Article Figures & Data
Figures
- Figure 1. The genome of T. gondii harbors an unusual heterodimeric GC conjugated to P-type ATPase domain.
(A) The primary and secondary topology of TgATPaseP-GC as predicted using TMHMM, SMART, TMpred, Phobius, and NCBI domain search tools. The model was constructed by consensus across algorithms regarding the position of domains and transmembrane spans. The N terminus (1–2,480 aa) containing 10 α-helices resembles P-type ATPase with at least four subdomains (color-coded). The C terminus (2,481–4,367 aa) harbors two potential nucleotide cyclase catalytic regions, termed GC1 and GC2, each following six transmembrane helices. The question-marked (?) helix was predicted only by Phobius (probability score, 752). The color-coded signs on secondary structure show the position of highly conserved sequences in the ATPase and cyclase domains. The key residues involved in the base binding and catalysis of cyclases are also depicted in bold letters. (B, C) Tertiary structure of GC1 and GC2 domains based on homology modeling. The ribbon diagrams of GC1 and GC2 suggest a functional activation by pseudo-heterodimerization similar to tmAC. The model shows an antiparallel arrangement of GC1 and GC2, where each domain harbors a seven-stranded β-sheet surrounded by three α-helices. The image in panel (C) illustrates a GC1-GC2 heterodimer interface bound to GTPαS. The residues of GC2 labeled with asterisk (*) interact with the phosphate backbone of the nucleotide.
- Figure 2. TgATPaseP-GC is a constitutively expressed protein located at the apical end in the plasma membrane of T. gondii.
(A) Scheme for the genomic tagging of TgATPaseP-GC with a 3′-end HA epitope. The SacI-linearized plasmid for 3′-insertional tagging (p3′IT-HXGPRT-TgATPaseP-GC-COS-HA3’IT) was transfected into parental (RHΔku80-hxgprt−) strain followed by drug selection. Intracellular parasites of the resulting transgenic strain (Pnative-TgATPaseP-GC-HA3’IT-TgGra1-3′UTR) were subjected to staining by specific antibodies (24 h post-infection). Arrows indicate the location of the residual body. The host-cell and parasite nuclei were stained by DAPI. Scale bars represent 2 μm. (B) Immunofluorescence staining of extracellular parasites expressing TgATPaseP-GC-HA3’IT. The α-HA immunostaining of the free parasites was performed before or after membrane permeabilization either using PBS without additives or detergent-supplemented PBS with BSA, respectively. The appearance of TgGap45 signal (located in the IMC) only after permeabilization confirms functionality of the assay. TgSag1 is located in the plasma membrane and, thus, visible under both conditions. Scale bars represent 2 μm. (C) Immunostaining of extracellular parasites encoding TgATPaseP-GC-HA3’IT after drug-induced splitting of the IMC from the plasma membrane. Tachyzoites were incubated with α-toxin (20 nM, 2 h) before immunostaining with α-HA antibody in combination with primary antibodies recognizing IMC (α-TgGap45) or plasmalemma (α-TgSag1), respectively. Scale bars represent 2 μm. (D, E) Immunoblots of tachyzoites expressing TgATPaseP-GC-HA3’IT and of the parental strain (RHΔku80-hxgprt−, negative control). The protein samples prepared from extracellular parasites (107) were directly loaded onto membrane blot, followed by staining with α-HA and α-TgGap45 antibodies. Samples in panel (E) were collected at different time periods during the lytic cycle and stained with α-HA and α-TgGap45 (loading control) antibodies. COS, crossover sequence; S.C., selection cassette.
- Figure S1. Phylogenetic analysis reveals a protozoan-specific clading of TgATPaseP-GC.
