Fig 1.
Structure of the apical complex in Toxoplasma.
A. Left and middle: Drawings depicting some of the tubulin-containing structures (red) in T. gondii, including the 22 cortical microtubules, a pair of intra-conoid microtubules, as well as the 14 fibers that make up the conoid. IMC: Inner Membrane Complex. Right: Transmission electron microscopy (TEM) image of a negatively stained TritonX-100 (TX-100) extracted parasite. B. TEM images of the apical parasite cytoskeleton negatively stained after detergent extraction and protease treatment. Left: end-on view of a parasite apical cytoskeleton. Most cortical microtubules and conoid fibers have detached, which allows a clear view of the preconoidal rings lying inside the apical polar ring. Arrowheads indicate the periodic "spikes" in the preconoidal ring. Middle and right: disassembled conoids with attached preconoidal rings.
Fig 2.
Identification of candidate apical complex proteins via immunoprecipitation using GFP-Trap and eGFP-CEN2 knock-in parasites.
A. Deconvolved wide-field images of intracellular eGFP-CEN2 knock-in parasites [55] expressing mCherryFP tagged tubulin (red). Insets (shown at 2X) include the preconoidal region (arrowhead). PA: peripheral annuli; C: centrioles. B. Table showing the protein length, fitness score, average unique spectral counts, peptide counts, and sequence coverage for TGGT1_274160, TGGT1_257370, and TGGT1_231840, identified by MuDPIT in 4 replicates of immunoprecipitation using GFP-Trap and eGFP-CEN2 knock-in parasites. See S1 Table for the complete list of identified proteins. C. Deconvolved wide-field images of intracellular parasites expressing mCherryFP tagged tubulin (red) and mEmeraldFP (green) tagged Pcr1 (TGGT1_274160), Pcr2 (TGGT1_257370), or Pcr3 (TGGT1_231840) with expression driven by a T. gondii tubulin promoter. As predicted for preconoidal proteins, Pcr1, Pcr2, and Pcr3 are localized to a structure (green, insets) that is apical and smaller in diameter than the conoid (red, insets). Insets (shown at 2X) include the preconoidal region (arrowheads).
Fig 3.
Pcr2 is recruited to the preconoidal region at an early stage of daughter formation.
A. Projection of deconvolved wide-field images of intracellular Pcr2-mNeonGreen 3’ endogenous tag parasites (green) labeled with a mouse anti-IMC1 antibody and a secondary goat anti-mouse Alexa350 antibody (cyan). B. Projection of deconvolved wide-field images of TX-100 extracted extracellular Pcr2-mNeonGreen 3’ endogenous tag parasites (green) labeled with rat anti-CEN2 antibody and a secondary goat anti-rat Cy3 antibody (red). Insets (shown at 2X, contrast enhanced to display weaker signals) include the preconoidal region (arrow). Ce: centrioles. PA: peripheral annuli. C. Drawing depicting a dividing parasite with daughters developing inside the mother cell. For simplification, the cortical microtubules of the mother parasite are not shown. D. Montage showing projections of deconvolved wide-field images of intracellular Pcr2-mNeonGreen 3’ endogenous tag parasites transiently expressing mAppleFP-β1-tubulin (TUBB1, red) from a T. gondii tubulin promoter. The cortical microtubules in the mother parasites are present and clearly seen in the single slices of the 3-D stack, but not clearly visible in these projections due to the decreased contrast in the maximum intensity projection for weaker signals. Pcr2 (green) is recruited to the newly formed apical cytoskeleton as soon as the daughters are detectable. Top row: interphase parasites. Row 2–4: parasites with daughters from early to mid-stage of assembly. Insets (shown at 2X, contrast enhanced to display weaker signals) include the apical region of one of the daughter parasites indicated by arrows. Ce: centrioles. Arrowhead: a Pcr2-mNeonGreen concentration is occasionally seen in the basal region of these parasites.
Fig 4.
Generation of mEmeraldFP-Pcr2 knock-in and Δpcr2 parasites and assessment of their plaquing efficiency.
