Significance
Host cell invasion is a key step for apicomplexan infection and survival. This process was widely studied in Toxoplasma tachyzoites and to a much lesser extent in sporozoites. However, it remains uncharacterized in the bradyzoite and merozoite stages. In this study, we show that bradyzoites invade host cells in a moving-junction (MJ)–dependent fashion, which is enriched in AMA2 and AM4 proteins. We demonstrated that AMA4 does not contribute to invasion of tachyzoite and has no role during acute infection. However, AMA4 is required for cyst burden, supporting the notion that cyst-forming bradyzoites can disseminate to contribute to cyst burden. Furthermore, we designed an efficient vaccine strategy targeting MJ proteins that further enhanced our understanding of their function during toxoplasmosis.
Keywords: Toxoplasma, bradyzoite, invasion, moving-junction, vaccine
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.
Toxoplasmosis is one of the most common zoonotic diseases and is caused by the intracellular apicomplexan parasite Toxoplasma gondii. This parasitic infection has a prevalence ranging between 25 and 80% of the worldwide population, according to regions (1). The complete life cycle of T. gondii involves a sexual stage that is restricted to the feline intestinal epithelium and an asexual stage that occurs within any warm-blooded animals. The asexual life cycle can sustain the parasite’s transmission between intermediate hosts, via oral transmission of tissue cysts containing bradyzoites (2, 3). The ingestion of sporulated oocysts (containing sporozoites) shed in the environment by felids represents another route of transmission (4, 5). Bradyzoites and sporozoites invade enterocytes and rapidly convert to tachyzoites, which are the fast-replicating asexual forms causing acute toxoplasmosis. This phase is usually controlled by the host immune response, unless tachyzoites cross the placental barrier in primary infection in pregnant women, leading to severe outcomes, ranging from abortion to stillbirth with congenital neurological defects. Tachyzoites can differentiate into bradyzoites, which encyst in skeletal muscles and in the central nervous system, establishing latent chronic toxoplasmosis. Bradyzoites are thought to be dormant parasites, but they are active and can constantly remodel cysts (6, 7). Whilst chronic toxoplasmosis is considered asymptomatic in immunocompetent hosts, it may be associated with chorioretinitis and behavioral and mental health disorders (8, 9). However, the influence of T. gondii on human behavior remains under debate (10). In immunocompromised patients (AIDS or cancer), reactivation of chronic toxoplasmosis correlates with severe morbidity and mortality (11). Drugs currently used to treat toxoplasmosis are effective only on tachyzoites; therefore, bradyzoites can persist in cysts for prolonged periods. A vaccine could prevent infection and reactivation of cysts. However, the only available vaccine, Toxovax, is an attenuated live vaccine restricted to veterinary use (12) and suffers from possible reversion of the attenuated strain into the virulent one.
The technical ease of propagating tachyzoites in vitro combined with the availability of the genetically tractable RH strain has contributed significantly to our knowledge of T. gondii biology. In contrast, the two other transmissible stages, bradyzoites and sporozoites, are technically challenging to work with and remain poorly studied. As an obligate intracellular parasite, T. gondii survival and persistence depend on invading host cells. Invasion by tachyzoites involves the formation of a tight connection between the parasite and host cell membranes (13), referred to as the moving junction (MJ). The formation of the MJ depends on the sequential secretion of the microneme and rhoptry secretory organelles (13) and is critically driven by the assembly of a parasite complex at the parasite/host cell interface (14, 15). Following microneme secretion, apical membrane antigen 1 (AMA1) is displayed on the parasite surface with its ectodomain comprising three subdomains referred to as D1, D2, and D3 (16). Subsequently, a macromolecular complex comprising RON2, RON4, RON4L1, RON5, and RON8 is secreted from the rhoptries and targeted to the host cell (17, 18) with RON2 spanning the host cell membrane. Despite being a large protein, the ectodomain of RON2 is quite small and incorporates a short region of approximately 40 amino acids (D3 domain), that interacts with AMA1 D1 (19). Importantly, the AMA1–RON2 complex is equally conserved and is critical for invasion of many apicomplexan parasites including Plasmodium spp. (20, 21), the etiological agent of malaria. A widely accepted model suggests that the AMA1–RON2 complex serves as a bridge between the parasite and host cell (13, 14, 22) and is now considered a potential target to block MJ formation and therefore invasion and infection (23–25). Recent studies showed that immunization with recombinant Plasmodium falciparum AMA1 D1-D2-D3 in complex with the D3 fragment of RON2 induced complete protection in mice upon challenge with a lethal Plasmodium yoelii strain and induced protection of Aotus monkeys against a virulent P. falciparum infection, while vaccination with AMA1 alone conferred no protection or only partial protection in the two models (26, 27).
