Identification of new components of the basal pole of Toxoplasma gondii provides novel insights into its molecular organization and functions

Abstract

The Toxoplasma gondii tachyzoite is a singled-cell obligate intracellular parasite responsible for the acute phase of toxoplasmosis. This polarized cell exhibits an apical complex, a hallmark of the phylum Apicomplexa, essential for motility, invasion, and egress from the host cell. Located on the opposite end of the cell is the basal complex, an elaborated cytoskeletal structure that also plays critical roles in the lytic cycle of the parasite, being involved in motility, cell division, constriction and cytokinesis, as well as intravacuolar cell-cell communication. Nevertheless, only a few proteins of this structure have been described and functionally assessed. In this study, we used spatial proteomics to identify new basal complex components (BCC), and in situ imaging, including ultrastructure expansion microscopy, to position them. We thus confirmed the localization of nine BCCs out of the 12 selected candidates and assigned them to different sub-compartments of the basal complex, including two new domains located above the basal ring and below the posterior cup. Their functional investigation revealed that none of these BCCs are essential for parasite growth in vitro. However, one BCC is critical for constricting of the basal complex, likely through direct interaction with the class VI myosin heavy chain J (MyoJ), and for gliding motility. Four other BCCs, including a phosphatase and a guanylate-binding protein, are involved in the formation and/or maintenance of the intravacuolar parasite connection, which is required for the rosette organization and synchronicity of cell division.

Keywords: Apicomplexa; Toxoplasma gondii; basal complex; cell-cell communication; constriction; cytoskeleton; expansion microscopy; myosin heavy chain (MHC).

Figure 1

Identification of putative MyoJ-interacting proteins using TurboID. Immuno-detection of MyoJ endogenously tagged with TurboID-3Ty (MyoJ-TurboID-3Ty) in intracellular (A) and extracellular (B) tachyzoites also expressing endogenously tagged CEN2 (CEN2-YFP). MyoJ-TurboID-3Ty localizes at the basal end of the parasite (arrowhead). Biotinylated proteins are detected using a fluorophore-conjugated streptavidin (Strep). In absence of biotin, mainly the apicoplast (arrow) of each parasite is labelled with a weak signal at the posterior pole. In contrast, after incubation with biotin, mainly the posterior pole is stained. Magenta: mouse anti-Ty, Cyan: streptavidin-AlexaFluor 594, Yellow: CEN2-YFP. Scale bars: 2 μm. (C) Western blot of biotinylated proteins, isolated with streptavidin magnetic beads after incubation of egressing ΔKU80 and MyoJ-TurboID-3Ty parasites ± biotin for 7 h. Revelation was performed using streptavidin-HRP. (D) Scatter plot of the log2(ratio) of the proteins found in the two replicates (samples 1 and 2). The bait MyoJ is shown in red, the candidates investigated in this study (CX and BCCX) are in blue, the known basal complex proteins in orange, the known IMC proteins in green, and the other known proteins in black.

Figure 2

BCCs targeted to the basal complex very late or very early during cell division. (A) Endogenous tagging of CDPK6, BCC6, and BCC7 shows that these proteins appear at the basal complex in mature parasites only. Note that CDPK6 is also found at the apical pole of the parasites including in the daughter cells. (B) In contrast, BCC2 and BCC5 are observed very early during division, appearing as a ring before the IMC1 staining of the daughter cells. Magenta: rabbit anti-IMC1, Yellow: mouse anti-Ty. Scale bars: 2 μm.

Figure 3

BCCs present to the basal complex all along the IMC elongation. Immunofluorescence assays performed on parasites expressing the endogenously tagged version of BCC1, BCC8, BCC10, and BCC11 show that all these BCCs cap the basal end of the IMC during the development of the daughter cells and in the mature parasites. Magenta: rabbit anti-IMC1, Yellow: mouse anti-Ty. Scale bars: 2 μm.

Figure 4

Mapping of the BCCs in three sub-compartments of the basal complex, including two new ones. (A) Co-localization of BCC2, BCC6, and BCC7 with endogenously tagged MyoJ and MyoC showing that these three proteins delineate a new sub-compartment located between the basal end of the IMC and the basal ring. (B) BCC1, BCC5, and CDPK6 co-localize with endogenously tagged MyoJ at the posterior cup. (C) A comparison of BCC8, BCC10, and BCC11 staining with endogenously tagged MyoJ shows that these three proteins are located below the posterior cup. Magenta: IMC1, Yellow: BCC of interest, Cyan: MyoC or MyoJ. Scale bars: vacuoles, 2 μm; zooms, 1 μm.