A. Left, schematic for generating mEmeraldFP-Pcr2 knock-in, Δpcr2 parasites, and the Pcr2-mNeonGreen 3’ endogenous tagged line, and Southern blotting strategy. The positions of the restriction sites, CDS probe (purple bar) and the probe annealing downstream of the pcr2 coding sequence (“3’ UTR probe”, orange bar) used in Southern blotting analysis and the corresponding DNA fragment sizes expected are shown (also see Materials and Methods for expected hybridization patterns). Right, Southern blotting analysis of the pcr2 locus in RHΔhxΔku80 (WT), mEmeraldFP-Pcr2 knock-in (KI), Δpcr2 (KO), complemented (Comp) parasites and the Pcr2-mNeonGreen 3’ tagged line (“3’tag”). The box representing the pLIC tagging plasmid is not drawn to scale. Two sets (a and b) of independently generated knock-in, knockout, and complemented lines were analyzed. B. EM examination of the apical complex in the RHΔhxΔku80 parental (WT, left column) and Δpcr2 (right column) parasites that had been incubated with the calcium ionophore A23187 (which induces conoid protrusion), followed by TX-100 treatment. Arrowheads: preconoidal rings. C. Plaques formed by RHΔhxΔku80 (WT), mEmeraldFP-Pcr2 knock-in (KI), Δpcr2 (KO), and complemented (Complement) parasite lines. Plaque assays for two independent sets of knock-in, knockout and complemented lines are shown. Nine days after inoculation, the cultures were fixed with 70% ethanol and then stained with crystal violet. “plaques” are cleared spaces where the HFF monolayers were destroyed by recurring cycles of parasite invasion, replication and egress.
Table 1.
Quantification of plaque assay for the four T. gondii strains.
s.d.: Standard Deviation. Cytolytic efficiency (CE) = (Total area of the host cell monolayer lysed by a parasite line)/(Total area lysed by the WT parasite). P-values from unpaired Student’s t-tests are indicated on the right. Pn: P-values for comparison of number of plaques. Pa: P-values for comparison of average area of plaques.
Fig 5.
Parasite replication is not affected, but egress is impaired in Δpcr2 parasites.
A. The average number of replications at 12, 24 or 36 hrs after infection in four independent experiments for RHΔhxΔku80 (WT), mEmeraldFP-Pcr2 knock-in (mE-Pcr2 KI), knockout (Δpcr2), and complemented (Comp) parasites. Error bars: standard error. B. Dot plots of time taken to disperse after treated with 5 μM A23187 for intracellular RHΔhxΔku80 (WT), mEmeraldFP-Pcr2 knock-in (mE-Pcr2 KI), knockout (Δpcr2), and complemented parasites. N: total number of vacuoles analyzed in 4 experiments, in which the parasite egress was monitored by time-lapse microscopy with 10-second intervals. *: Δpcr2 parasites in 37 out of 49 vacuoles failed to disperse from the parasitophorous vacuole during the 600 sec observation period. C. Images selected from time-lapse experiments of intracellular WT, mE-Pcr2 KI, Δpcr2, and complemented parasites treated with 5 μM A23187 (also see S2 Video). A23187 was added immediately before the beginning of the time-lapse. Red arrows indicate the positions of the parasitophorous vacuole in each image. Cyan arrows indicate some of the egressed parasites that have invaded into a new host cell.
Table 2.
Quantification of invasion for the four T. gondii strains.
The number of intracellular parasites per field was counted in ten fields per strain, in each of three independent biological replicates. s.e.: Standard error of the mean. P-values from unpaired Student’s t-tests are indicated on the right.
Fig 6.
Calcium ionophore-induced micronemal secretion is not significantly affected in Δpcr2 parasites.
A. Images selected from time-lapse experiments of intracellular RHΔhxΔku80 (WT), mEmeraldFP-Pcr2 knock-in (mE-Pcr2 KI), and knockout (Δpcr2) treated with 5 μM A23187 (also see S3 Video). The cell-impermeant DNA-binding dye, DAPI, was added to the medium to monitor the permeabilization of the host cell. Δpcr2 parasites are able to secrete effectors that lyse the host cell upon A23187 treatment, indicated by DAPI entering the host cell nucleus and binding to DNA, as well as by the dramatic change in the morphology of the host cell (see S2 and S3 Videos). Insets are DAPI images of the nuclear region of the host cell shown at 0.5X. Brackets in the mE-Pcr2 KI panels indicate the host cell nucleus included in the insets. Contrast was adjusted so that the DAPI labeling at the rim of the nucleus is easily visible. The nuclei of uninfected fibroblasts (marked by dashed circles) remained unlabeled by DAPI ~19 min after A23187 treatment as shown in the larger field of view images in the right-hand column. B. Projections of deconvolved wide-field fluorescence images of intracellular WT, mE-Pcr2 KI, Δpcr2, and complemented (Comp) parasites labeled with a mouse anti-MIC2 (red), a rat anti-GAP45 (cyan) and corresponding secondary antibodies. C. Western blots of the secreted (supernatant, S) and unsecreted (pellet, P) fractions of WT, mE-Pcr2 KI, Δpcr2, and complemented (Comp) parasites after A23187 or BAPTA-AM (a calcium chelator; negative control) treatment. The blots were probed by antibodies against MIC2 and GRA8. M: molecular weight markers, the masses of which are indicated in kDa by the numbers on the left. D. Levels of MIC2 in the secreted fractions relative to that from the wild-type in 3 independent biological replicates. For each sample, the MIC2 secretion upon A23187 stimulation is normalized against GRA8 in the pellet from the same sample. Error bars: standard error.
