A conserved ankyrin repeat-containing protein regulates conoid stability, motility and cell invasion in Toxoplasma gondii

Abstract

Apicomplexan parasites are typified by an apical complex that contains a unique microtubule-organizing center (MTOC) that organizes the cytoskeleton. In apicomplexan parasites such as Toxoplasma gondii, the apical complex includes a spiral cap of tubulin-rich fibers called the conoid. Although described ultrastructurally, the composition and functions of the conoid are largely unknown. Here, we localize 11 previously undescribed apical proteins in T. gondii and identify an essential component named conoid protein hub 1 (CPH1), which is conserved in apicomplexan parasites. CPH1 contains ankyrin repeats that are required for structural integrity of the conoid, parasite motility, and host cell invasion. Proximity labeling and protein interaction network analysis reveal that CPH1 functions as a hub linking key motor and structural proteins that contain intrinsically disordered regions and coiled coil domains. Our findings highlight the importance of essential protein hubs in controlling biological networks of MTOCs in early-branching protozoan parasites.

Fig. 1

Discovery of new proteins localized to the apical complex in T. gondii. a Comparisons for known proteins previously localized to the apical complex (left), new hypothetical proteins predicted (middle), and proteins identified to be at the apical complex (right). Patterns based on expression pattern during the cell cycle. Known proteins included: RNG1, RNG2, AKMT, MyoH, CaM1, CaM2, CaM3, MLC3, MLC5, MLC7, SAS6L. b Workflow used for identification, confirmation, and functional analysis of new apical proteins with protein numbers shown at each step. a, b See also Supplementary Table 1 and Supplementary Figs. 1, 2. c Super-resolution imaging of the essential protein CPH1 at the conoid. IFA was performed with mouse anti-Ty (green), rat anti-HA (red), and rabbit anti-MLC1 (blue) followed by secondary antibodies conjugated to Alexa Fluor dyes. Scale bar = 1 µm. d Localization of CPH1 at the apical complex by immunoelectron microscopy. Red arrows indicate distribution of gold particles along the microtubules. Scale bar = 100 nm. c, d Extracellular parasites were stimulated with 3 μm A23187 for 10 min to extend the conoid prior to processing. Images are representative of three or more experiments with similar outcomes. e Conservation of CPH1 and the apical complex in apicomplexan parasites. CPH1 orthologs (Supplementary Table 1 lists genes and species names) were used to generate the dendrogram shown (left). The chart summarizes the presence (+) and absence (−) of the conoid or polar ring and ankyrin repeats in CPH1 (present study)

Fig. 2

CPH1 is essential for parasite motility, invasion, and egress but not for microneme secretion. a Western blot analysis of CPH1 endogenously tagged with AID-3HA (α-HA) in the parental TIR1 line (shown for comparison). Aldolase (α-ALD) loading control. b Degradation of CPH1-AID triggered by addition of 500 µM auxin (+IAA) for different times (h), vs. mock, 0.1% ethanol (−IAA). Western blot performed as in a (Supplementary Fig. 3A). c Immunofluorescence microscopy of CPH1-AID parasites grown ± IAA for 24 h stained using mouse anti-HA (green) and rabbit anti-GAP45 (red). Scale bar = 2 µm. d Plaque formation with TIR1 and CPH1-AID lines grown ± IAA for 7 days. Scale bar = 0.5 cm. a–d Representative of three or more experiments with similar outcomes. e Microneme secretion following stimulation with bovine serum albumin (BSA) plus 1% ethanol for 10 min. f Parasite motility was recorded by video microscopy, manually scored, and plotted as percentage of total. ***P < 0.0001. g Parasite invasion into HFF cells was examined after 20 min challenge. ***P < 0.0001. e–g Parasite lines were grown in ±IAA for 40–44 h and extracellular parasites were collected and immediately used for assays. Mean ± SEM (n = 3 experiments, each with 3 technical replicates, n = 9). One-way ANOVA with Kruskal–Wallis test (e) or with Tukey’s multiple comparison (g) or two-way ANOVA (f) with Tukey’s multiple comparison. h–j Parasite lines were grown in ±IAA for 36 h and video microscopy was performed after addition of 2 μm A23187. h Representative example of images from three independent experiments showing time lapse recordings of DsRED diffusion (time stamp min:s). Scale bar = 20 µm. i Mean of replicates (n = 8 vacuoles) from each experiment plotted as separate lines (n = 3 experiments). j Mean of replicates (n = 44 −IAA, 53 +IAA in total) from each experiment plotted as separate lines (n = 3 experiments). ***P < 0.0001. Two-way ANOVA with Tukey’s multiple comparison. RLU relative luminescence units, ns not significant

Fig. 3

CPH1 is required for conoid stability in extracellular parasites. a Conoid morphology of intracellular parasites examined by TEM. CPH1-AID parasites grown in ±IAA for 24 h. Microtubule spirals seen in tangential sections (blue arrowhead) or cross-section (white arrow). Scale bar = 200 nm. b Conoid protrusion in extracellular parasites as assessed under phase contrast microscopy. Mean ± SEM for 3 experiments with 3 replicates for each (n = 9). DMSO served as a control. ns not significant, one-way ANOVA. c Conoid morphology of extracellular parasites examined by negative staining and TEM. Image in left bottom shows measurements for the conoid width and length (labeled as a–d). The line with arrow points to the measurements in d. Scale bar = 250 nm. d Measurement of conoid width and length as shown in c. ***P < 0.0001, mean ± SEM (n = 3 experiments, each with 3 technical replicates), n = 35 conoids were measured for each line (−IAA vs. +IAA). b–d Parasites grown in ±IAA for 40–44 h were collected and stimulated with 3 µM A23187 for 10 min prior to processing. a, c Representative of three or more experiments with similar outcomes. b, d Statistics were performed with one-way ANOVA with Tukey’s multiple comparison

