Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii

Abstract

Toxoplasma gondii encodes three protein kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 blocks parasite division. Conversely, down-regulation of PKAc1 or stabilisation of a dominant-negative PKAr isoform that does not bind cAMP triggers premature parasite egress from infected cells followed by serial invasion attempts leading to host cell lysis. This untimely egress depends on host cell acidification. A phosphoproteome analysis suggested the interplay between cAMP and cGMP signalling as PKAc1 inactivation changes the phosphorylation profile of a putative cGMP-phosphodiesterase. Concordantly, inhibition of the cGMP-dependent protein kinase G (PKG) blocks egress induced by PKAc1 inactivation or environmental acidification, while a cGMP-phosphodiesterase inhibitor circumvents egress repression by PKAc1 or pH neutralisation. This indicates that pH and PKAc1 act as balancing regulators of cGMP metabolism to control egress. These results reveal a crosstalk between PKA and PKG pathways to govern egress in T. gondii.

Keywords: Toxoplasma gondii; acylation; cAMP‐dependent protein kinase A; cGMP‐dependent protein kinase G; egress.

Figure EV1. Generation of transgenic lines expressing tagged alleles of PKAr or PKAc1

1. Cloning strategy for the introduction of 3Ty or TyDD at the C‐terminus of PKAr or PKAc1 at their respective endogenous locus.

2. PCR analysis showing correct insertion of the constructs coding for the epitope tags.

3. Multiple sequence alignment of the PKAr genes in apicomplexan parasites. In all analysed apicomplexan orthologues (T. gondii TGGT1_242070, Neospora caninum NCLIV_017370, Eimeria tenella ETH_00011940, Hammondia hamondi HHA_242070, Sarcocystis neurona SN3_01200890, Plasmodium falciparum PF3D7_1223100, P. berghei PBANKA_143800 and Cryptosporidium cgd7_120), the N‐terminus of PKAr contains the putative myristoylation and palmitoylation sites and the C‐terminal domain contains two cAMP‐binding sites/red boxes). The star in the B site indicates glycine 321 that was mutated in this study.

4. Expression of a second copy of PKArWT‐Ty or PKArG2A‐Ty does not affect parasite division. Error bars represent ±SD for 100 vacuoles counted in triplicate from three biological replicates.

Figure 1. PKA1 is targeted to the inner membrane complex (IMC) by dual acylation of PKAr

1. A, B

   Western blot analysis of ΔKu80, PKAc1‐3Ty (A) and PKAr‐3Ty (B) parasite lysates probed with αTy antibodies. Profilin (αPRF) served as loading control.

2. C, D

   Peripheral localisation of PKAc1‐3Ty (C) and PKAr‐3Ty (D) shown by IFA using anti‐Ty antibodies. Antibodies against GAP45 and IMC1 were used as markers of plasma membrane and IMC, respectively.

3. E

   Plasma membrane stained with an αSAG1 antibody is separated from the IMC stained with anti‐Ty after aerolysin treatment, indicating that PKAr‐3Ty localises at the IMC.

4. F

   The N‐terminus putative acylation site of PKAr is essential for its IMC targeting.

5. G

   A second copy of DDmyc‐PKAc1 is stabilised after 1 h in presence of Shld‐1.

6. H

   A second copy of DDmyc‐PKAc1 stabilised with Shld‐1 is targeted to the IMC in presence of a second copy of PKArWT‐Ty. When of a second copy of PKArG2A‐Ty is provided, stabilised DDmyc‐PKAc1 does not localise at the cell periphery.

Data information: Scale bars = 2 μm.

Figure 2. Down‐regulation of PKAr or overexpression of PKAc1 blocks parasite division

1. A

   Lytic growth of intracellular wild‐type Toxoplasma gondii tachyzoite stages over a period of 8 days results in plaques within monolayers of host cells. Destabilisation of PKAr‐TyDD leads to the absence of plaque formation.

2. B

   A drop in PKAr‐TyDD expression is detected 5 h after Shld‐1 removal and a significant parasite growth defect is observed as early as 12 h later. Profilin (αPRF) served as loading control.

3. C

   IFA analysis showed that the down‐regulation of PKAr‐TyDD leads to the abnormal segregation of the apicoplast‐associated thioredoxin family protein (ATrx1) 24 h after Shld‐1 withdrawal.

4. D

   Replication assay of PKAr‐TyDD parasites grown for 24 h +/‐ Shld‐1 indicates that no more than four parasites are observed per vacuole upon PKAr destabilisation (100 parasites were counted in three independent replicates).

