doi: 10.1073/pnas.2219533120.
Epub 2023 Jan 24.
Invasion of Toxoplasma gondii bradyzoites: Molecular dissection of the moving junction proteins and effective vaccination targets
Affiliations
- PMID: 36693095
- PMCID: PMC9945962
- DOI: 10.1073/pnas.2219533120
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Invasion of Toxoplasma gondii bradyzoites: Molecular dissection of the moving junction proteins and effective vaccination targets
Rania Najm et al.
Proc Natl Acad Sci U S A.
.
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Abstract
Toxoplasmosis is a neglected parasitic disease necessitating public health control. Host cell invasion by Toxoplasma occurs at different stages of the parasite's life cycle and is crucial for survival and establishment of infection. In tachyzoites, which are responsible for acute toxoplasmosis, invasion involves the formation of a molecular bridge between the parasite and host cell membranes, referred to as the moving junction (MJ). The MJ is shaped by the assembly of AMA1 and RON2, as part of a complex involving additional RONs. While this essential process is well characterized in tachyzoites, the invasion process remains unexplored in bradyzoites, which form cysts and are responsible for chronic toxoplasmosis and contribute to the dissemination of the parasite between hosts. Here, we show that bradyzoites invade host cells in an MJ-dependent fashion but differ in protein composition from the tachyzoite MJ, relying instead on the paralogs AMA2 and AMA4. Functional characterization of AMA4 reveals its key role for cysts burden during the onset of chronic infection, while being dispensable for the acute phase. Immunizations with AMA1 and AMA4, alone or in complex with their rhoptry neck respective partners RON2 and RON2L1, showed that the AMA1-RON2 pair induces strong protection against acute and chronic infection, while the AMA4-RON2L1 complex targets more selectively the chronic form. Our study provides important insights into the molecular players of bradyzoite invasion and indicates that invasion of cyst-forming bradyzoites contributes to cyst burden. Furthermore, we validate AMA-RON complexes as potential vaccine candidates to protect against toxoplasmosis.
Keywords: Toxoplasma; bradyzoite; invasion; moving-junction; vaccine.
Conflict of interest statement
The authors declare no competing interest.
Figures
RON2, RON4, RON4L1, RON5, and RON8 decorate the MJ of bradyzoites. A. In vivo transcriptome analysis of T. gondii during acute and chronic infection and prediction of T. gondii ron2, ron2L1, ron2L2, ron4, ron4L1, ron5, ron8, ama1, ama2, and ama4 genes expression during acute and chronic infection (31). B. IFA on Pru bradyzoite cysts following in vitro induction of conversion, using anti-AMA1, anti-RON2, anti-RON4, anti-RON5, anti-RON8 (red) antibodies, anti-p21 (cyan), and lectin DBA-FITC labeling cyst wall. DIC, differential interference contrast. (Scale bars, 5 μm.) C. Staining of HA3-RON4L1 with anti-HA antibodies (red) and DBA-FITC (green) (Left). Colocalization of RON4L1 (green) with RON2 (red) (Right). DIC, differential Interference contrast. (Scale bars, 5 μm.) D. IFAs performed on 76 K bradyzoites invading HFF cells using anti-RON2 (red), anti-RON4 (red), anti-RON5 (green), and anti-RON8 (green) antibodies. For IFA of RON4L1, bradyzoites are extracted from the brains of mice infected with the Pru HA3-RON4L1-tagged line and RON4L1 revealed with the anti-HA3 antibody (green). The arrows point to the ring of the MJ when they are halfway the invasion process, and the arrowheads point the residual dot of the MJ when the parasites are at the end of the invasion process. The letter "a" shows the location of the apical end of the parasite. DAPI stains the nucleus, which is very basal in bradyzoite. E. IFAs on newly invaded Pru tachyzoites (Upper) and bradyzoites extracted from brain cysts (Lower) in HFF monolayer using anti-AMA1 (red) and anti-RON5 (green) antibodies. Same exposure time for tachyzoites and bradyzoites. The asterisk marks the bulk of unsecreted AMA1 present in micronemes at the apical end of parasite F and G. IFAs of intracellular 76 K bradyzoites after 5 min (F) and 30 min (G) of contact with the HFF monolayer using anti-RON5 (green) and anti-RON2 (red), and anti-RON5 (green) and anti-ROP1 (red). Higher exposure of ROP1 staining is labeled "ROP1exp". Arrows correspond to trails at the posterior end of invaded bradyzoites. (D–G) Scale bars, 2 μm.