Fig. 4

Ankyrin repeats target CPH1 to the conoid and are critical for function. a Model of CPH1 complementation constructs. The ankyrin repeat 1 (48–91) or ankyrin repeat 2 (429–463) were deleted from the wild-type copy CPH1-Ty to create copies of CPH1^∆ank1-Ty and CPH1^∆ank2-Ty, respectively. b Expression of stable complementing lines in the CPH1-AID line. Localization by IFA (left) following culture in ±IAA for 24 h, scale bar = 2 µm. Red arrows point to the partial conoid localization of CPH1^∆ank1-Ty. Plaque formation (right) following growth in ±IAA for 7 days (right). Scale bar = 0.5 cm. c Transient expression of CPH1^∆ank2-Ty, the CPH1-AID line. Localization as in b. d Conoid integrity in extracellular parasites examined by negative staining and TEM. Parasites were grown in ±IAA for 40–44 h, collected, and stimulated with 3 µM A23187 for 10 min to protrude the conoid. Scale bar = 250 nm. b–d Representative of three or more experiments with similar outcomes

Fig. 5

CPH1 is a central hub interacting with multiple proteins at the conoid. a Replicate mass spectrometry data sets generated with BirA fusion lines were analyzed using SFINX (http://sfinx.ugent.be/) to generate a proximity-based protein interaction network. Interactors of CPH1 and MyoH completely overlapped, and were partially shared with those for RNG2. Baits (BirA fusions)—deep blue nodes; prey (interactors)—light blue nodes. See Supplementary Data 2. b Localizations for the CPH1-MyoH-RNG2 interactome were analyzed for annotated and hypothetical proteins identified in the CPH1-MyoH-RNG2 interactome. Pie chart shows percentages of each category. The number in each category indicates protein number. N = 24 interactors. c Domains were analyzed for apical proteins and microtubule in the CPH1-MyoH-RNG2 interactome with InterPro. N = 17 interactors. b, c Protein localizations and domains for proteins in the CPH1-MyoH-RNG2 interactome were determined as defined in Supplementary Data 2. See also Supplementary Data 4, 5 and Supplementary Fig. 5 for GO term analysis. CC coiled coil, DCX double cortin microtubule-binding domain, myosin myosin A and H, prichextensin proline-rich motif present in plant cell wall, IDRs intrinsically disordered regions

Fig. 6

CPH1 depletion has a minimal effect on protein stability of known conoid proteins. a Domain arrangements of RNG2, DCX, and MyoH as determined by InterPro. CRISPR fitness scores were derived from ref. . b Intracellular parasites grown in ±IAA for 24 h were stained with mouse anti-Ty (green) to detect the tagged proteins and rabbit anti-GAP45 (red) to outline the parasite followed by secondary antibodies conjugated to Alexa Fluor dyes. Scale = 2 μm. c Extracellular parasites collected after natural or mechanical egress from cultures grown in ±IAA for 40–44 h were stained with mouse anti-Ty (green) and rabbit anti-MLC1 (red) followed by secondary antibodies conjugated to Alexa Fluor dyes. Red arrow indicates position of the conoid. Scale = 2 μm. d Extracellular parasites were treated with 3 μM A23187 to protrude the conoid, and stained with rabbit anti-HA and mouse anti-Ty antibodies, and followed with reagents for proximity ligation assay (PLA). S6SL is a pre-conoid protein. Phase, DAPI (blue) and PLA (red) are shown. Scale = 2 μm. b–d Representative of three or more experiments with similar outcomes

Fig. 7

CPH1 mediates important interaction with hypothetical unknown conoid proteins. a Analyses of interactions with CIP1, CIP2, and CIP3. Left panel: domain analysis as determined by InterPro, and domain names as shown in Fig. 5c. CRISPR fitness scores were derived from ref.. Right panels: intracellular and extracellular parasites were treated, fixed, and stained as described in Fig. 6b, c. Scale = 2 μm. b PLA for interaction of CPH1 with the CIPs was performed as described in Fig. 6d. Scale = 2 μm. a, b Representative of three or more experiments with similar outcomes. c CIP1, CIP2, and CIP3 were sequentially deleted, generating double and triple knockouts. Plaque sizes and numbers are plotted (N > 50 plaques). Mean ± SEM for 3 experiments with 3 replicates for each (n = 9). ***P < 0.0001, Kruskal–Wallis test with Dunn’s correction for multiple comparison. For details on generation of the parasite lines, see Supplementary Fig. 7. d Parasites were treated with 3 µM A23187 to protrude the conoid, prior to EM processing. Four major categories of conoid structure were identified with examples shown on left: (1) normal conoid from TIR1 parental line; (2) loss of the pre-conoid ring from ∆cip1∆cip2; (3) disrupted conoid, and (4) collapsed conoid from ∆cip1∆cip2∆cip3 (colored for the category numbers). Ratios of different categories were calculated from two independent experiments, and averages were shown (N = 20 conoids for each group). Scale = 500 nm. ns not significant