Figure 5

The basal complex is organized in four ring-shaped structures. (A) Co-staining of BCC1, BCC2, and BCC8 with endogenously tagged MyoJ or MyoC confirms their localization into the basal complex. BCC1 co-localizes with MyoJ at the posterior cup, while and BCC2 is located in a new sub-compartment above the basal ring (MyoC staining). Finally, co-labeling of BCC8 in a MyoJ-tagged strain corroborates the presence of a ring structure under the basal posterior cup that appears highly constricted in extracellular parasites compared to intracellular. Magenta: IMC1 or acetylated tubulin (Ac-tub), Yellow: BCC of interest, Cyan: MyoC or MyoJ. Scale bars: vacuole, 10 μm; zooms, 1 μm. (B) Co-localization of BCC10 within intravacuolar connections marked by F-actin chromobodies (Cb-ACT-GFPTy) and parasite periphery marked by GAP45. Magenta: GAP45, Yellow: BCC10, Cyan: F-actin. Scale bars: vacuole, 2 μm; zooms, 1 μm. (C) Model of the basal pole of the tachyzoite with the location of the BCCs. According to the mapping performed in this study, the basal complex is organized in at least four superimposed ring-shaped sub-compartments. The previously described basal ring and posterior cup are orange and pink, respectively.

Figure 6

BCC1 is involved in the constriction of the basal complex. (A) Localization of endogenously tagged MyoJ (MyoJ-2Ty) in the auxin-inducible knockdown of BCC1 (BCC1-mAID-3HA) assessed by immunofluorescence (IFA) after a 24 h treatment with or without 500 μM of auxin (IAA). Magenta: IMC1, Yellow: BCC1, Cyan: MyoJ. Scale bars: 2 μm. (B) Diameter of the basal pole of BCC1-mAID-3HA/MyoJ-2Ty tachyzoites ± IAA, measured on IFA pictures using the IMC1 staining. The distance between the basal edges of the IMC has been measured in 53 non-treated and 108 treated parasites. The results are represented as mean ± SD, and their significance was assessed using an unpaired t-test, with the two-tailed p-value written on the graph. (C) Electron microscopy pictures of BCC1-mAID-3HA cell line non-treated and treated with IAA for 24 h. On the panel in the middle, zoom on the basal pole. Asterisks show the parasites apical pole. C, intracellular connection; HM, host mitochondria; N, nucleus; NTN, nanotubular network. (D) The expression of BCC1-2Ty and MyoJ-2Ty was assessed in the MyoJ-mAID-3HA and BCC1-mAID-3HA cell lines, respectively, after 0, 24, and 48 h of IAA treatment. (E) Localization of endogenously tagged BCC1 (BCC1-2Ty) in the auxin-inducible knockdown of MyoJ (MyoJ-mAID-3HA) assessed by IF after a 24 h treatment with or without 500 μM of IAA. Magenta: IMC1, Yellow: BCC1, Cyan: MyoJ. Scale bars: 2 μm.

Figure 7

BCC1 depletion impairs parasite fitness and motility in vitro. (A) Plaque assays performed with BCC1-mAID-3HA/MyoJ-2Ty and the parental TIR1 strains over 7 days treated or not with auxin (IAA). (B) Competition assay performed over 5 passages (14 days) with BCC1-mAID-3HA/MyoJ-2Ty treated or not with IAA. The experiment has been done in triplicate, and the results are presented as mean ± SD. (C) Trail deposition staining after gliding of control (TIR1), BCC1-mAID-3HA/MyoJ-2Ty, AND MyoJ-mAID-3HA/BCC1-2Ty parasites treated or not for 24 h with 500 μM of IAA. Parasites were allowed to glide on poly-L-lysine-coated coverslips for 10 min in saline buffer, and trails were stained with anti-SAG1 (green) Scale bars: 10 μm. The arrows point towards helical gliding while arrowheads point to circular gliding trails. (D) Quantification of the overall parasite motility from trail deposition assays. The total number of trails (circular, helical and short) and parasites were determined for 6 to 10 fields per sample, in duplicates, and for four independent experiments. (E) Quantification of the types of motility performed by the parasites from the same fields and categorized in two groups, helical or circular. (F) Measure of the length of the helical trails on the same fields. For (D, E), the results are presented as mean ± SD; for (F) the results are presented as mean ± SEM. Their significance was assessed using an unpaired t-test with the two-tailed p-value written on the graphs. * for p<0.05, ** for p<0.01, *** for p<0.001.

Figure 8

BCC5, BCC8, BCC10, and BCC11 depletion impacts the rosette organization of the parasites and their intravacuolar connection. (A) Immuno-detection of MyoI endogenously tagged (MyoI-2Ty) in an auxin-inducible knockdown of BCC5, BCC8, BCC10, and BCC11 assessed by IFA after 30 h treatment with or without 500μM of IAA. Magenta: IMC1, Yellow: BCC of interest, Cyan: MyoI. Scale bars: 10μm. (B) Percentage of vacuoles in which the parasites were connected to each other’s. This was assessed by FRAP experiment using the soluble GFP as a reporter of diffusion. See Supplementary Figure 5 for the significance of the results (representative vacuoles for “connected” and “non-connected” as well as for the number of bleached vacuoles).