Protein kinase A negatively regulates Ca2+ signalling in Toxoplasma gondii

Abstract

The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating stages and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is complete to allow for replication to begin is not understood. Here, we show that the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that PKAc1 is sequestered to the parasite periphery by dual acylation of PKA regulatory subunit 1 (PKAr1). Upon genetic depletion of PKAc1 we show that newly invaded parasites exit host cells shortly thereafter, in a perforin-like protein 1 (PLP-1)-dependent fashion. Furthermore, we demonstrate that loss of PKAc1 prevents rapid down-regulation of cytosolic [Ca2+] levels shortly after invasion. We also provide evidence that loss of PKAc1 sensitises parasites to cyclic GMP (cGMP)-induced Ca2+ signalling, thus demonstrating a functional link between cAMP and these other signalling modalities. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.

Fig 1. Toxoplasma PKAc1 localises to the tachyzoite periphery likely by dual acylation of its regulatory subunit PKAr1.

(A) Localisation of HA-tagged PKAc1 by IFA, co-staining with the peripheral marker IMC1 (i) and the apical IMC marker ISP1 [30] (ii). (B) Identification of PKAc1-interacting proteins by anti-HA immunoprecipitation, whole eluate tryptic digestion, mass spectrometry, and label-free quantification. Red and green lines signify 2-fold and 4-fold enrichment, as compared to the parental line (see S1 Table for full list of identified proteins). (C) Identification of PKAc1-interacting proteins by anti-HA immunoprecipitation, SYPRO Ruby staining of eluates followed by in-gel digest and mass spectrometry. Table denotes proteins and coverage identified in each band. (D) Western blot of Ty epitope-tagged PKAr1 (TGME49_242070). (E) Localisation of Ty epitope tagged PKAr1 by IFA, co-stained with either (i) IMC1 antibodies or (ii) the antibodies to the apical IMC protein ISP1, outlining localisation to the developing daughter cells. (F) Mutational analysis of putative myristoylation and palmitoylation sites at the N-terminus of PKAr1. (i) The first 15 amino acids of PKAr1 (PKAr1(1–15)_WT) were fused to GFP and colocalised with GAP45 and (ii) ISP1, showing localisation to the periphery and to the developing daughter cells, respectively. (iii) Putative myristoylation site—glycine at position 2—was mutated to alanine (PKAr1(1–15)_G2A), (iv) putative palmitoylated cysteine residues at positions 5 and 7 were mutated to alanine (PKAr1(1–15)_C5AC7A). (v) Both putative myristoylation and palmitoylation sites were mutated (PKAr1(1–15)_G2AC5AC7A). In each case, localisation of GFP fusions were compared to the peripheral marker GAP45 and counterstained with DAPI. (G) Western blot on PKAr1(1–15)_WT-GFP and point mutants. C5AC7A, cysteine-to-alanine-mutations at amino acid 5 and 7; GAP45, glideosome-associated protein 45; GFP, green fluorescent protein; G2A, glycine to alanine mutation at amino acid 2; HA, haemagglutinin; IFA, immunofluorescence assay; IMC, inner membrane complex; IP, immunoprecipitation; ISP1, IMC subcompartment protein 1; PKAc1, protein kinase A catalytic subunit 1; PKAr1, protein kinase A regulatory subunit 1; WT, wild type.

Fig 2. Generation and characterisation of a conditional knockdown of Toxoplasma PKAc1.

(A) Schematic representation of tet-off promoter replacement-based conditional knockdown of PKAc1 (PKAc1 cKD). Details of genetic strategy and validation of PKAc1 cKD are available in S1 Fig. (B) Western blot of down-regulation of PKAc1 levels upon ATc treatment at 24 hours and 48 hours and comparison to parental line. HA antibodies were used to detect PKAc1 and GAP45 antibodies were used as a loading control. (C) IFA of PKAc1 upon ATc treatment for 24 hours and 48 hours. (D) Plaque assay of parental and PKAc1 cKD and parental lines ± ATc upon infection of confluent HFF monolayers. (E) Images of in vitro cultures of HFFs infected with PKAc1 cKD tachyzoites after for 24 hours growth ± ATc. White arrows point to intact intracellular parasite vacuoles and black arrows show examples of unhealthy and detached HFFs. Scale bars = 50 μm. More images are available in S2 Fig. ATc, anhydrotetracycline; cKD, conditional knockdown; DHFR, dihydrofolate reductase; GAP45, glideosome-associated protein 45; HA, haemagglutinin; HFF, human foreskin fibroblast; IFA, immunofluorescence assay; PKAc1, protein kinase A catalytic subunit 1.

Fig 3. PKAc1-deficient Toxoplasma tachyzoites damage host cells upon invasion.