The tree shows the evolutionary relationship of TgATPaseP-GC from T. gondii with orthologs from the listed organisms signifying various domains of life. The image represents a single most parsimonious cladogram generated by maximum likelihood method. Sequence alignment and construction of the tree were performed by CLC Genomics Workbench v12.0, followed by visualization using Figtree v1.4.3. The colored dots on branching nodes show the bootstrap values. Organism abbreviations and accession numbers: Arabidopsis thaliana, AAM51559.1; Ascaris suum (receptor type), PRJNA80881; Bos taurus (retinal type), NP_776973.2; B. taurus (soluble α1-subunit), P19687.1; B. taurus (soluble β1-subunit), P16068.1; Caenorhabditis elegans (receptor type), NP_494995.2; C. elegans (soluble), NP_510557.3; Drosophila melanogaster (receptor type), AAA85858.1; D. melanogaster (soluble β-subunit), NP_524603.2; E. tenella (soluble α), XP_013229212.1; E. tenella (particulate β), XP_013235760.1; Hammondia hammondi, HHA_254370; Homo sapiens (retinal type), NP_000171.1; H. sapiens (soluble α1-subunit), NP_001124157.1; H. sapiens (soluble β1-subunit), NP_001278880.1; Musca domestica (receptor type), XP_005177218.1; M. domestica (soluble), XP_019895151.1; Mus musculus (retinal type), NP_001007577.1; M. musculus (soluble α1-subunit), AAG17446.1; M. musculus (soluble β1-subunit), AAG17447.1; Oryza sativa, ABD18448.1; Oxytricha trifallax, EJY85073.1; Paramecium tetraurelia, XP_001346995.1; P. falciparum (soluble α), AJ245435.1; P. falciparum (particulate β), AJ249165.1; Prunus persica, AGN29346.1; Tetrahymena pyriformis, AJ238858.1; T. gondii, EPR59074.1; Toxocara canis (receptor-type), KHN81453.1; and T. canis (soluble), KHN85312.1.
- Figure S2. The alignment of P-type ATPase-like domains of TgATPaseP-GC, PfGCα, and PfGCβ with the members of human P4-ATPases.
The human P4-ATPases were aligned by MAFFT alignment server (v7) (Katoh & Standley, 2013), and then merged with the aligned ATPase domains of TgATPaseP-GC, PfGCα, and PfGCβ using MAFFT merge. The alignment was trimmed, as indicated by horizontal lines, and signature residues were color-coded with the Clustal Omega program (>30% sequence conservation). Selected amino acids that are essential for ATP binding and phosphorylation, and transmembrane helices are boxed in black. Transmembrane topology of ATP8A1 retrieved from TMHMM Server (v2) (Sonnhammer et al, 1998) was used as a reference. Organism abbreviations and accession numbers: P-type ATPase-like domain of TgATPaseP-GC, T. gondii (EPR59074.1); PfGCα, P. falciparum (AJ245435.1); PfGCβ, P. falciparum (AJ249165.1); and P4-ATPases of Homo sapiens: ATP8A1 (Q9Y2Q0.1), ATP8B4 (Q8TF62.3), ATP9A (O75110.3), ATP10A (O60312.2), and ATP11C (Q8NB49.3).
- Figure S3. The sequence alignment of GC1 and GC2 domains from TgATPaseP-GC with other representative cyclases identifies signature residues.
Amino acid sequences were aligned with the Clustal Omega program. The color-coding refers to conserved residues. The secondary structures depicted underneath the alignment correspond to the type III tmAC (UniProt-Protein Data Bank ID: 1AZS, Oryctolagus cuniculus for GC1, Rattus norvegicus for GC2; DSSP hydrogen bond estimation algorithm). In type III cyclases, seven residues are involved in cofactor binding for the catalysis, which are indicated above the alignments as “Me” for metal, “B” for base, “Pγ” for phosphate, “R” for ribose, and “Tr” for transition state binding. Note that alignment of GC1 and GC2 domains from TgATPaseP-GC to their orthologous GCs/ACs showed that unlike other cyclases, a 74-residue-long segment (3,033–3,107 aa) is inserted between α3 and β4 of GC1 (not shown). Organism abbreviations and accession numbers: TgATPaseP-GC, T. gondii (UniProt; S7VVK4); PfGCα, P. falciparum (AJ245435.1); PfGCβ, P. falciparum (AJ249165.1); PtGC, Paramecium tetraurelia (XP_001346995.1); TpGC, Tetrahymena pyriformis (AJ238858.1); soluble MmGCα, Mus musculus (AAG17446.1); soluble MmGCβ, M. musculus (AAG17447.1); retinal type MmGC2, M. musculus (NP_001007577.1); type II RntmAC, Rattus norvegicus (P26769.1); and type V OctmAC, Oryctolagus cuniculus (CAA82562.1).
- Figure S4. Expression of TgATPaseP-GC1 and GC2 domains in the M15 and BTH101 strains of E. coli.