5. E, F

   Stabilisation of a second copy of DDmyc‐PKAc1 leads to the complete block of cell growth indicated by plaque assay (E) and Western blot analysis (F).

6. G, H

   Stabilisation of a second copy of DDmyc‐PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld‐1 (100 parasites were counted in three independent replicates).

Data information: In (D, H), error bars represent ±SD for 100 vacuoles counted in triplicate from three biological replicates. Scale bars = 2 μm (C, G), 3 mm (A, E).Source data are available online for this figure.

Figure EV2. Expression of a second copy of PKArWT or mistargeted PKArG321E rescues a conditional knockdown of PKAr‐TyDD

1. A

   Conditional destabilisation of PKAr‐TyDD can be rescued by re‐addition of Shld‐1 up to 8 h after initial Shld‐1 removal (data are from three independent biological replicates).

2. B, C

   The conditional partial destabilisation of PKAr‐TyDD is successfully rescued by introduction a second copy of PKArWT‐Ty, PKArG2A‐Ty or NtGAP45PKAr‐Ty as shown by plaque assays (B) and cell division assays (C) in the absence of Shld‐1 (100 parasites were counted in three independent replicates).

3. D

   Western blot analysis of PKAr‐TyDD in presence of Shld‐1 and its derivatives complemented with the above‐mentioned PKAr variants ± Shld‐1 for 24 h. In the presence of a second copy, a partial drop of endogenous PKAr‐TyDD expression was observed upon removal of Shld‐1 while the expression levels of the second copies were significantly higher than their endogenous counterpart. Profilin (PRF) served as a loading control.

4. E

   Immunofluorescence assay indicate that PKArWT‐Ty is targeted to the IMC, PKArG2A‐Ty to the cytosol and NtGAP45PKAr‐Ty to the plasma membrane.

Data information: In (A, C), data are presented as mean ± SEM. Scale bars = 3 mm (B), 2 μm (E). Source data are available online for this figure.

Figure EV3. Generation, genotyping and phenotyping of PKAc1‐iKD parasites

1. Schematic representation of the strategy used to replace the endogenous promoter of PKAc1 by a tet‐repressive promoter to generate a PKAc1 inducible knockdown (PKAc1‐iKD).

2. PCRs performed on gDNA extracted from a clone showing the correct integration of the construct.

3. Peripheral localisation of PKAc1‐Ty‐iKD shown by IFA using an anti‐Ty antibody. An antibody against GAP45 is used as a marker of the plasma membrane. Scale bar = 2 μm.

4. Replication assay of PKAc1‐iKD. Parasites grown for 24 h ± ATc did not show any defect in intracellular growth.

5. The number of intracellular PKAc1‐Ty‐iKD parasites 30 min after invasion was not affected in presence or absence of ATc.

Data information: In (D, E), error bars represent ±SD for 100 vacuoles counted in triplicate from three biological replicates.

Figure 3. Down‐regulation of PKAc1 leads to premature egress and “restless invasion”

1. A

   A drop in PKAc1‐Ty‐iKD expression is detected as early as 24 h upon ATc treatment with almost no protein detectable after 36 h.

2. B

   Conditional down‐regulation of PKAc1‐Ty‐iKD leads to the absence of plaque formation.

3. C, D

   Down‐regulation of PKAc1‐Ty‐iKD upon ATc treatment leads to increased parasite dispersion after 40 h and premature egress (data are from three independent biological replicates).

4. E

   Prematurely egressed PKAc1‐Ty‐iKD parasites after 24 h of ATc treatment invade and exit fully lysing the monolayer of HFF cells, while the non‐treated parasites invade and initiate a new lytic cycle.

Data information: In (D), data are presented as mean ± SD. Scale bars = 1 mm (B), 20 μm (C), 2.5 mm (E). Source data are available online for this figure.

Figure 4. Overexpression of a non‐cAMP‐binding PKArG21E isoform prevents release of active PKAc1 causing premature egress and “restless invasion”

1. PKAr is predicted to possess two cAMP‐binding sites. A single glycine to glutamic acid change in the site B of the cAMP‐binding domain is known to abolish cAMP binding and to prevent activation of the holoenzyme in the presence of cAMP. In PKAr, the conserved glycine residue is Gly321.

2. Addition of Shld‐1 leads to the rapid stabilisation of DDmyc‐PKArWT‐Ty and DDmyc‐PKArG321E‐Ty.

3. PKAc1 is co‐immunoprecipitated with DDmyc‐PKArWT‐Ty in absence of cAMP but is not recovered in presence of 20 μM cAMP as revealed with [35S]‐labelled methionine/cysteine metabolic labelling. Conversely, PKAc1 is co‐immunoprecipitated with DDmyc‐PKArG321E‐Ty in presence or absence of cAMP indicating the G321E substitution prevents the release of active PKAc1 in presence of cAMP. The autoradiogram shown is representative of two independent biological replicates.

