Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma
- Department of Biochemistry, University of Cambridge, United Kingdom
Figures
Figure 1 with 1 supplement
TgLMBD3 is a conserved protein in the plasma membrane at apical annuli sites in T. gondii.
(A) Wide-field immunofluorescence assay imaging of cells expressing TgLMBD3-6xHA (magenta) and eGFP-Centrin2 (green) and immunostained inner membrane complex (IMC)1 (blue) in either the intracellular stage in hosts or extracellular tachyzoites. Scale bar=5 µm. (B) Membrane topology of TgLMBD3 by DeepTMHMM (Hallgren et al., 2022) with numbers indicating amino acid domain lengths. (C) Trypsin-shaving sensitivity over 4 hr, visualised on western blots, of TgLMBD3-6xHA and markers of the exterior leaflet of the plasma membrane (PM), IMC, cytosol (PRF, profilin), and mitochondrion (mito.). kDa, kilodalton. (D) Maximum likelihood phylogeny of limb development membrane protein 1 domain (LMBD) proteins resolving as four major orthogroup clades. Node support values are bootstraps followed by aLRT SH-like supports. (E) Expanded Alveolata clade from LMBD orthogroup III showing major groups. Black dots indicate aLRT SH-like support >0.95. See Figure 1—figure supplement 1 for full phylogenies.
Figure 1—figure supplement 1
Maximum likelihood phylogenies of the LMBR1 domain-containing proteins from Figure 1.
(A) All LMBR1 domain-containing proteins can be divided into four different orthogroups. Numbers at branches are bootstrap/aLRT SH-like supports for the monophylies of the four orthogroups. Sequences of the Plasmodium spp. from the orthogroup I are at the base of the orthogroup and outside of the cohort of sequences that represent the other Apicomplexa, including TgLMBD3. This is likely caused by a long-branch attraction (LBA) artefact, which pulls the fast-evolving Plasmodium sequences towards the sequences from the other orthogroups. Nevertheless, the placement of these Plasmodium sequences within the orthogroup I is well supported. The LBA artefact is diminished by computing the phylogenetic tree from an alignment of the sequences solely from the orthogroup I (B). Here, Plasmodium spp. is within the monophyletic Apicomplexa. Furthermore, monophyly of Alveolata (Ciliates+Myzozoa[Apicomplexa, Chrompodelids, Perkinsids, Syndiniales, Dinoflagellates]) and monophyly of SAR (Stramenopiles, Alveolata, Rhizaria) clade are also supported, suggesting vertical evolution of the LMBD3 gene from LECA (last eukaryotic common ancestor) into current eukaryotic lineages. aLRT SH-like supports are shown only for the relevant branches that constitute higher order taxa annotated in the figure.
Figure 2
Apical annuli occur at gaps in the inner membrane complex (IMC).
Three-dimensional structured illumination microscopy (3D-SIM) imaging of immunofluorescence assays of intracellular tachyzoites in host cells. (A) TgLMBD3-6xHA (magenta) and eGFP-Centrin2 (green) expressing cells with side (s) and top (t) projections of apical annuli shown in zoom. IMC1 (blue). (B) Suture protein ISC3-3xV5 (green) co-expressed with TgLMBD3-6xHA (magenta) showing apical annuli positioned at the apical cap suture where it intersects with longitudinal sutures (arrows). IMC1 (blue). (C, D) Optical sections showing GAP45 or IMC1 (green) with TgLMBD3-6xHA (magenta) showing gaps in these IMC proteins where apical annuli occur. All scale bars=2 µm or 200 nm for zoomed panels.
Figure 3
TgLMBD3 is recruited to the apical annuli late in cytokinesis.
Three-dimensional structured illumination microscopy (3D-SIM) imaging of immunofluorescence assays of intracellular tachyzoites at various stages of daughter cell formation and emergence from the mother cell. Maternal annuli are indicated with arrows. TgLMBD3-6xHA (magenta) co-labelled with apical cap marker ISP1 (green) and inner membrane complex (IMC)1 (blue). Scale bar=2 µm.
