol, 100 mM NaCl, 20 mM NaF, 10 mM MgCl2, 1 µM CaCl2, 2 mM (5.4 mol%) 1,2-dioleoyl-sn-glycerol (Sigma-Aldrich), 0.2 mM ATP, and up to 1-2 µg of the 293T cell lysates expressing the 3xFLAG-tagged DGKs and/or 3xFLAG-MAG1. Measurement was performed at a final time point of 30 min using the ADP-Glo™ Kinase Assay (Promega) following manufacturer instructions.
Flippase Assay
NBD-phospholipid incorporation was assessed by flow cytometry as described in9. Briefly, 5x106 extracellular parasites were washed in Hanks balanced salt solution (pH 7.4) containing 1 g/L glucose. 1µM NBD-phospholopid was incubated at room temperature. At the designated time point, 20mM DPX was added to quench fluorescence of outer leaflet localize lipid. 10,000 cells were analyzed with a Gallios 4 lasers. The mean of fluorescence intensities per cells was calculated.
Co-immunoprecipitation
Intracellular tachyzoites were harvested, washed in PBS, and lysed in coIP buffer (0.2%
[v/v] Triton X-100, 50 mM Tris-HCl [pH 8], 150 mM NaCl) in presence of a protease inhibitor cocktail (Roche). Cells were sonicated on ice and centrifuged at 14,000 rpm for 30 minutes at 4°C. Supernatants were then subjected to IP as already described10. Briefly, Co-IP experiments were performed on 1% paraformaldehyde cross-linked intracellular parasites. Parasites were suspended in 100 µL of 6 M Urea in 50 mM ammonium bicarbonate (AB). To this solution, 2 µl of DTT (50 mM in LC-MS grade water) were added and the reduction was carried out at 37°C for 1 hour. Alkylation was performed by adding 2 µL of iodoacetamide (400 mM in distilled water) for 1 hour at room temperature in the dark. Urea concentration was lowered to 1M with 50 mM AB, and protein digestion was performed overnight at 37 °C with 15 µL of freshly prepared trypsin Promega (0.2 µg/µL in AB). After beads removal, the sample was desalted with a C18 microspin column (Harvard Apparatus, Holliston, MA, USA), dried under speed-vacuum, and re-dissolved in H2O/CH3CN/FA 94.9/5/0.1 before LC-ESI-MS/MS analysis. LC-ESI- MS/MS was performed on a Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) equipped with an Easy nLC 1000 system (Thermo Fisher Scientific). Peptides were trapped on an Acclaim pepmap100, C18, 3µm, 75 µm x 20mm nano trap-column (Thermo Fisher Scientific) and separated on a 75 µm x 500 mm, C18, 2µm Easy-Spray column (Thermo Fisher Scientific). The analytical separation was run for 90 min using a gradient of H2O/FA 99.9%/0.1% (solvent A) and CH3CN/FA 99.9%/0.1% (solvent B). The gradient was run as follows: 0-5 min 95 % A and 5 % B, then to 65 % A and 35 % B in 60 min, then to 10 % A and 90 % B in 10 min, and finally
stay at 10 % A and 90 % B for 15 min. The entire run was at a flow rate of 250 nL/min.
ESI was performed in positive mode. For MS survey scans, the resolution was set to 70 000, the ion population was set to 3 × 106 with a maximum injection time of 100 ms and a scan range window from 400 to 2000 m/z. For MS2 data-dependent acquisition, up to fifteen precursor ions were selected for higher-energy collisional dissociation (HCD).
The resolution was set to 17 500, the ion population was set to 1 × 105 with a maximum injection time of 50 ms and an isolation width of 1.6 m/z units. The normalized collision energies were set to 27%. Peak lists were generated from raw data using the MS Convert conversion tool from ProteoWizard. The peaklist files were searched against the Toxoplasma gondii TGGT1 database from ToxoDB using Mascot search engine (Matrix Science, London, UK; version 2.5.1). Trypsin was selected as the enzyme, with one potential missed cleavage. Fragment ion mass tolerance was set to 0.020 Da and parent ion tolerance to 10.0 ppm. Variable amino acid modification was oxidized methionine and fixed amino acid modification was carbamidomethyl cysteine. The mascot search was validated using Scaffold 4.7.3 (Proteome Software Inc., Portland, OR). Protein identifications were accepted if they could be established at greater than 95.0 % probability and contained at least 3 identified peptides. Fifty-five proteins were recovered. We manually filtered putative interactors by selecting proteins with more than five identified peptides and excluding secreted proteins or proteins frequently recovered in our previous immunoprecipitation experiments. With these criteria, the four following proteins were selected for further analysis: TGGT1_238390, TGGT1_208420, TGGT1_230820, TGGT1_233220.