(A) Scheme depicting the molecular cloning of GC1, GC2, and GC1+GC2 domains in the pQE60 expression vector. The open reading frames of indicated domains were amplified starting from the first upstream start codon (ATG) using the tachyzoite-derived RNA and ligated into BglII-digested pQE60 plasmid. Proteins were fused with a C-terminal 6xHis-tag for subsequent detection by immunoblot and purification by virtue of the Ni-NTA column. (B) PCR screening of transgenic M15 strains showing ORF-specific amplicons of GC1 (1,185 bp), GC2 (927 bp), and GC1+GC2 (4,176 bp). (C) Immunoblot of purified GC1-6xHis and GC2-6xHis proteins (5 μg) using the mouse α-His antibody. The protein bands of 47 and 38 kD correspond to GC1 and GC2 domains, respectively. Purification was performed under denaturing conditions from the M15 strain, GC1+GC2 could not be purified. (D) HPLC elution profile of cGMP and GTP after GC assay performed with bacterial lysates expressing GC1, GC2, or GC1+GC2 domains. Elution profiles of GTP and cGMP standards are also depicted (retention time for GTP ∼5 min, cGMP ∼7.5 min). Analyte elution was recorded by absorbance at 260 nm (mAU; milli-Absorbance Units). HPLC was performed on C18 reversed-phase column (3 μm particle size, 15 cm × 4.6 mm; SUPELCOSIL LC-18-T; Sigma-Aldrich) with a flow rate of 1 ml/min (sample injection volume, 20 μl). (E) Functional testing of GC1, GC2, and GC1+GC2 domains in the BTH101 strain, deficient in cAMP signaling. Transgenic bacterial strains expressing GC1, GC2, GC1+GC2, EcCyaA (positive control), or harboring the empty pQE60 vector (negative control) were grown in LB medium (OD600, 1.6; 37°C), and then dilution plated on MacConkey agar containing 1% maltose (carbon source) and 200 μM IPTG (inducer) followed by incubation at 30°C (∼32 h). The appearance of red colonies indicates cAMP-dependent catabolism of disaccharide after a functional expression of EcCyaA, but not others.
- Figure 3. Genetic disruption of TgATPaseP-GC is lethal to tachyzoites of T. gondii.
(A) Scheme for the CRISPR/Cas9-mediated disruption of the gene in parasites expressing TgATPaseP-GC-HA3’IT. The guide RNA was designed to target the nucleotides between 145 and 164 bp in the progenitor strain (Pnative-TgATPaseP-GC-HA3’IT-TgGra1-3′UTR). Image shows the loss of HA signal in some vacuoles after CRISPR/Cas9-cleavage. Parasites were transfected with the pU6-TgATPaseP-GCsgRNA-Cas9 vector and then stained with α-HA and α-TgGap45 antibodies at specified periods. Scale bars represent 20 μm. (B) Quantitative illustration of TgATPaseP-GC-HA3’IT–disrupted mutant parasites from panel (A). The HA-negative vacuoles harboring at least two parasites were scored during successive passages (P1–P3). (C) The replication rates of the HA+ and HA− tachyzoites, as evaluated by immunostaining (panel A). About 500–600 vacuoles were enumerated for the parasite numbers per vacuole (n = 3 assays).
- Figure 4. Cre recombinase–mediated down-regulation of cGMP synthesis impairs the lytic cycle of T. gondii.
(A) Schematics for making the parasite mutant (Pnative-TgATPaseP-GC-HA3’IT-3′UTRexcised). A vector expressing Cre recombinase was transfected into the progenitor strain (Pnative-TgATPaseP-GC-HA3’IT-3′UTRfloxed), in which 3′UTR of TgATPaseP-GC was flanked with Cre/loxP sites. Parasites transfected with a vector expressing Cre recombinase were selected for the loss of HXGPRT selection cassette (S.C.) using 6-thioxanthine. (B) Genomic screening of the TgATPaseP-GC mutant confirming Cre-mediated excision of 3′UTR and HXGPRT. Primers, indicated as red arrows in panel (A), were used to PCR-screen the gDNA isolated from four different mutant clones (C1–C4) along with the progenitor strain. (C) Immunoblot showing repression of TgATPaseP-GC-HA3’IT in parasites with excised 3′UTR with respect to the progenitor and parental (RHΔku80-hxgprt−) strains. Parasites (107) were subjected to the dot blot analysis using α-HA and α-TgGap45 (loading control) antibodies. (D) Immunostaining of the mutant (TgATPaseP-GC-HA3’IT-3′UTRexcised) and progenitor parasites revealing loss of HA signal in the former strain. Parental strain was used as a negative control for the background staining. Parasites were stained with α-HA and α-TgGap45 antibodies 24 h postinfection. Scale bars represent 2 μm. (E) Changes in the steady-state cGMP level of the mutant compared with the parental and progenitor strains. Fresh syringe-released parasites (5 × 106) were subjected to ELISA-based cGMP measurements (n = 4 assays). (F) Plaque assays using the TgATPaseP-GC mutant, progenitor, and parental strains. The dotted white areas and blue staining signify plaques and intact host-cell monolayers, respectively (left). The area of each plaque (arbitrary units, a. u.) embodies the growth fitness of indicated strains. 150–200 plaques of each strain were evaluated (right) from three assays. **P ≤ 0.01; ****P ≤ 0.0001.