Figure 4
Three SNARE proteins likely form a complex at the inner side of the plasma membrane at the apical annuli.
(A) Schematic of the SNARE complex which facilitates fusion of a secretory vesicle with target membrane (Jahn and Scheller, 2006). (B) Wide-field fluorescence microscopy localisation of TgStxPM, TgNPSN, and TgSyp7 SNAREs expressed in T. gondii (as 3xV5 N-terminal fusions) and co-stained for eGFP-Centrin2. All panels are in the same magnification with scale bar=5 μm. (C) Three-dimensional structured illumination microscopy (3D-SIM) image of TgNPSN with Centrin2. The zoomed panels show the apical annuli either in side (s) or top (t) projection as indicated. The scale bars for large and small (zoomed) panels are 2 μm and 200 nm, respectively.
Figure 5 with 1 supplement
Depletion of apical annuli plasma membrane proteins impairs replication rates of T. gondii.
(A). Depletion of each apical annuli protein shown in hours of 3-indolacetic acid (IAA) auxin treatment observed by anti-V5 western blots. Histone H3 serves as a loading control, and molecular weight markers in kDa are shown. (B) Plaque assays in host cell monolayers showing plaque development over 8 days in knockdown cell lines for each apical annuli protein without (control) or with IAA-induced protein knockdown. (C) Replication states of T. gondii parasitophorous vacuoles 24 hr post invasion scored according to parasite number per vacuole. Each protein knockdown cell line is assayed either without (control) or with IAA-induced protein knockdown. Significant statistical differences between vacuole types are indicated by p-values *<0.05; **<0.01; ***<0.001, error bars = standard deviation.
Figure 5—figure supplement 1
Western blot and PCR analysis validating correct epitope-tag integration.
(A) Western blots of cell lysates using anti-v5 antibody shows TgNPSN, TgStxPM, and TgSyp7 were successfully tagged with 3xV5. (B) PCR showing successful integration of epitope tag in the intended locus for each apical annuli SNARE protein. For TgNPSN, TgStxPM, TgSyp7, a universal forward primer that anneals in the promoter region is used together with a gene-specific reverse primer to obtain a PCR product. For TgLMBD3, a gene-specific forward primer is used together with a reverse primer annealing in the terminator region. Products obtained are the expected size for each reaction. Parental cell lines for the transfection are used as a negative control.
Figure 6 with 2 supplements
Secretion of dense granule proteins into the parasitophorous vacuole is inhibited when apical annuli membrane proteins are knocked down.
Wide-field immunofluorescence assays of (A) GRA5, (B) GRA1, and (C) GRA2 without (control) or with auxin-induced protein knockdown for the four apical annuli membrane proteins. Assays for GRA5 were performed using digitonin permeabilisation to preferentially stain the secreted protein only (see Figure 6—figure supplement 1 for further examples). Assays for GRA1 and GRA2 were performed with Triton X-100 permeabilisation to visualise the non-secreted dense granules as well as the secreted GRAs. Scale bar = 5 µm.
Figure 6—figure supplement 1
Wide-field images of parasitophorous vacuoles stained for GRA5 after depletion of the four apical annuli proteins.
Immunofluorescence assays for GRA5 without (control) or with auxin-induced protein knockdown for the four apical annuli membrane proteins. Cells were pre-treated with auxin to induce complete annuli protein knockdown then seeded onto human foreskin fibroblast (HFF) monolayer on coverslips and allowed to proliferate for 24 hr. Coverslips were permeabilised with digitonin. Scale bar=10 µm.
Figure 6—figure supplement 2
Abundance and distribution of rhoptry and microneme proteins are unaffected when apical annuli membrane proteins are knocked down.
Wide-field immunofluorescence assays of MIC2 and ROP1 without (control) or with auxin-induced protein knockdown for the four apical annuli membrane proteins. Assays for MIC2 and ROP1 were done 24 hr after invasion. Scale bar=5 µm.