Cyclic GMP Measurement by ELISA
Extracellular parasites were resuspended in IC buffer and treated with either BIPPO, ePA or DMSO for 10 minutes at 37 °C. Reactions were stopped by placing tubes to ice.
Parasite lysis was achieved by freeze and thaw 5 times. cGMP levels were determined with a cGMP ELISA Detection Kit (GenScript) according to the manufacturer's instructions.
Phylogenetic analysis
Protein sequence alignment was peformed using MUSCLE11,12. Alignments were curated manually utilizing BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html) to identify conserved domains and trim uninformative positions. Curated alignments were fed into PhyML using the web service platform Phylogeny.fr13, to generate the phylogenetic tree using LG model of amino acids substitution with NNI topology search. Sequences used were obtained from EuPathDB. Alignment of the full-length proteins is deposited as Supplementary Information Fig. S4.
Statistics and Reproducibility
All data are presented as mean value of 3 independent biological replicates (n = 3) ± standard deviation, unless otherwise stated in the figure. The mean of each independent biological replicate was generated by counting 100 vacuoles. All data analysis was done with Graphpad Prism. Null hypothesis (α=0.05) was tested using the unpaired two-tailed Student’s t-test, except for figures 2e, 3l, 3p, 3q where ANOVA was applied and p-values significant for data interpretation have been represented on graph.
2e. One way ANOVA (α=0.05) with Tukey’s multiple comparison test. Statistically significant differences in egress were found upon treatment with BIPPO (F=268.8, dFn=4 dFd=10, p= 1.0E-08), A23187 (F=24.44, dFn=4 dFd=10, p= 3.8E-05), Propranolol
(F=108.6, dFn=4 dFd=10, p= 3.0E-08), and disconnection (F=58.66, dFn=4 dFd=10, p=
6.6E-07), but not DMSO (F=1.8, dFn=4 dFd=10, p= 0.2046).
3l. One way ANOVA (α=0.05) with Tukey’s multiple comparison test. Statistically significant differences in egress were found upon treatment with BIPPO (F=189.6, dFn=4 dFd=10, p= 1.0E-08), A23187 (F=13.86, dFn=4 dFd=10, p= 4.4E-04), Propranolol (F=45.09, dFn=4 dFd=10, p= 2.3E-06), and disconnection (F=97.92, dFn=4 dFd=10, p=
6.0E-08), but not DMSO (F=0.67, dFn=4 dFd=10, p= 0.6247).
3p. One way ANOVA (α=0.05) with Tukey’s multiple comparison test. Statistically significant differences were found upon treatment with BIPPO (F=9.876, dFn=6 dFd=14, p= 2.3E-04), ePA (F=19.24, dFn=6 dFd=14, p= 5.1E-06), and DMSO (F=15.66, dFn=6
dFd=14, p= 1.7E-05).
F3q. Two-way ANOVA (α=0.05) with Tukey’s multiple comparison test. pH changes (F=540.31, dFn=2 dFd=32) account for 48.6% of the variance (p = 1.0E-08), GC, CDC50.1 and UGO depletion (F=77.84, dFn=7 dFd=16) account for 30.42% of the variance (p = 1.0E-08) and the interaction between the two factors (F=29.61, dFn=14 dFd=32) account for 18.64% of the variance (p = 1.0E-08).
Methods References
1 Meissner, M., Schluter, D. & Soldati, D. Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298, 837-840, doi:10.1126/science.1074553 (2002).
2 Soldati, D. & Boothroyd, J. C. Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science 260, 349-352 (1993).
3 Donald, R. G., Carter, D., Ullman, B. & Roos, D. S. Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation. J Biol Chem 271, 14010-14019 (1996).
4 Kim, K., Soldati, D. & Boothroyd, J. C. Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker. Science 262, 911-914 (1993).
5 Donald, R. G. & Roos, D. S. Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria. Proc Natl Acad Sci U S A 90, 11703-11707 (1993).