(A) Static invasion assay of parental and PKAc1cKD tachyzoites. Data are the mean ± SEM of three biological replicates. For each replicate, parasites in 16 panels, each with an area of 211.3 × 198.1 μm, were counted at 63× magnification. P values are calculated using an unpaired two-tailed t test, where ** ≤ 0.005. (Bi and ii) Live cell imaging of parental strain ± ATc and (iii) PKAc1 cKD without ATc treatment before, during, and after invasion of host HFFs. White arrowheads point to invading tachyzoites and dashed white lines signify the border of the target HFF before and after invasion. Time in minutes and seconds is displayed across the bottom of panels. Stills correspond to S1, S2, and S3 Movies, respectively. Scale bar = 10 μm. (Ci and ii) Live cell imaging of two representative PKAc1-depleted tachyzoites (+ATc) before, during, and after invasion of HFFs. As above, white dashed lines outline host cell border pre-invasion, whilst red border denotes HFF outline post-invasion. White arrowheads signify tracked tachyzoites. Time in minutes and seconds is displayed across the bottom of panels. Stills correspond to S4 and S5 Movies. Scale bar = 10 μm. (D) Quantification of time to host cell collapse in Parental and PKAc1-depleted lines. Greater than 600 signifies that host cell collapse was not observed over the 10-minute (600-second) filming period. Data represent mean ± 95% confidence intervals; Parental + ATc, n = 20; PKAc1-ATc, n = 12; PKAc1 + ATc, n = 26 across at least three experiments. P values are calculated using an unpaired two-tailed t test, where **** ≤ 0.0001. (E) Integrity of host cells invaded for 1 hour by parental and PKAc1cKD lines, ±ATc as measured as a function of tachyzoite number versus crystal violet absorbance. Data represent mean ± SEM of four technical replicates. P values are calculated using a two-way ANOVA, where * ≤ 0.05 only at highest tachyzoite concentration. Individual numerical values underlying (A), (D), and (E) may be found in S1 Data. ATc, anhydrotetracycline; cKD, conditional knockdown; HFF, human foreskin fibroblast; PKAc1, protein kinase A catalytic subunit 1; PKAc1cKD, protein kinase A catalytic subunit 1 conditional knockdown.

Fig 4. PLP-1 is required for host cell damage in PKAc1-deficient tachyzoites.

(A) Genetic deletion of PLP-1 (Δplp1) in PKAc1 cKD, as confirmed by western blot analysis. See S5 Fig for genetic strategy and genotyping. (B) Morphology of PKAc1 and PKAc1/PLP-1-deficient tachyzoites during in vitro growth in HFF after 24 hours of ATc treatment. White arrows point to intracellular tachyzoites after several rounds of replication. Black arrow point to examples of collapsed host cells. More images in S2 Fig. (C) Static invasion assay (after 10 minutes) of PKAc1 cKD/Δplp1 and Δplp1 tachyzoites as compared to PKAc1 cKD (reproduced from Fig 3A). (D) Effect of treatment with DCCD on host cell invasion of parental and PKAc1 cKD +ATc. Data from C and D represent mean ± SEM from three biological replicates. P values are calculated using an unpaired two-tailed t test, where * ≤ 0.05, ** ≤ 0.005, **** ≤ 0.00005, and ns = not significant. (E) Quantification of time to host cell collapse in Parental and PKAc1 cKD and PKAc1 cKD/Δplp1 tachyzoites ± ATc and ± 50 μM DCCD. Greater than 600 signifies that host cell collapse was not observed over the 10-minute (600-second) filming period. Parental + ATc, PKAc1 ± ATc data are reproduced from Fig 3 for purposes of comparison. Data represent mean ± 95% confidence intervals over at least three independent experiments, in which PKAc1 + ATc + 50 μM DCCD, n = 16; PKAc1/Δplp1 − ATc, n = 7; PKAc1/Δplp1 + ATc, n = 12. Individual numerical values underlying (C), (D), and (E) may be found in S1 Data. ATc, anhydrotetracycline; cKD, conditional knockdown; DCCD, N,N′-Dicyclohexylcarbodiimide; HA, haemagglutinin; HFF, human foreskin fibroblast; ns, not significant; PKAc1, protein kinase A catalytic subunit 1; PLP-1, perforin-like protein 1; SAG1, surface antigen 1.

Fig 5. PKAc1 controls the rapid down-regulation of cytosolic Ca²⁺ shortly after invasion.