While the MJ-dependent invasion process has been extensively characterized in tachyzoites, the molecular composition and the role of MJ in other parasitic stages remain largely unexplored. Paralogs of AMA1 (sporoAMA1 or AMA3) and RON2 (sporoRON2 or RON2L2) are exclusively expressed at the apical region of sporozoites, where they form a complex for invasion (28). Two additional AMA1 homologs were discovered by analyzing the mechanism of adaptation of the RH strain following AMA1 deletion (29). Indeed, in the absence of AMA1, Toxoplasma up-regulated AMA2, which retained the ability to interact with RON2, albeit with reduced affinity. It also up-regulated the expression of a highly divergent AMA1 paralog, AMA4, that forms a functional complex with its coevolved partner TgRON2L1 (16, 29). The AMA4–RON2L1 complex is expressed at very low levels in tachyzoites but highly expressed in sporozoites (16). The least studied stages remain the merozoite and bradyzoite. Only one report documented the invasion process of mature bradyzoites, which, based on electron microscopy images, concluded that no MJ is formed during bradyzoite invasion (30). Yet, bradyzoites invasion is a critical step in the life cycle of Toxoplasma. It can take place in the intestinal epithelium when they are released from ingested cysts, upon ingestion of undercooked or raw infected meat. Invasion of bradyzoites also occurs in the context of chronic toxoplasmosis when bradyzoites exit cysts and reinvade new cells to form new cysts (7), a step likely responsible for the increase in cyst burden over time. Finally, invasion of bradyzoites is also a critical first step upon reactivation of chronic toxoplasmosis in immunocompromised individuals.
Here, we dissected the invasion process of bradyzoites and detected all tachyzoite MJ RON proteins in invading bradyzoites; however, AMA1 was not visible at the MJ. In contrast, AMA2 and AMA4 were not detectable at the MJ of tachyzoites but are highly expressed in bradyzoites and decorate their MJ during invasion. We generated AMA4 knockout (KO) parasites in a cystogenic strain and showed that AMA4 does not play any role during acute infection but contributes to the establishment of chronic toxoplasmosis. We next explored the protective efficacy of Toxoplasma recombinant AMA proteins alone or in complex with their respective RON2-D3-binding domains against acute and chronic toxoplasmosis. The immunization with the AMA1–RON2 complexes proved highly protective after an oral challenge of mice with encysted bradyzoites (≈80% reduction of cyst burden in the brain) or intraperitoneal challenge with a lethal dose of tachyzoites (80% increased mouse survival). In contrast, the AMA4–RON2L1 complex mainly protected against chronic toxoplasmosis. Moreover, we showed that the sera produced by immunized mice target the invasion process. Altogether, our findings reveal important insights into the process of bradyzoites invasion and highlight the AMA–RON complexes as potential vaccine candidates for protection against toxoplasmosis.
Results
Bradyzoites Use a MJ–Dependent Process to Invade.
To characterize the molecular mechanism of bradyzoite invasion, we first investigated whether the tachyzoite MJ proteins were also expressed in bradyzoites. In vivo transcriptome analysis of T. gondii during acute and chronic infection predicted the expression of RON2, RON4, RON4L1, RON5, RON8, and AMA1 proteins in bradyzoites (Fig. 1A) (31). To corroborate these results, we induced the differentiation of tachyzoites of the Prugnaud (Pru) strain into bradyzoites in vitro, by alkaline stress, and performed immunofluorescence assay (IFA) staining with anti-MJ antibodies, antibodies recognizing the late bradyzoite marker p21 protein, and fluorescein-labeled Dolichos biflorus agglutinin (DBA), which specifically binds to the wall of cysts containing bradyzoites (32). Since we did not have specific sera for RON4L1, the endogenous locus of RON4L1 was epitope-tagged with a triple hemagglutinin (Pru–HA3–RON4L1) (SI Appendix, Fig. S1). We found that AMA1, RON2, RON4, RON4L1, RON5, and RON8 were well expressed in the bradyzoites of the Pru strain (Fig. 1 B and C). This prompted us to test their expression during bradyzoite invasion. To avoid any confounding results with tachyzoites which persist after the in vitro differentiation, we used purified bradyzoites from the brains of infected mice with the Pru strain to perform our invasion assay. Remarkably, typical MJ staining was observed for RON2, RON4, RON4L1, RON5, and RON8 on invading parasites that were fixed 3 min after contact with host cells (Fig. 1D), demonstrating that bradyzoites use an MJ-associated process to invade host cells. The visualization of AMA1 at the MJ of invading tachyzoites is particularly challenging (33, 34). We recently succeeded in observing AMA1 at the residual MJ of newly invaded tachyzoites, using a new batch of rabbit anti-AMA1 antibodies (34)(Fig. 1E). In bradyzoites, AMA1 appears to be expressed at reduced levels in the micronemes compared to tachyzoites under the same exposure time during imaging (Fig. 1E). Moreover, AMA1 was not visible at the MJ in invading bradyzoites (Fig. 1E).