- Figure S5. Efficiency of Cre recombinase–mediated 3′UTR excision in TgATPaseP-GC mutant.
Efficiency of 3′UTR excision was quantified by scoring the loss of HA signal in the TgATPaseP-GC-HA3’IT-3′UTRexcised mutant, which was generated as described in Fig 4, and immunostained with α-HA and α-TgGap45. 300 vacuoles containing parasites with or without HA signal were counted from three independent IFAs (mean with SEM).
- Figure 5. cGMP signaling governs the key events during the lytic cycle of T. gondii.
(A–D) In vitro phenotyping of the TgATPaseP-GC mutant, its progenitor, and parental strains. The intracellular replication (A), host-cell invasion (B), parasite egress (C), and gliding motility (D) were assessed using standard phenotyping methods. The progenitor and mutant strains were generated as shown in Figs 2A and 4A, respectively. The replication rates were analyzed 24 and 40 h postinfection by scoring the parasite numbers in a total of 500–600 vacuoles after staining with α-TgGap45 antibody (panel A, left) (n = 4 assays). The average parasite numbers per vacuole is also depicted (panel A, right). Invasion and egress rates were calculated by dual staining with α-TgGap45 and α-TgSag1 antibodies. In total, 1,000 parasites of each strain from four assays were examined to estimate the invasion efficiency. The natural egress of tachyzoites was measured after 40, 48, and 64 h by scoring 500–600 vacuoles of each strain (n = 3 assays). To estimate the gliding motility, fluorescent images stained with α-TgSag1 antibody were analyzed for the motile fraction (500 parasites of each strain), and 100–120 trail lengths per strain were measured (n = 3 assays). (E) Effect of PKG inhibitor compound 2 (2 μM) on the motility of TgATPaseP-GC mutant and its progenitor strain (500 parasites of each strain, n = 3 assays). A total of 100 trails in the progenitor, and 15 trails of the mutant (due to severe defect) were measured. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; and ****P ≤ 0.0001.
- Figure S6. Pharmacological inhibition of phosphodiesterases can repair phenotypic defects in the TgATPaseP-GC mutant.
(A) Gliding motility of the Pnative-TgATPaseP-GC-HA3’IT-3′UTRexcised mutant and progenitor (Pnative-TgATPaseP-GC-HA3’IT-3′UTRfloxed) strains in the presence of two known phosphodiesterase inhibitors, zaprinast (500 μM), and BIPPO (55 μM). Fresh syringe-released parasites were treated with drugs for 15 min during the assay, followed by fixation and staining with α-TgSag1 antibody. 500–600 parasites of each strain were evaluated for the motile fraction, and 100–120 trail lengths were measured by ImageJ (n = 3 assays, mean with SEM). (B, C) Egress and invasion rates of indicated parasite strains after exposure to zaprinast and BIPPO. Intracellular and extracellular parasites were differentially stained with α-TgGap45 and α-TgSag1 antibodies, as shown in the Materials and Methods section. For egress, parasitized cells (MOI, 1; 40 h postinfection) were stimulated with either zaprinast (500 μM) or BIPPO (55 μM) for 5 min 30 s before fixation and staining. Drug treatment during invasion assay was performed for 1 h. In total, 500–600 vacuoles and 1,000 parasites were scored for each strain in panels (B) and (C), respectively (n = 3 assays, mean with SEM). Statistics was performed for individual pair of columns using t test (*P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001).
- Figure S7. C-terminal epitope tagging and Cre recombinase–mediated knockdown of TgPKG in T. gondii.