(A) Live cell imaging of Parental tachyzoites with ATc treatment, (B) PKAc1 without ATc treatment, and (C) PKAc1 treated with ATc, all stably expressing GCaMP6f/mCherry at the uprt locus. In A, B, and C, (i) outlines images of individual movies, showing when parasites are extracellular, invading, and upon completion of invasion (blue arrow) at ‘baseline’ (defined as when GCaMP/mCherry ratio reaches 35%) (see accompanying S7, S8, and S9 Movies, respectively). In each case, (ii) shows a graphical representation of tracked parasites in (i), where blue line denotes the moment of completion of invasion and green arrow shows the moment of host cell egress. Dotted line marks 35% of maximum GCaMP/mCherry ratio, defined as ‘baseline’. In each case, (iii) shows overlays of all traced parasites. S6, S7 and S8 Figs show individual traces of each tracked parasite, respectively. (D) Graphical representation of each tracked parasite across all conditions showing time to ‘baseline’ fluorescence (defined as 35% of maximum). (E) Graphical representation of normalised GCaMP/mCherry ratio at t = 100 seconds across all parasites in all conditions, and in the case of PKAc1 cKD +ATc, segregated depending on time of egress. Data in D and E are represented as mean ± 95% confidence interval. P values are calculated pairwise, using an unpaired two-tailed t test, where * ≤ 0.05; **** ≤ 0.0001. Individual numerical values underlying (D) and (E) may be found in S1 Data. ATc, anhydrotetracycline; cKD, conditional knockdown; GCaMP, GFP-Calmodulin-M13-peptide-6; PKAc1, protein kinase A catalytic subunit 1.

Fig 6. PLP-1 activity does not affect defective Ca²⁺ dynamics upon loss of PKAc1.

(A) Live cell imaging of PKAc1 cKD/Δplp1 without and (B) with ATc. (C) PKAc1 cKD treated with ATc and 50 μM DCCD. All parasite lines are stably expressing GCaMP6/mCherry at the uprt locus. In A, B, and C, (i) shows images of the individual representative movie, showing stills when parasites are extracellular, invading, and upon completion of invasion (blue arrow) and then when the normalised GCaMP/mCherry ratio reaches 35% of maximum (see S10, S11 and S12 Movies, respectively). (ii) Shows, in each case, a graphical representation of tracked parasites in (i), where blue arrow denotes the moment of completion of invasion and green arrow shows the moment of host cell egress. Dotted line marks 35% of maximum GCaMP/mCherry ratio, defined as ‘baseline’. (iii) Shows, in each case, overlays of all traced parasites. S9 Fig shows individual traces of each tracked parasite for PKAc1 cKD/Δplp1, with and without ATc, and S10 Fig outlines individual traces of PKAc1 cKD + ATc + 50 uM DCCD. (D) Graphical representation of each tracked parasite across all conditions, showing time to reach 35% of maximum, which is defined as ‘baseline’. Parental and PKAc1 cKD lines are from Fig 5. (E) Graphical representation of normalised GCaMP6/mCherry ratio at t = 100 seconds across all parasites in all conditions. Data in D and E are represented as mean ± 95% confidence interval. P values are calculated pairwise, using an unpaired two-tailed t test, where **** ≤ 0.0001. Individual numerical values underlying (D) and (E) may be found in S1 Data. ATc, anhydrotetracycline; cKD, conditional knockdown; DCCD, N,N′-Dicyclohexylcarbodiimide; GCaMP, GFP-Calmodulin-M13-peptide-6; PKAc1, protein kinase A catalytic subunit 1; PLP-1, perforin-like protein 1.

Fig 7. Cytosolic [Ca²⁺] are negatively regulated by PKAc1 in extracellular tachyzoites.

(A) Measurements of intracellular Ca²⁺ using Fura-2 upon increasing concentration of extracellular Ca²⁺. Data represent mean ± SEM; statistical significance was assessed using unpaired t test. (B) FACS-based quantitation of GCaMP6/mCherry mean fluorescent intensity of Parental and PKAc1 cKD ± ATc in both IC and EC buffers. Values are from at least three independent replicates and are represented using box and whisker plots. P values are calculated pairwise using a paired two-tailed t test, where **** ≤ 0.0001. Individual numerical values underlying (A) and (B) may be found in S1 Data. ATc, anhydrotetracycline; cKD, conditional knockdown; EC, extracellular; FACS, fluorescence activated cell sorting; GCaMP6, GFP-Calmodulin-M13-peptide-6; IC, intracellular; PKAc1, protein kinase A catalytic subunit 1.

Fig 8. Loss of PKAc1 expression causes a greater sensitivity to cGMP-induced rise in cytosolic [Ca²⁺].

FACS-based analysis of GCaMP6-expressing (A) Parental and (B) PKAc1 cKD tachyzoites’ responses over time to a serial dilution of BIPPO in the absence (i) and presence (ii) of ATc. Data represent a single representative experiment. (C) Baseline-corrected dose–response curves at t = 50 seconds of GCaMP6-expressing Parental and PKAc1 cKD, ±ATc stimulated with either (i) BIPPO or (ii) A23187. Data represent mean ± SEM of at least three independent biological replicates. P values are calculated using a permutation test of the difference between two groups of growth curves. The P value is the proportion of permutations, where the mean t is greater in absolute value than the mean t for the original data set. Individual numerical values underlying (C) may be found in S1 Data. ATc, anhydrotetracycline; BIPPO, 5-benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one; cGMP, cyclic GMP; cKD, conditional knockdown; FACS, fluorescence-activated cell sorting; GCaMP6, GFP-Calmodulin-M13-peptide-6; MFI, mean fluorescent intensity; PKAc1, protein kinase A catalytic subunit 1; TATi, transactivator Toxoplasma inducible.