Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species

Abstract

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

Fig 1. Conoid complex features in Toxoplasma tachyzoites.

(A) Schematic of the recognised components of the conoid and their location within the apical structures of the cell pellicle in either retracted or protruded states. (B–E) Transmission electron micrographs of T. gondii tachyzoites with conoid either retracted (B, C) or protruded (D, E). Tubulin filaments of the conoid walls are evident in tangential section (E) and 2 CRs of the conoid canopy are evident at the conoid’s anterior end (D, E). The conoid is surrounded by 2 APRs (P1 and P2) formed by the anterior aspect of the IMC and anchoring the subpellicular microtubules (Mt), respectively. RDs can be seen running to the apex through conoid (E). Scale bars represent 1 μm (B, D) and 100 nm (C, E). A, apicoplast; APR, apical polar ring; C, conoid; CR, conoidal ring; G, Golgi apparatus; IMC, inner membrane complex; M, micronemes; Mi, mitochondrion; Mt, microtubule; Nu, nucleus; R, rhoptries; RD, rhoptry duct; V, plant-like vacuole.

Fig 2. Apically targeted BioID using baits RNG2, SAS6L, and new apical cap protein MORN3.

(A) Immunodetection of HA-tagged MORN3 in T. gondii intracellular parasites costained for apical cap-marker ISP1. Upper panels show mature cells; lower panels show internal daughter pellicle buds forming during the early stages of endodyogeny. Scale bar = 5 μm. (B) Steptavidin-detection of biotinylated proteins after 24 hours of growth with elevated biotin. Native biotinylated proteins ACC1 and pyruvate carboxylase are seen in the parental control (lacking BirA*) and in the BioID bait cell lines. Additional biotinylated proteins are seen in each of the bait cell lines grown with elevated biotin, including self-biotinylation of the bait fusion. ACC1, Acetyl-CoA carboxylase; BioID, proximity-dependent biotin identification; HA, hemagglutinin; MORN3, Membrane Occupation and Recognition Nexus 3; SAS6L, SAS6-like.

Fig 3. Super-resolution imaging of T. gondii proteins at the conoid body and base.

Immunodetection of HA-tagged conoid proteins (green) in cells coexpressing either APR marker RNG2 or conoid marker SAS6L (magenta) imaged either with conoids retracted with parasites within the host cell, or with conoids protruded in extracellular parasites. (A) Example of protein specific to the conoid body and (B) examples of proteins specific to the conoid base. See S2 and S3 Figs for further examples. All panels are at the same scale, scale bar = 5 μm, with zoomed inset from white boxed regions (inset scale bar = 0.5 μm). Dashed white lines indicate the cell boundary. APR, apical polar ring; HA, hemagglutinin; SAS6L, SAS6-like.

Fig 4. Super-resolution imaging of T. gondii proteins at the conoid canopy rings and MORN2 at the plasma membrane.

(A) Examples of proteins specific to the conoid canopy rings. (B) Peripheral membrane protein (cytosolic leaflet) MORN2 in intracellular parasites. Immunodetection of HA-tagged proteins as for Fig 3. See S3 Fig for further examples. All panels are at the same scale, scale bar = 5 μm, with zoomed inset from white boxed regions (inset scale bar = 0.5 μm). HA, hemagglutinin; MORN2, Membrane Occupation and Recognition Nexus 2.

Fig 5. Super-resolution imaging of T. gondii proteins at conoid canopy puncta and the apical polar rings.

Immunodetection of HA-tagged proteins as for Fig 3. (A) Examples of protein specific to the conoid canopy puncta. (B) Examples of proteins specific to the apical polar rings in the vicinity of APR1 (TGME49_208340) and APR2 (TGME49_320030). See S3 and S4 Figs for further examples. All panels are at the same scale, scale bar = 5 μm, with zoomed inset from white boxed regions (inset scale bar = 0.5 μm). APR1, apical polar ring 1; APR2, apical polar ring 2; HA, hemagglutinin.

Fig 6. Heatmap indicating conservation of conoid-associated proteins among Alveolata.

Presence (red, orange) and absence (white) of putative orthologues of 54 T. gondii conoid-associated proteins (Table 1) in 157 surveyed Alveolata species (see S5 Fig for taxa, S3 Table for orthologue numbers, and S1 Data for orthologue sequences). ToxoDB protein numbers (left) and existing protein names (right) are shown. In case of a presence, the taxon either contains at least one homologous sequence that has the T. gondii protein as its best BLASTp match (red) or it has only homologous sequences that were obtained via sensitive HMMer searches but that did not retrieve a T. gondii match by BLASTp (orange), indicative of more divergent homologues (see Methods). The proteins are shown clustered according to their binary (presence-absence) patterns across the Alveolata. Known protein locations in T. gondii are indicated by colour (see key) where “apex” indicates low-resolution imaging of an apical punctum only. The species tree (top) shows phylogenetic relationships and major clades: Piropl., Piroplasmida; Crypt., Cryptosporidium; Greg., Gregarinasina; green shading, Perkinsozoa; brown shading, Colponemidia. Columns for species of interest are darkened and indicated by a triangle at the bottom of the figure (A–P)–A: Toxoplasma gondii; B: Sarcocystis neurona; C: Eimeria tenella; D: Plasmodium berghei; E: Plasmodium falciparum; F: Babesia bovis; G: Theileria parva; H: Nephromyces sp.; I: Cryptosporidium parvum; J: Chromera velia; K: Vitrella brassicaformis; L: Symbiodinium microadriaticum; M: Perkinsus marinus; N: Tetrahymena thermophila; O: Stentor coeruleus; P: Colponemid sp.. For each species the source of the protein predictions is indicated: genome (DNA, green) or transcriptome (RNA, dark red), along with BUSCO score as estimates of percentage completeness. AA, apical annuli; APR, apical polar ring; CCP, conoid canopy punctum; CCR, conoid canopy ring.