6 Roiko, M. S., Svezhova, N. & Carruthers, V. B. Acidification Activates Toxoplasma gondii Motility and Egress by Enhancing Protein Secretion and Cytolytic Activity. PLoS Pathog 10, e1004488, doi:10.1371/journal.ppat.1004488 (2014).
7 Millholland, M. G. et al. A host GPCR signaling network required for the cytolysis of infected cells facilitates release of apicomplexan parasites. Cell Host Microbe 13, 15-28,
doi:10.1016/j.chom.2012.12.001 (2013).
8 Sato, M. et al. Evaluations of the selectivities of the diacylglycerol kinase inhibitors R59022 and R59949 among diacylglycerol kinase isozymes using a new non-radioactive assay method.
Pharmacology 92, 99-107, doi:10.1159/000351849 (2013).
9 Takatsu, H. et al. Phospholipid flippase activities and substrate specificities of human type IV P- type ATPases localized to the plasma membrane. J Biol Chem 289, 33543-33556,
doi:10.1074/jbc.M114.593012 (2014).
10 Gaskins, E. et al. Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii. J Cell Biol 165, 383-393, doi:10.1083/jcb.200311137 (2004).
11 Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput.
Nucleic Acids Res 32, 1792-1797, doi:10.1093/nar/gkh340 (2004).
12 Edgar, R. C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113, doi:10.1186/1471-2105-5-113 (2004).
13 Dereeper, A. et al. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36, W465-469, doi:10.1093/nar/gkn180 (2008).
Data availability
The data that support the findings of this study are available from the corresponding author upon request. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011692 and 10.6019/PXD011692.
Acknowledgements
This research was supported by Swiss National Science Foundation (FN3100A0-116722 to DSF and BSSGI0-155852 to MB) and HB is the recipient of a Swiss Government Excellence Scholarship with Uruguay. We gratefully thank Natacha Klages, Jean Baptiste Marq their instrumental technical contributions to the project. We are grateful to Hayley Bullen and Nicolo Tosetti for the preliminary investigations on DGKs. We thank David Sibley and Kevin Brown for sharing the mAID system prior to publication and for helpful advices. We thank the excellent service at the proteomics, bioimaging and flow- cytometry core facilities at the Faculty of Medicine of the University of Geneva. Thanks to all the meBOP students of 2018 for repeating some of the presented experiments and critically challenging the proposed model.
Author contributions
D. S-F. and H.B. conceived the project. M.B. provided insightful discussions and constructive suggestions. H.B. and M.L. designed, performed and interpreted the
experimental work. D. S-F supervised the research. H.B. and D.S.-F wrote the paper with editorial support from M.L.
Competing interests
The authors declare no competing financial interests.
Figure
Figure 1. DGK2 intrinsically regulates natural egress in Toxoplasma gondii
a. Schematic representation of the T. gondii lytic cycle in natural egress conditions.
Parasites typically complete one lytic cycle and egress in 48 hours. HC = Host Cell, P = parasite, PV = Parasitophorous Vacuole. Numbers indicate represented time-points. b.
DGK2-Ty migrates at the expected size on western blot (70kDa). Western blot was probed using αTy and αDGK2 specific antibodies. Catalase: loading control. Black and red asterisk mark the migration size of the tagged or unmodified version of the protein. c.
Endogenously C-terminally tagged DGK2 localize to dense granules and accumulates in the parasitophorous vacuole. GRA1: dense granules. GAP45: parasite periphery. Scale bar 3 μm. d. DGK2-iKD parasites are regulated by ATc in 24 hr. Black and red asterisk mark specific and aspecific bands, respectively. Catalase: loading control. e. DGK2-iKD but not parental ΔKU80 parasites display a growth defect in the presence of ATc. Scale bar 2mm f. Egress induced by BIPPO (a phosphodiesterase inhibitor) or propranolol (a phosphatidic acid phosphatase inhibitor) was not impaired in the DGK2-iKD parasites. g and h. DGK2-iKD displayed a delay of natural egress timing and show mostly larger intact vacuoles at 60 hours post infection (hpi). Representative images of DGK2-iKD parasites vacuoles at 44 and 60 hpi are shown in g (scale bar 7 µm) while global quantification is shown in h. i. Intracellular vacuole growth is not affected by depletion of DGK2. j. Premature egress phenotype induced by PKAc1-iKD is considerably delayed by DGK2 deletion. k. DGK2-iKD parasites egress normally in response to acidic pH.