Fig 7.
The Δpcr2 parasite moves spasmodically in Matrigel.
A. Four examples each of wild-type and Δpcr2 parasites that displayed constriction during movement in 50% Matrigel. B. Table showing percentage of motile RHΔhxΔku80 (WT), mE-Pcr2 Knock-in (KI), Δpcr2, complemented (Comp), and Δakmt parasites in the 3-D motility assay. C. Bar graphs that show the path length and average run length of the trajectories highlighted in D. Average run length is calculated by dividing the path length by the number of runs within each trajectory. The number of runs is defined as 1+ number of sustained (> ~11 sec) pauses. D. Movement tracks for WT, Δpcr2, complemented (Comp), and Δakmt parasites generated by projections of 3-D motility timelapses. See S6 and S7 Videos. To make these 2-D images of parasites’ paths through the 3-D gel, a projection of the 3-D stack at each time point was first generated by Stack focuser (ImageJ/Fiji), then 150 consecutive timeframes in the projected sequence were compressed into a single frame. Six traces each for WT, Δpcr2, and complemented parasites are highlighted by traces drawn by hand. Some of the pauses in the parasite movement that lasted for 7 frames (~ 11 sec) or longer in these traces are indicated with arrows of the same color.
Fig 8.
Pcr2 functions differently from another motility regulator, AKMT, and Pcr2 knockout does not block actomyosin activity.
A. Images selected from time-lapse experiments of intracellular Δakmt and Δpcr2 parasites treated with 5 μM A23187. Note that Δakmt parasites, which are largely immobile, maintained their organization within the vacuole after lysis of the host cell. However the position and orientation of the Δpcr2 parasites shifted during the time lapse due to sporadic parasite movement (see S4 Video). B. Actin-chromobody-mEmerald (actin-Cb-mE) distribution before and after A23187 treatment in RHΔhxΔku80 parental (WT), Δakmt and Δpcr2 parasites. The grayscale actin-Cb-mE fluorescence images are projections of the image stack at the corresponding time point. C. Localization of AKMT in the RHΔhxΔku80 parental (WT) and Δpcr2 parasites before and after ~ 5 min 5 μM A23187 treatment. Before exposure to A23187, AKMT is concentrated at the apical end (arrows) of intracellular WT and Δpcr2 parasites. The increase in intra-parasite [Ca2+] caused by A23187 treatment triggers the dispersal of AKMT from the parasite apical end in both the parental and the Δpcr2 parasites. AKMT was labeled by immunofluorescence using a rat anti-AKMT antibody and a secondary goat anti-rat Cy3 antibody. The grayscale anti-AKMT fluorescence images are projections of deconvolved image stacks.
Fig 9.
The Δpcr2 parasite moves fitfully during invasion.
Images selected from time-lapse recording of RHΔhxΔku80 (WT, A), mEmeraldFP-Pcr2 knock-in (KI, B), knockout (Δpcr2, C-E), and complemented (Comp, F) parasites in the process of invasion or attempted invasion. D and E show two examples of abortive invasion by the Δpcr2 parasite. The frames where the invasion has completed are marked in blue. Green arrows: constrictions formed during invasion. Also see S8 Video.
Fig 10.
Δpcr2 parasites are defective in assembling the moving junction.
A. Projections of deconvolved wide-field fluorescence images of intracellular mE-Pcr2 KI (KI) and Δpcr2 parasites labeled with a mouse anti-RON4 (red), a rat anti-GAP45 (cyan) and corresponding secondary antibodies. B. Outline of a pulse invasion assay to analyze the assembly of the moving junction (marked by anti-RON4 labeling), and the differential accessibility of the intracellular vs extracellular portion of the invading parasites to antibody labeling of the SAG1 surface antigen. C. DIC and projections of deconvolved wide-field fluorescence images of RHΔhxΔku80 (WT), mEmeraldFP-Pcr2 knock-in (KI), knockout (Δpcr2) and complemented (Comp) parasites, in which SAG1 (green) RON4 (red), and GAP45 (cyan) were labeled by immunofluorescence in the pulse invasion assay described in B. Two predominant patterns are included. D. Quantification of all four SAG1 and RON4 labeling patterns observed in WT, KI, Δpcr2 and complemented (Comp) parasites from three independent biological replicates. Error bars: standard error. * P value <0.05 (unpaired Student’s t-tests), when compared with WT parasites.
Fig 11.
Simplified schematics of an invading parasite with the "engine" and "transmission" modules in the motility apparatus (inset) illustrated.
PV: parasitophorous vacuole. N: nucleus. IMC: the Inner Membrane Complex.