AMA2 and AMA4 are associated with the MJ of invading bradyzoites. A. IFAs on in vitro Pru cysts using anti-AMA4 (red), anti-HA (red) for staining of AMA2-HA3, and anti-p21 (cyan) antibodies. Cyst wall was stained using DBA-FITC (green). (Scale bars, 5 μm.) B. IFAs on newly invaded Pru tachyzoites using anti-AMA4 (red), anti-HA (red) for staining of AMA2-HA3 and anti-RON4 (green) antibodies. C. IFAs on Pru AMA2-HA3 tagged bradyzoites at the beginning (Upper), halfway point (Middle), or end of the invasion (Lower) using anti-AMA2 (green), and anti-RON5 (red) antibodies. D. IFAs on 76 K bradyzoites at the beginning (Upper), halfway point (Middle), or end of the invasion (Lower) using anti-AMA4 (red), and anti-RON5 (green) antibodies. (B–D) IFAs were performed after 3 min of contact of parasites with HFF cells, except for Lower panels of C and D that correspond to 10-min invasion. The asterisk marks the bulk of unsecreted AMA proteins present in micronemes at the apical end of parasite. The arrows point to the ring of the MJ when they are in the process of invasion, and the white arrowheads point the residual dot of the MJ when the parasites are at the end of the invasion process. The letter "a" shows the location of the apical end of the parasite. Yellow arrows show the trails of proteins behind the parasite. (B–D) Scale bars, 2 μm.
AMA4 is involved in chronic toxoplasmosis in cystogenic Me49∆ku80 strain. A. Immunoblot with anti-AMA4 antibodies of lysates from Me49∆ku80 (Ctrl), Me49∆ku80 KO-AMA4, and Me49∆ku80 KO-AMA4 complemented with a copy of ama4 (Compl.). Actin was used as a loading control. B. Plaque assays of HFFs monolayers infected with either Me49∆ku80 (Ctrl) or Me49∆ku80 KO-AMA4 tachyzoites and grown for 14 d. Quantification of the size of the lysis plaques. Student’s t test, no difference. Results are presented as mean ± SD (n = 3 biological replicates, each with two technical replicates). The biological replicates are represented by different symbols. C. Invasion assay for 5 min of contact of Me49∆ku80 (Ctrl) or Me49∆ku80 KO-AMA4 tachyzoites with fibroblast monolayers. Student’s t test, no significant difference. Results are presented as mean ± SD of three independent experiments, each with three technical replicates. The biological replicates are represented by different symbols. D. In vivo virulence of Me49∆ku80 (Ctrl) or Me49∆ku80 KO-AMA4 after intraperitoneal injection of 200 tachyzoites in BALB/c mice. Results are presented as the pool of two independent experiments, each with five mice per group. E. Percentage of cyst load in brains of Swiss mice 1 mo after intraperitoneal infection of 200 tachyzoites of Me49∆ku80 (Ctrl), Me49∆ku80 KO-AMA4, and complemented KO-AMA4 (Compl.). Results are presented as mean ± SD of cyst brains (n = 3 biological replicates for control and KO-AMA4 and n = 2 biological replicates for complemented line). The biological replicates are represented by different symbols. Student’s t test, ****P < 0.0001 (10 mice per group).