Buffers at pH 7.4, 6.4 and 5.4 were used. l and m. Heterologous expression of DGK2 in mammalian cells shows that it is an active DAG kinase. Expression levels in cell lysates are shown in l. n. ePA acts an inducer of microneme secretion and is blocked by
Compound 1.All data is representative or mean of 3 independent biological experiments (n = 3) ± standard deviation, unless otherwise stated in the figure. Refer to full methods for details on statistical analysis.
Figure 2. ePA stimulates microneme secretion in a GC-dependent manner
a. As previously reported12 endogenously C-terminally tagged GC localizes at the plasma membrane but restricted to the apical pole and residual body in intracellular parasites.
GAP45: parasite periphery. Scale bar 2µm. b. GC-mAID-HA parasites are regulated by IAA in 6 hr. Actin: loading control. c. GC-mAID-HA but not parental parasites display a severe growth defect in the presence of IAA. Scale bar 3mm d. Parasites lacking GC are
significantly impaired in invasion. e, f. Induced egress is significantly impaired in GC- mAID-HA parasites. Representative pictures of vacuoles containing parasites that failed to secrete micronemes (IN), that secreted micronemes but failed to disconnect (clumped) and that egressed normally parasites are shown in f (GRA3 staining is shown in green (parasitophorous vacuole marker) and GAP45 in red (parasite periphery marker)). The Arrowhead indicates places where PVM have been discontinued and parasites appear outside of the PV. Scale bars 6µm. Global quantification of egress events is shown in e.
Red p-values refer to the quantification of clumping. g. BIPPO (5µM), ePA (50nM) or Ethanol (EtOH, 2%) stimulation of microneme secretion is blocked in parasites lacking GC. GRA1 is used as control for parasite viability. All data is representative or mean of 3 independent biological experiments (n = 3) ± standard deviation.
Figure 3. GC assembly involved two co-factor proteins
a. C-terminal tagging of CDC50.1 with mAID-HA at the endogenous locus (CDC50.1- mAID-HA) partially co-localizes with GC-Ty to the apical pole and residual body within intracellular parasites. Scale bar 2 µm. b. GC-Ty co-immunoprecipitates with CDC50.1- mAID-HA c. CDC50.1-mAID-HA parasites are regulated by IAA and concomitantly (lower panel) GC-Ty signal decreases. Catalase loading control d. CDC50.1-mAID-HA downregulation leads to the formation of small plaques. Scale bar 2mm. e. Quantification of natural egress defect in CDC50.1 depleted parasites. f. UGO and GC partially colocalize at the apical end and residual body of intracellular parasites. Scale bar 2 µm. g.
UGO-mAID-HA but not parental parasites display a severe growth defect in the presence of IAA. Scale bar 2mm. h. Regulation of UGO-mAID-HA upon 24h IAA addition. Scale bar 2 µm. i, j. GC-Ty miss localizes through the secretory pathway in absence of CDC50.1 (i) and UGO (j). Scale bar 2µm. k. Abundance and stability of GC-Ty is affected in UGO-mAID-HA. l. Induced egress is significantly impaired in UGO and CDC50.1 depleted parasites. p-values in red refer to the quantification of clumping. m.
Parasites lacking UGO but not CDC50.1 are significantly impaired in invasion n. BIPPO (5µM) and ePA (50nM) stimulation of microneme secretion is blocked in parasites lacking UGO. GRA1: control for parasite viability. o. ePA (50nM), but not BIPPO (5µM), stimulation of microneme secretion is reduced in parasites lacking CDC50.1.
GRA1: control for parasite viability. p. BIPPO (5µM) and ePA (50nM) stimulated increase in cGMP level is blocked in parasites lacking GC and UGO. Parasites depleted in CDC50.1 only show a defect responding to ePA. p-values in black for BIPPO analysis, in red for ePA, in green for DMSO, in blue unpaired two tailed Student’s t-test. q. GC, CDC50.1 and UGO depleted parasites egress poorly in response to acidic pH. Buffers at
7.4, 6.4 and 5.4 were used. All data is representative or mean of 3 independent biological experiments (n = 3) ± standard deviation, unless otherwise stated in the figure.