Figure 7
Knockdown of apical annuli membrane proteins results in accumulation of dense granule proteins in the parasite.
(A) Volcano plots showing the changes of abundance of cell proteins with three apical annuli proteins depleted over 24 hr of auxin treatment compared to untreated controls (N=3). Black dots represent measures for individual cell proteins other than dense granule proteins, red and orange dots indicate dense granule proteins assigned by hyperLOPIT (red=statistically significant changes, orange=non-significant changes, using adjusted p-values) (Barylyuk et al., 2020). (B) The 15 most significantly increased proteins with each apical annuli protein knockdown amount to 26 common proteins randomly sampled by the shotgun proteomics, all but 6 of which are known dense granule proteins. Red bracketed numbers indicate GRAs, black bracketed numbers non-GRAs. FC, fold change; protein nb., VEuPathdb gene identifiers; hypoth., hypothetical protein; DG, dense granule; EV, endomembrane vesicles; PM, plasma membrane; NA, not assigned a location by hyperLOPIT. See Supplementary file 2 for full quantitative proteomics data.
Additional files
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Supplementary file 1
BioID supplementary data file.
Sheet 1: BioID_significant_changes: Columns show ToxoDB accession number, log2-fold change (logFC) with left and right confidence intervals (CI.L, CI.R), the average log2-abundance value of this protein across treatments and their replicates (AveExpr), the moderated t-statistics value (t), the raw p-value (P.Value), the adjusted p-value (adj.P.Val), and the log-odds that the protein is differentially abundant (B). Sheet 2: Normalised TMT intensity values for three LMBD3-BirA* biological replicates (RUN1-3) each with three parental control samples.
- https://cdn.elifesciences.org/articles/94201/elife-94201-supp1-v2.xlsx
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Supplementary file 2
Quantitative proteomics supplementary data file.
Sheets 1–3 give data for the three cell lines: TgLMBD3-mAID-3xV5, mAID-3xV5-TgNPSN, and mAID-3xV5-TgSyp7. Columns show ToxoDB accession number, number of peptides, log2-median-aligned protein abundances for all replicates in knockdown (KD_rep1-rep3) and control treatment (control_rep1-rep3), treatment means and standard deviation (SD), effect size (Cohen’s D), statistical power for two-sided t-test at p=0.01, log2-fold change between treatment and control (logFC), the average log2-abundance value of this protein across treatments and their replicates (AveExpr), the moderated t-statistics value (t), the raw p-value (P.Value), the adjusted p-value (adj.P.Val), the log-odds that the protein is differentially abundant (B), the protein description (Description), and the TAGM-predicted subcellular location according to the ToxoLOPIT map of Barylyuk et al., 2020.
- https://cdn.elifesciences.org/articles/94201/elife-94201-supp2-v2.xlsx
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Supplementary file 3
Primers and plasmids for genetic modifications.
- https://cdn.elifesciences.org/articles/94201/elife-94201-supp3-v2.xlsx
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Supplementary file 4
Antibodies used for immunofluorescence assays (IFAs) and Western blots.
- https://cdn.elifesciences.org/articles/94201/elife-94201-supp4-v2.xlsx
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Supplementary file 5
R markdown file of analytical workflow of quantitative proteomics data.
The markdown file contains the pipeline from the peptide-to-spectrum match (PSM)-level input data obtained from Proteome Discoverer. It provides an overview on structure and quality of the raw data (chunk 1–5), explores missing data structure and protein coverage across experiments (chunk 6–7), aggregates the psm-level data to proteins (chunk 8), and creates the linear model fits using Limma Bayes algorithms (chunk 10). The last two chunks (11+12) create the output data files and Volcano plots submitted with this manuscript.
- https://cdn.elifesciences.org/articles/94201/elife-94201-supp5-v2.pdf
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MDAR checklist
- https://cdn.elifesciences.org/articles/94201/elife-94201-mdarchecklist1-v2.pdf
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Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma
eLife 13:e94201.
https://doi.org/10.7554/eLife.94201