Fig. 1.
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.
Intriguingly, when the bradyzoites are intracellular, RON stainings appeared as a trail at the posterior end of the parasite (Fig. 1F). This observation was absent in tachyzoites (17) and shared similarities with the trails observed at the posterior end of invading sporozoites (35). These trails were always characterized by a straight line, marked by a more intense fluorescent dot at the end (Fig. 1F, arrows). To determine whether these trails corresponded to an extension of the parasitophorous vacuole membrane (PVM), we stained the invading parasites with ROP1, a rhoptry protein translocated to the PVM at early steps of its formation (36). Interestingly, ROP1 decorated a trail with RON5 at the posterior end on bradyzoite 30 min after invasion (Fig. 1G). Similar to sporozoites (35), this observation supports the conclusion that these trails correspond to the PVM, which might be translocated away from the original site of invasion due to bradyzoite high motility (7).
AMA2 and AMA4 Are Up-Regulated in the Bradyzoite Stage and Decorate the MJ of Invading Bradyzoites.
Proteomic and transcriptomic analyses of acute and late-chronic T. gondii infection indicated a stage-specific increase in transcription in bradyzoites of AMA4 and its partner RON2L1 but also AMA2 (Fig. 1A). An alternative isoform of AMA3 (sporoAMA1) is also predicted to be expressed during late-chronic infection (37). However, the transcript encodes a truncated version of AMA3, predicted to be nonfunctional. Moreover, there are almost no transcripts corresponding to its partner RON2L2 (37). Thus, we focused our analysis on AMA2, AMA4, and RON2L1. We no longer have anti-RON2L1 antibodies (16), and we failed to obtain a tagged RON2L1 parasite in Pru strain. Therefore, we could not evaluate its expression in bradyzoites. Using specific antibodies against AMA4, and a tagged AMA2 line that we generated in Pru-AMA2-HA3 (SI Appendix, Fig. S2), we showed that AMA2 and AMA4 are both well expressed in bradyzoites (Fig. 2A). Strikingly, while we could never see AMA2 and AMA4 at the MJ of invading tachyzoites (Fig. 2B), where they are poorly expressed, both were associated with the MJ of invading bradyzoites (Fig. 2 C and D). In some parasites, AMA2 and AMA4 also appeared as a trail, dragging behind newly invaded bradyzoites (Fig. 2 C and D, Lower), and this trail colocalized with RON5.
Fig. 2.
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.
Altogether, these results show that bradyzoites form an MJ during invasion, albeit with a different molecular composition or abundance of proteins from that of invading tachyzoites.
AMA4 Plays a Role in Establishing Chronic Infection.
We next tested the role of AMA4 during acute and chronic infection. We generated an AMA4 KO in a cystogenic type II Me49∆ku80 strain (Fig. 3A and SI Appendix, Fig. S3 A–B). Tachyzoites depleted of AMA4 showed smaller lysis plaques, as compared to control parasites and a small reduction of invasion after 5 min of contact with fibroblasts, but both were not statistically significant (Fig. 3 B and C). Following intraperitoneal inoculation of 1,000 tachyzoites, mice infected with control or KO-AMA4 succumbed to infection within the same timeframe, indicating that AMA4 does not play a major role during the acute phase of toxoplasmosis in a mouse model (Fig. 3D). However, mice infected with 200 KO-AMA4 tachyzoites exhibited a significantly reduced cyst burden (approximately 70%) in their brains, as compared to mice infected with the wild-type (WT) parasites (Fig. 3E). To verify that this phenotype was due to the lack of the ama4 gene, we generated a complemented line (Compl.) by reintroducing the Ty3-tagged ama4 gene under its own promotor in the UPRT locus of the KO-AMA4 line (Fig. 3A and SI Appendix, Fig. S3 C and D). This restored the number of cysts to that observed in the WT strain (Fig. 3E), demonstrating that AMA4 is critically important in establishing chronic infection in the Me49 cystogenic strain.