Figure 4. Model of signaling cascade leading to natural egress
a. Schematic of the T. gondii lytic cycle growing as a connected parasites (coenocyte) sheltered in the parasitophorous vacuole. Egress requires the coordination of microneme secretion and parasite fission. Both processes are controlled by cGMP production via guanylate cyclase (GC). b. In absence of external signals, DGK2 limits the intracellular growth timing and induces egress. GC in presence of two interacting partners, UGO and
CDC50.1, senses vacuolar phosphatidic acid (PA) produced by DGK2 and produces cGMP in the cytoplasm of the parasite. The concentration of cGMP depends on production by GC and degradation by PDEs, which are regulated by PKAc1. cGMP activates PKG which in turn increases the production of PI(4,5)P2 and its hydrolysis by PI-PLC to produce diacylglycerol (DAG) and IP3. IP3 stimulates calcium release from an unknown store and concomitantly activates CDPKs while DAG is further converted in PA by DGK1 to promote microneme secretion by binding to APH. Secretion of micronemes leads to egress, gliding and invasion. Microneme secretion can be stimulated or inhibited by several chemicals shown in red c. The GC receptor is composed of one P4-ATPase and two GC catalytic domains. The P4-ATPase domain is predicted to interact with the co-factor CDC50.1 and to be responsible for sensing of vacuolar levels of PA and pH. The two GC domains are responsible of cGMP production and their functionality is critically dependent on the presence of UGO.
Supplementary materials
Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform
Supplementary Figure 1
a. Extracellular parasites were incubated in intracellular buffer for one hour and excretory secretory antigens were analyzed by western blot. b. PCR shows correct integration for generation of DGK2- KO strain. Primers are listed in Table S1. Expected size 5088/4787: 1.3Kb, 5088/p30A: 1.3Kb. c.
PCR shows correct integration for generation of DGK2-iKD strain. Primers are listed in Table S1.
Expected size 1935/4787: 1.1Kb, 5242/p30A: 1.6Kb, 5242/4767: 1.8Kb. d. DGK2 is absent in the DGK2-KO parasites. Catalase: loading control. e. DGK2-KO parasites display a growth defect in plaque assay. f. DGK2-iKD parasites were not impacted in their ability to replicate intracellularly. g.
DGK2-KO parasites were not impacted in their ability to replicate intracellularly. h. DGK2-iKD parasites were not impacted in their ability to invade host cells. i. DGK2 KO parasites were not impacted in their ability to invade host cells. j. Egress induced by BIPPO, A23187 or propranolol was not impaired in DGK2-KO parasites. k and l. DGK2-iKD and DGK2-KO display a delay of natural egress timing and show enlarged populated vacuoles at 50-60 hpi. Representative images of DGK2-KO parasites vacuoles at 36-44 and 50-60 hpi are shown infecting HFF cells in k (scale bar 6 µm). Global quantification of natural egress in HeLa and VERO cells is shown in l. m. DGK2 protein is not expressed in the PKAc1-iKD/DGK2-KO parasites. Actin: loading control. n and o. ePA stimulates microneme secretion in the nanomolar range. p. ePA but not phosphatidylcholine and phosphatidylethanolamine stimulates microneme secretion. All data is representative or mean of 3 independent biological experiments (n = 3) ± standard deviation.
Domain a
Flippase-like domain guanylate cyclase
domain guanylate cyclase domain TMD
1
b
GC- mAID-HA Tir1
1.5 2
6693/70816693/66946693/70816693/66943
g
0 5 10 15 20 25 30
0 25 50 75 100 125 150 175 200
time (minutes)
Arbitraty units
PS PA PC
c
100 80 60 40
% of egressed vacuoles 20
GC-mAID-HA GC-mAID-HA MyoI-KO
+ +
+ Tir1
DMSO
IN OUT
IAA:
0.0 0.5 1.0 1.5
Relative incorporation
PS PA PC
GC-mAID-HA Tir1
+ +
h
IAA:
f
1 2 3 4 5
Tir1 +IAA GC-mAID-HA GC-mAID-HA +IAA
Relative cGMP increase
BIPPO ePA DMSO
Parasite/Vacuole
60
20 40
2 4 8 16
% of Vacuoles
Parental GC-mAID-HA GC-mAID-HA +IAA d
e
GC-mAID-HA MyoI-KO
GC-mAID-HA
1.5 2 1
6077/60786077/20176078/20186077/60786077/20176078/2018
0.5
Supplementary Figure 2
a. Schematic representation of T. gondii guanylate cyclase. Blue boxes represent predicted transmembrane domains. b. PCR shows correct integration for generation of GC-mAID-HA strain.