(A) 3′-insertional tagging of the TgPKG gene with an HA epitope (step 1) and subsequent deletion of loxP-flanked (floxed) 3′UTR by Cre recombinase (step 2). The construct for 3′-insertional tagging (3′IT) via shown crossover sequence (COS) was transfected into the parental strain (RHΔku80-hxgprt−) and drug-selected for the HXGPRT selection cassette (S.C.). The eventual progenitor strain (Pnative-TgPKG-HA3’IT-3′UTRfloxed) expressed TgPKG-HA3’IT under its own regulatory elements. In the second step, the progenitor strain was transfected with a vector expressing Cre recombinase (pSag1-Cre) to cutoff the floxed 3′UTR and HXGPRT by negative selection, resulting in repression of TgPKG. (B) Genomic screening PCR validating the integrity of the TgPKG mutant generated by the excision of 3′UTR. Primers indicated as red-colored arrows in panel (A) were used to test gDNAs isolated from the mutant clones (C1–C3) and progenitor strain. The mutagenesis was further confirmed by sequencing of amplicons (1.9 kb). (C) Efficiency of 3′UTR excision in the Pnative-TgPKG-HA3’IT-3′UTRexcised mutant. The parasite vacuoles with or without HA signal were evaluated after staining of the mutant with α-HA and α-TgGap45, as shown in Fig 6A (350 vacuoles from n = 3 assays; mean with SEM).
- Figure 6. Mutagenesis of TgPKG recapitulates the phenotype of the TgATPaseP-GC mutant.
(A–F) Phenotyping of the TgPKG mutant in comparison with its progenitor and parental strains. For making of the mutant, refer to Fig S7. (A) Images demonstrating the expression of TgPKG-HA3’IT in the progenitor strain, and its down-regulation in the mutant. The parental strain served as a negative control. Intracellular parasites (24 h postinfection) were stained with α-HA and α-TgGap45 antibodies. The merged image includes DAPI-stained host and parasite nuclei in blue. Scale bars represent 2 µm. (B) Immunoblot depicting the expression of TgPKG isoforms in clonal mutants (C1 and C2) along with the progenitor and parental strains. Extracellular tachyzoites (2 × 107) of each strain were subjected to protein isolation followed by immunoblotting with α-HA antibody. Expression of 112 and 135-kD isoforms in the progenitor and mutants, but not in the parental strain, confirms efficient 3′-HA tagging and successful knockdown of the protein, respectively. TgRop2 served as loading control. (C) Plaque assay revealing comparative growth of the mutant, progenitor, and parental strains. The plaque area is shown in arbitrary units (a. u.). A total of 140–170 plaques of each strain were scored from three assays. (D) Replication rates of indicated strains during early (24 h) and late (40 h) cultures. Tachyzoites proliferating in their vacuoles were stained with α-TgGap45 antibody and counted from 400 to 500 vacuoles for each strain (n = 3 assays). (E, F) Invasion and egress of the designated parasites as judged by dual-color staining. Intracellular tachyzoites were immunostained red using α-TgGap45 antibody, whereas extracellular ones appeared two-colored (red and green), stained with both α-TgGap45 and α-TgSag1 antibodies. In total, 1,000–1,200 parasites were evaluated to score the invasion rate of each strain (n = 5 assays). The percentage of ruptured vacuoles at indicated periods was determined by observing 400–500 vacuoles of each strain from three experiments. (G) Motile fraction and trail lengths of the indicated parasite strains. Extracellular parasites immunostained with α-TgSag1 were analyzed for the motile fraction (600 parasites) and trail lengths (100 parasites) (n = 3 assays). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; and ****P ≤ 0.0001.
- Figure S8. Inhibition of residual activity in the TgPKG-HA3’IT mutant by compound 2 augments the motility defect.
Motile fraction and trail lengths of the Pnative-TgPKG-HA3’IT-3′UTRexcised mutant and progenitor (Pnative-TgPKG-HA3’IT-3′UTRfloxed) strains, generated according to Fig S7, were measured by staining with α-TgSag1 antibody after compound two treatment (2 μM, 15 min). A total of 500 parasites for each strain were analyzed to calculate the motile fraction. In total, 100 trails were measured for the progenitor strain, whereas only 30 trails could be evaluated for the mutant because of severe defect (n = 3 assays, mean with SEM). Statistical significance was calculated for each pair of column individually by t test. (*P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001).
Supplementary Materials