4. Conditional overexpression of a dominant‐negative DDmyc‐PKArG321E‐Ty that cannot bind cAMP leads a dramatic reduction in plaque formation while overexpression of DDmyc‐PKArWT‐Ty is well tolerated by the parasite.

5. Overexpression of the dominant‐negative DDmyc‐PKArG321E‐Ty triggers premature egress from infected host cells and prevents initiation of a new lytic cycle as revealed by IFA. Images are representative of ˜80% of the parasite population. Blue = DAPI; red = GAP45; green = GRA3.

6. In heavily infected cells, addition of Shld‐1 at 32 h post‐inoculation for 4 h leads to premature egress of DDmyc‐PKArG321E‐Ty parasites but not of the DDmyc‐PKAr‐Ty control line as assessed by IFA.

7. Quantification of extracellular DDmyc‐PKArG321E‐Ty parasites released from heavily infected cells after addition of Shld‐1 at 32 h post‐inoculation for 4 h (100 parasites were counted in three independent replicates).

8. A non‐selective PKA inhibitor, KT5270, significantly enhances the premature egress of DDmyc‐PKArWT‐Ty parasites in presence of Shld‐1 (data are from three independent biological replicates; statistical analysis was done by two‐tailed t‐test).

9. Prematurely egressed DDmyc‐PKArG321E‐Ty parasites after 4 h of Shld‐1 treatment invade and exit host cells, fully lysing the monolayer of HFF cells while the non‐treated parasites invade and initiate a new lytic cycle.

Data information: In (G, H), data are presented as mean ± SEM. Scale bars = 3 mm (D), 10 μm (E), 5 μm (F) and 2.5 mm (I). Source data are available online for this figure.

Figure EV4. Phenotypic analysis of DDmyc‐PKArG321E‐Ty and DDmyc‐PKArWT‐Ty parasites

1. Intracellular growth in presence of Shld‐1 for 24 h is not impaired in both DDmyc‐PKArG321E‐Ty and DDmyc‐PKArWT‐Ty parasites (100 parasites were counted in three independent replicates).

2. Immunoprecipitation of DDmyc‐PKArG321E‐Ty parasites. Mass spectrometry‐based analysis of the band migrating at 40 kDa recovered three peptides from PKAc1 only.

3. Conditional stabilisation of DDmyc‐PKArG321E‐Ty can be rescued by washing Shld‐1 off up to 2 h after initial Shld‐1 addition.

4. A pharmacological PKA inhibitor, H89, significantly enhances the premature egress of DDmyc‐PKArG321E‐Ty parasites in presence of Shld‐1 (data are from three independent biological replicates).

Data information: In (A, D), error bars represent ±SD for 100 vacuoles counted in triplicate from three biological replicates.Source data are available online for this figure.

Figure 5. Premature egress induced by PKA genetic inhibition is blocked by PKG chemical inhibition

1. GO term enrichment analysis of proteins differentially phosphorylated upon DDmyc‐PKArG321E‐Ty stabilisation compared with its wild‐type counterpart DDmyc‐PKArWT‐Ty. Bonferroni corrected P‐values are indicated.

2. Upon Shld‐1 addition, a putative cGMP‐specific PDE, PDE2 was more phosphorylated at Serine 1317 in DDmyc‐PKArG321E‐Ty parasites compared with DDmyc‐PKArWT‐Ty parasites suggesting a crosstalk between cAMP and cGMP signalling.

3. Chemical inhibition of the cGMP‐dependent PKG by Compound 1 and Compound 2 blocks egress induced by 5 μM BIPPO, a PDE inhibitor, or by stabilisation of DDmyc‐PKArG321E‐Ty in presence of Shld‐1. A PKGT761M substitution that renders PKG resistant to both compounds indicates that the block in egress in only mediated by PKG when 0.2 μM Compound 1 is used while the block in egress associated with C2 is not specific to PKG inhibition only (data are from three independent biological replicates).

4. Genetic inhibition of PKA triggers MIC2 secretion in intracellular buffer. Anti‐GRA1 antibodies were used as a loading control and anti‐catalase antibodies served as a lysis control (data are from three independent biological replicates; statistical analysis was done by two‐tailed t‐test).

Data information: In (C, D), data are presented as mean ± SD. Source data are available online for this figure.