Fig 7. Live-cell widefield and super-resolution imaging of P. berghei ookinetes expressing GFP fusions of conoid complex orthologues.

T. gondii orthologue locations are shown in Figs 3–5. (A) Widefield fluorescence imaging showing GFP (green), Hoechst 33342-stained DNA (grey), and live cy3-conjugated antibody staining of ookinete surface protein P28 (magenta). (B, C) 3D-SIM imaging of fixed GFP-tagged cell lines for conoid orthologues (B) or APR orthologues (C) with same colours as before (A). Inset for APR protein (1334800) shows rotation of the 3D-reconstruction to view the parasite apex face on. All panels are at the same scale, scale bar = 5 μm, with zoomed inset from yellow boxes (inset scale bar = 0.5 μm or 1 μm for 1334800). 3D-SIM, three-dimensional structured illumination microscopy; APR, apical polar ring; DIC, differential interference contrast; GFP, green fluorescent protein.

Fig 8. Ultrastructure of conoid complexes of P. berghei zoites.

Transmission electron micrographs of P. berghei zoites: ookinetes (A–F), sporozoites (G–J), and bloodstream merozoites (K–M). (A) Longitudinal section through an ookinete showing the apical complex with micronemes (M) plus the crystalline body (Cr). Insert: Detail of the apical cytoplasm showing a microneme (M) with a duct running towards the anterior (arrows). (B–E) Details of longitudinal and tangential sections through the apical complex with either 2 or 3 CRs evident with the anterior collar consisting of an outer electron-dense layer (cd) closely adhering to the IMC which forms the anterior polar ring (P1) and an inner electron-lucent layer (cl) which is closely associated with subpellicular microtubules (Mt) which forms the inner polar ring (P2). Underlying micronemes (M) with ducts (D) extend to the cell apex. F. Cross section through part of the apical collar showing the ookinete plasma membrane (pl) with the underlying IMC closely adhering to the electron-dense layer of the collar (cd) with the more electron-lucent region (cl) closely associated with subpellicular microtubules (Mt). (G) Longitudinal section through a sporozoite showing the anteriorly located rhoptries (R) and micronemes (M) and the central nucleus (Nu). (H, I). Detail of the anterior of the mature sporozoites showing the CRs and the in-folding of the IMC to form the first APR (P1) with second APR beneath (P2) associated with the subpellicular microtubules (Mt). Note the angle formed by the apical polar rings relative to the longitudinal cell axis. (J) Longitudinal section of an early stage in sporozoite formation showing apical CRs and the perpendicular projection of the CRs and APRs. (K) Longitudinal section through a spherical-shaped merozoite released from an erythrocyte showing the rhoptries (R), micronemes (M), and nucleus (Nu). (L, M) Enlargement of the apical region showing the CRs and the closely positioned polar rings (P1, P2). Scale bars represent 1 μm (A, G, K) and 100 nm in all others. See also S6 and S7 Figs. APR, apical polar ring; CR, conoidal ring; IMC, inner membrane complex.

Fig 9. Live-cell widefield and super-resolution imaging of P. berghei sporozoites expressing GFP fusions of conoid complex orthologues.

(A) Widefield fluorescence imaging showing GFP (green) and Hoechst 33342-stained DNA (grey). All panels are at the same scale, scale bar = 5 μm, with the exception of zoomed images from white boxed regions in the merge. (B, C) Super-resolution imaging of GFP-fused conoid complex proteins (green) in fixed cells shown with the cell surface stained for sporozoite surface protein CSP (magenta). All panels are at the same scale, scale bar = 5 μm, with zoomed inset from white boxed regions (inset scale bar = 0.5 μm). CSP, circumsporozoite protein; DIC, differential interference contrast; GFP, green fluorescent protein.

Fig 10. Live-cell imaging of P. berghei merozoites expressing GFP fusions of conoid complex orthologues.

Widefield fluorescence imaging showing GFP (green) and Hoechst 33342-stained DNA (grey) with some parasites seen pre-egress from the erythrocyte and others post egress. All panels are at the same scale, scale bar = 5 μm shown, with zoomed inset from white boxed regions (inset scale bar = 2 μm). DIC, differential interference contrast; GFP, green fluorescent protein.

Fig 11. Conservation and variability of the conoid complex in apicomplexan zoite forms.

Schematics of cell apices from Toxoplasma and Plasmodium showing presence of common structures but displaying variability in their size and arrangement. Toxoplasma is shown with either the conoid retracted or protruded. A row of vesicles of unknown function lines the intraconoidal microtubules in Toxoplasma and other coccidians. Schematics draw on both TEM and EM tomography data that is presented or cited throughout the report. APR1, apical polar ring 1; APR2, apical polar ring 2; EM, electron microscopy; IMC, inner membrane complex; TEM, transmission electron micrograph.