Fig. 3.
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).
AMA1 in Complex with RON2 Exhibits a Strong Protective Effect against Acute and Chronic Infection with T. gondii.
We next interrogated the efficacy of targeting Toxoplasma AMA1 alone or in complex with RON2, as a vaccination approach against toxoplasmosis. C57BL/6 mice were immunized with recombinant AMA1 D1-D2-D3, hereafter referred to as rAMA1, or with rAMA1 in complex with the D3 domain of RON2 (rAMA1–RON2D3) (Fig. 4A). An Enzyme-Linked Immunosorbent Assay (ELISA) using sera collected 14 days after the last immunization showed that both rAMA1 and rAMA1–RON2D3 induced a similar high IgG response (Fig. 4B and SI Appendix, Fig. S4 A and C). IFAs showed that anti-rAMA1 and anti-rAMA1–RON2D3 antibodies displayed a microneme staining, which was absent in control sera (Fig. 4C). The specificity of the sera for AMA1 was verified by western blot (Fig. 4D) using parasites that did not express AMA1 (29). No signal was observed in IFAs on RH KO-AMA1 (SI Appendix, Fig. S5), thus excluding a reactivity with the RON2 peptide.
Fig. 4.
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.
To investigate the efficacy of the vaccine strategy against acute toxoplasmosis, mice were challenged with a lethal dose of 1,000 tachyzoites of type II Pru and monitored daily for their survival (Fig. 4A). A significant but slight delay in mortality was observed between mice immunized with rAMA-1 alone compared to the control adjuvant (Fig. 4E). Strikingly, immunization with the rAMA1–RON2D3 complex yielded consistent protection of more than 70% of mice against the lethal challenge (Fig. 4E). These observations were repeated in an independent vaccination experiment (SI Appendix, Fig. S4 B and D) illustrating the high protective efficacy of this complex over AMA1 alone against acute toxoplasmosis. We then examined whether this complex yielded a protective response against a natural route of infection. Animals were challenged with 15 cysts of the 76 K strain by oral gavage, and the establishment of chronic toxoplasmosis was assessed by quantifying the cyst burden in the brain 1 mo later. Immunization with rAMA1 induced a significant decrease of 56% in the number of cysts in the brains of immunized mice, as compared to the control group (Fig. 4F). Notably, rAMA1–RON2D3 resulted in stronger protection against chronic toxoplasmosis (80% decrease) (Fig. 4F), a result repeated in another independent experiment (Fig. 4J). Altogether, these results validate the efficacy of using AMA1 in complex with RON2 to protect against both acute and chronic toxoplasmosis.
The AMA4–RON2L1 Complex Is Also a Vaccine Candidate against Chronic Toxoplasmosis.
The role we identified for AMA4 in chronic Toxoplasma infection (Fig. 3E) offers the potential for targeting a broader range of AMA–RON2 pairs of Toxoplasma invasive stages. We therefore conducted similar vaccine experiments with recombinant AMA4 containing D1, D2, and D3 domains (rAMA4), alone or in complex with the D3 domain of RON2L1 (rAMA4–RON2L1D3) (Fig. 4A). Both rAMA4 and rAMA4–RON2L1D3 induced significant immune responses by ELISA (SI Appendix, Fig. S4 A and C), specific for AMA4 as noted by the absence of reactivity on KO-AMA4 by western blot (Fig. 4G). rAMA4 alone did not induce any protection against acute infection (Fig. 4H), yet it consistently slightly delayed the mortality of mice when in complex with RON2L1D3 (Fig. 4H and SI Appendix, Fig. S4 B and D). This protection was considerably less important than that observed with rAMA1–RON2D3 (Fig. 4E), supporting a minor role of AMA4 for invasion of tachyzoites during acute infection. Strikingly, immunization with rAMA4–RON2L1D3 led to 50% significant decrease of cyst burden in the brains of mice challenged orally with bradyzoite cysts (Fig. 4 I and J). Only a slight increase of protection against chronic infection was conferred when mice were immunized with both complexes, but this was not significant (Fig. 4J). Collectively, these results point to a less critical role of AMA4–RON2L1 than AMA1–RON2 in tachyzoite invasion but position the two complexes as important targets to protect against toxoplasmosis.
The AMA–RON Complexes Induce Invasion Inhibitory Antibodies.