Figure 6. PKA represses PKG‐dependent egress triggered by environmental acidification

1. The premature egress induced by PKA genetic inhibition is exacerbated when host cells are heavily infected (data are from three independent biological replicates).

2. Transferring supernatants of Shld‐1‐treated cultures from cells infected with DDmyc‐PKArG321E‐Ty parasites at a MOI of 8 induces egress of parasites from cells infected at a MOI of 2 within 3 h (data are from three independent biological replicates; statistical analysis was done by two‐tailed t‐test).

3. Acidification of the culture medium induces parasite egress in < 10 min. The effect is inhibited in presence of Compound 1 (data are from two independent biological replicates).

4. Neutralisation of cultures with the weak base NH₄Cl blocks premature egress induced by DDmyc‐PKArG321E‐Ty stabilisation. This block is circumvented by addition of the Ca²⁺ ionophore A23187 or the PDE inhibitor BIPPO (data are from three independent biological replicates).

Data information: Data are presented as mean ± SD.

Figure EV5. Generation and characterisation of PDE1‐3Ty, PDE2‐3Ty, GCα‐3Ty, PKG‐3Ty, ACα‐KO, Myc‐ACβ‐iKD and ACα‐KO/Myc‐ACβ‐iKD parasites

1. PCR analysis showing correct insertion of the constructs coding for the epitope tags.

2. Schematic representation of the strategy used to replace the endogenous promoter of ACβ by a Tet‐inducible promoter.

3. PCRs performed on gDNA extracted from a clone showing the correct integration of the construct.

4. Down‐regulation of Myc‐ACβ‐iKD after 8 days in presence of ATc leads to the formation of normal plaques.

5. Cloning strategy for the deletion of ACα in the Myc‐ACβ‐iKD background.

6. PCR analysis showing correct deletion of ACα in the Myc‐ACβ‐iKD background.

7. Down‐regulation of Myc‐ACβ‐iKD in the absence of ACα after 8 days in presence of ATc leads to the formation of smaller plaques compared with non‐treated parasites or ACα‐KO parasites.

Data information: Scale bars = 2.5 mm.

Figure 7. Cellular localisation of cAMP and cGMP signalling components possibly involved in egress

1. A–D

   Localisation of PDE1‐3Ty (A), PDE2‐3Ty (B), GCα‐3Ty (C) and PKG‐3Ty (D) shown by IFA using an anti‐Ty antibody. Antibodies against GAP45, IMC1, cb‐GFP and myc‐GAP70 were used as markers of the plasma membrane, IMC, residual body and apical pole, respectively.

2. E

   Western blot analysis indicates that GCα‐3Ty is present in three distinct isoforms. A PKG‐3Ty Western blot also shows three immune reactive isoforms. Catalase or actin was used as loading controls and ΔKu80 as the parental strain.

3. F

   PKG‐3Ty parasites were solubilised in either PBS or 1% Triton X‐100 (TX100) and split into soluble (S) and pellet (P) fractions. Total lysate is also shown.

4. G, H

   The signal of Myc‐ACβ‐iKD followed by IFA (G) or Western blot (H) after 48 h ± ATc.

5. I

   Invasion of ACα‐KO tachyzoites or Myc‐Acβ‐iKD after 48 h ± ATc is not affected. However, addition of ATc for 48 h reduces the invasion efficiency of ACα‐KO/Myc‐Acβ‐iKD (data are from three independent biological replicates). Error bars represent ±SD for 100 vacuoles counted in triplicate from three biological replicates.

6. J

   As for destabilisation of PKAc1‐iKD or stabilisation of DDmyc‐PKArG321E‐Ty, ACα‐KO/Myc‐Acβ‐iKD tachyzoites treated with ATc for 33 or 40 h, invade and exit the monolayer of HFF cells leading to lysis, while the non‐treated parasites invade and initiate a new lytic cycle.

Data information: Scale bars = 2 μm.Source data are available online for this figure.

Figure 8. Geography of cyclic nucleotide signalling and functional relationship between cAMP and cGMP signalling to control pH‐dependent egress

In presence of cAMP produced by ACα and ACβ, active PKAc1 is released from its regulatory subunit PKAr anchored at the inner membrane complex by dual acylation. At low pH, PKAc1 activates an unidentified PDE leading to the degradation of cGMP and down‐regulation of PKG preventing parasite egress. Upon unidentified stimuli, PKAc1 activity is repressed and/or GCα activity stimulated to increase cGMP levels and activate PKG. PKG stimulates Ca²⁺ mobilisation from intracellular store and PLC activity to produce IP₃ and DAG leading to microneme secretion and gliding motility necessary for parasite egress and invasion.