Primers are listed in Table S1. Expected size 6693/7081: 1.3Kb, 6693/6694: 1.3Kb. c. DMSO does not induce egress in the GC-mAID-HA or parental strain parasites. d. GC-mAID-HA depleted parasites were not impacted in their ability to replicate intracellularly. e. PCR shows correct integration for generation of MyoI-KO strain in the GC-mAID-HA background. Primers are listed in Table S1. Expected size 6077/6078: 1.2Kb, 6077/2017: 650Kb, 6078/2018: 700Kb. f. BIPPO (5µM) and ePA (50nM) stimulated increase in cGMP level is blocked in parasites lacking GC. g. PA is flipping inside the parasites based on flipping assay comparing PA, PS and PC. h. Bulk flipping of PS, PA or PC is not catalyzed by GC. All data is representative or mean of 3 independent biological experiments (n = 3) ± standard deviation.
Supplementary Figure 3
a. PCR shows correct integration for generation of CDC50.1-mAID-HA, UGO-mAID-HA, 233280- mAID-HA and 208420-mAID-HA strains. Primers are listed in Table S1. Expected size 8073/7081:
950bp and 8073/8072: 1.2Kb, 8085/7081: 800bp and 8085/8084: 1.2Kb, 8081/7081:750bp and 8081/8080: 900bp, 8083/7081: 900bp and 8083/8082: 1.3Kb. b. 233280-mAID-HA parasites are regulated by IAA. Actin is shown in red. Scale bar 2 µm. c. 233280-mAID-HA and 208420-mAID- HA parasites do not display a growth defect in the presence of IAA. Scale bar 5 mm. d. CDC50.1- mAID-HA co-immunoprecipitates with GC-Ty. e. CDC50.1-mAID-HA parasites are regulated by IAA. Actin is shown in red. Scale bar 2 µm. f. GC, UGO and CDC50.1 depleted parasites displayed a delay of natural egress timing and show mostly larger intact vacuoles at 55 hours post infection (hpi). Representative images of parasites vacuoles at 44 and 55 hpi are shown. Scale bar 30 µm. g and h Phylogenetic analysis of the N- and C- terminal regions of UGO, respectively. All data is representative or mean of 3 independent biological experiments (n = 3) ± standard deviation.
Supplementary Figure 4
Alignment of full length UGO orthologue genes. Output of the sequence alignment obtained with MUSCLE was curated manually utilizing BioEdit.
Supplementary Figure 5
a. Parasites lacking UGO or CDC50.1 are not impacted in microneme (MIC2), rhoptry (ROP2/3/4), dense granule (GRA3) or constitutive secreted (SAG1) protein localization. GAP45 is used as a marker of parasite periphery. Scale bar 2 µm. b. UGO-Ty and CDC50.1-mAID-HA partially colocalize. Scale bar 2 µm. c. UGO-Ty miss localizes through the secretory pathway in absence of CDC50.1. Scale bar 2 µm. d. UGO-mAID-HA or CDC50.1-mAID-HA parasites were not impacted in their ability to replicate intracellularly. e. DMSO does not induce egress in the UGO-mAID-HA, CDC50.1-mAID-HA or parental strain parasites. f. Acidification of the extracellular environment does not induce egress in absence of digitonin. g and h. Low potassium (extracellular buffer, g) and serum (5%, h) stimulation of microneme secretion is blocked in parasites lacking GC, UGO or CDC50.1. GRA1: control for parasite viability. All data is representative or mean of 3 independent biological experiments (n = 3) ± standard deviation.
Supplementary Table 3. Antibody list
antibody Origin dilution
GAP45 Frenal et al. PlOS Pat 2014 1:10000
CATALASE Dind et al. JCS 2000 1:3000
DGK2 In house* 1:1000
GRA1 Kind gift from M.F. Lebrun 1:10
GRA3 Kind gift from JF. Dubremetz 1:10
MIC2 Kind gift from JF. Dubremetz 1:10
ACTIN Gotz et al. EMBO J 2002 1:10
TY Bastin et al. Mol Biochem Par.
1996
1:10