Immunization of C57BL/6 mice with rAMA–ROND3 complexes protect against toxoplasmosis. A. Schematic representation illustrating the vaccination strategy of C57BL/6 mice receiving 40 μg of rAMA peptides or 40 μg of the rAMA–RON2D3 complex with the adjuvant or the adjuvant alone as a control. Mice were immunized subcutaneously three times, at a 2-wk interval between injections. The first immunization was performed in Freund’s complete adjuvant, followed by two injections in Freund’s incomplete adjuvant. At week 5 (W5), mice were challenged intraperitoneally by a lethal dose of 1,000 Pru tachyzoites and their mortality was monitored until week 10 (W10) or challenged by oral route with 15 cysts of the type II 76 K strain and brain cyst was quantified 1 mo after challenge. B. ELISA showing levels of total IgG in mice immunized with rAMA1 or rAMA1–RON2D3 as compared to nonimmunized mice. Student’s t test, ***P < 0.001; results are presented as mean ± SD of one experiment (sera of nine individual mice per group), representative of two independent immunizations (the second experiment is shown in SI Appendix, Fig. S4A ). C. IFAs on intracellular RH strain using control sera or sera from mice immunized with rAMA1 or rAMA1–RON2D3 (green), or anti-AMA1 (red) antibodies. D. Western blot of RH (WT) and RH KO-AMA1 tachyzoites using control sera or sera from mice immunized with rAMA1 or rAMA1–RON2D3. Antibodies to Dictyostelium actin recognizing T. gondii actin was used as loading control. (Scale bars, 4 μm.) E. Percentage of survival in C57BL/6 mice immunized with rAMA1 and rAMA1–RON2D3 as compared to control mice upon an intraperitoneal challenge with a lethal dose of tachyzoites. Log-rank test. *P < 0.05, ***P < 0.001. These results are representative of one experiment out of two independent experiments (seven mice per group). The second experiment is shown in SI Appendix, Fig. S4 . F. Percentage of cyst load in brains of C57BL/6 mice immunized with rAMA1 and rAMA1–RON2D3 as compared to control mice after 1 mo of challenging with 15 cysts by gavage. Two brains were pooled for quantification of cysts. Control: out of 20 immunized mice, 14 survived the challenge. rAMA1: out of 10 immunized mice, 6 survived. rAMA1–RON2D3: out of 10 immunized mice, all survived. Results are presented as mean ± SD of one experiment for immunization with rAMA1 and representative of two individual immunizations with rAMA1–RON2D3. The second experiment is shown in panel J. Student’s t test, **P < 0.01, ***P < 0.001. G. Western blot of Me49∆ku80 (WT), Me49∆ku80 KO-AMA1, and KO-AMA1 complemented (Compl.) tachyzoites using control sera or sera from mice immunized with rAMA4, rAMA4–RON2L1D3. Antibodies to Dictyostelium actin recognizing T. gondii actin was used loading control. H. Percentage of survival in C57BL/6 mice immunized with rAMA4 and rAMA4–RON2L1D3 compared to control mice upon an intraperitoneal challenge with a lethal dose of tachyzoites. Log-rank test *P < 0.05. The depicted results are those of one representative experiment out of two independents (seven mice per group). The second experiment is shown in SI Appendix, Fig. S4 . I. Percentage of cyst load in brains of C57BL/6 mice immunized with rAMA4, and rAMA4–RON2L1D3 compared to control mice after 1 mo of challenging with 15 cysts by gavage. Two brains were pooled for quantification of cysts. Control is the same as in panel F. rAMA4: out of 10 mice immunized, six survived. rAMA4–RON2L1D3: out of 10 mice immunized, eight survived. Results are presented as mean ± SD of one experiment for immunization with rAMA4 and representative of two individual immunizations with rAMA4–RON2L1D3. The second experiment is shown in panel J. Student’s t test, ***P < 0.001. J. Percentage of cyst load in brains of C57BL/6 mice immunized with rAMA1–RON2D3, rAMA4–RON2L1D3 or both complexes compared to control mice after 1 mo of challenging with 15 cysts by gavage. Control: out of 10 immunized mice eight survived to challenge. rAMA1–RON2D3: out of 10 immunized mice, eight survived. rAMA4–RON2L1D3: out of 10 immunized mice, eight survived. rAMA1–RON2D3 + rAMA4–RON2L1D3: out of 10 immunized mice, all survived. Student’s t test, ***P < 0.001; results are presented as mean ± SD (10 mice per group). Two brains were pooled for quantification of cysts.
Antibodies generated upon immunization with rAMA–ROND3 inhibit parasites’ invasion. A. Invasion inhibition assay of RH tachyzoites incubated with 1 mg/mL total IgG extracted from sera of mice immunized with rAMA or rAMA–RON2D3 during 5 min invasion of HFF monolayers. The percentage of inhibition was calculated with respect to relative control sera. Results are presented as mean ± SD representing average of three independent experiments. Student’s t test, ***P < 0.001. B. Invasion inhibition assay of KO-AMA1 tachyzoites treated with 1 mg/mL total IgG extracted from sera of mice immunized with rAMA or rAMA in complex and allowed to invade HFFs monolayers for 5 min. The percentage of inhibition was calculated with respect to their relative control sera. Results are presented as mean ± SD representing average of three independent experiments. C. IFAs on intracellular RH tachyzoites and KO-AMA1 tachyzoites using control sera or sera from mice immunized with rAMA4 or rAMA4–RON2L1D3. (Scale bars, 4 μm.)
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