To investigate the mechanism of protection induced by immunization with complexes, we tested the capacity of antibodies against the respective AMA–RON complex to inhibit invasion. The anti-rAMA1 antibodies significantly inhibited the invasion of RH tachyzoites (Fig. 5A), in a dose-dependent manner (SI Appendix, Fig. S6), and this was further enhanced by 35% by complexing rAMA1 with RON2D3 (Fig. 5A). Moreover, this effect was specific to AMA1 because no inhibition was observed in the RH KO-AMA1 strain (Fig. 5B). Conversely, the sera against rAMA4–RON2L1D3 only slightly inhibited the invasion of WT RH tachyzoites at 2 mg/mL (SI Appendix, Fig. S6), again supporting a predominant role of AMA1 for invasion of RH tachyzoites.
Fig. 5.
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.)
We did not succeed in developing a quantitative invasion assay for bradyzoites; this is due to the low number of bradyzoites extracted from cysts, both after infection of mice or in vitro switch. To quantify the invasion inhibitory effect of sera against rAMA4–RON2L1D3, we therefore took advantage of KO-AMA1 parasites, that overexpress AMA4 upon removal of AMA1 (29). Indeed, the sera against rAMA4 and rAMA4–RON2L1D3 gave a microneme staining in KO-AMA1 and not in WT parasites (Fig. 5C). Moreover, we obtained 15.5% and 50% reduction of invasion of KO-AMA1 parasites when incubated with sera against rAMA4 and rAMA4–RON2L1D3, respectively (Fig. 5B). These results suggest that one plausible mechanism of protection may be conferred in immunized mice through the inhibition of parasite invasion.
Discussion
Invasion is a key step for all Toxoplasma stages. The fast-replicative tachyzoite stage invades all nucleated cell types, including immune cells. In contrast, sporozoites and bradyzoites use enterocytes as a route for infection after ingestion of undercooked meat containing cysts or contaminated vegetable/water with oocysts. Invasion by bradyzoites can also take place after rupture of cysts or following reactivation of chronic infection. Merozoites exclusively invade the feline intestinal epithelium. Although invasion has been extensively studied in tachyzoites, and to a lesser extent in sporozoites, bradyzoite and merozoite invasion remains largely unexplored. Here, we show that bradyzoites form an MJ during invasion and that the composition differs from that of tachyzoites. We also designed a vaccine procedure to target two MJ complexes, rAMA1–RON2D3 and rAMA4- RON2L1D3. This strategy yielded efficient protection against acute and chronic toxoplasmosis, and the resulting data provided a greater understanding of the role of several invasion effectors during different parasite stages.
We showed that all tachyzoite MJ proteins are expressed in bradyzoites and that RONs form the characteristic ring-shaped structure in invading bradyzoites. We could not visualize AMA1 at the MJ of invading bradyzoites. This might be due to the lower expression of this isoform in bradyzoites as compared to tachyzoites, and the notorious difficulty of detecting AMA1 at the MJ (33, 34). In contrast, the AMA1 paralogs, AMA2 and AMA4, which are both highly expressed in bradyzoites but only expressed at low levels in tachyzoites, are clearly visible at the MJ of bradyzoites. AMA2 is known to interact with RON2 in the absence of AMA1 (29). Bradyzoites may therefore rely on AMA2, instead of AMA1, and as a consequence, the bradyzoite MJ might be shaped by a complex comprising AMA2/RON2/RON5/RON4/RON4L1/RON8. However, the difficulty in obtaining sufficient amounts of bradyzoites limited our ability to fully test this hypothesis and to determine whether AMA4 and its partner RON2L1 are also part of this or an independent complex.
We and others had previously established AMA1 as a crucial factor for invasion of tachyzoites (29, 38) and a key determinant for acute infection in a mouse model (39). Here, we obtained evidence that AMA4 is also an invasion factor, with a predominant role in bradyzoites. These conclusions are based on the following convergent observations. First, AMA4 is highly expressed in bradyzoites and is present at their MJ, strongly arguing for a critical role of AMA4 in this stage. Second, the sera against rAMA4 and rAMA4–RON2L1D3 inhibit the invasion of KO-AMA1 tachyzoites (high expression of AMA4), but not the invasion of WT tachyzoites (low expression of AMA4). Finally, we observed a significant reduction in the numbers of cysts in the brains of mice infected intraperitoneally with KO-AMA4 tachyzoites (Fig. 3G). Importantly, this reduction is not likely due to a failure of the mutant to enter the acute phase, as the mutant showed no defect in the lytic cycle in vitro (Fig. 3B), and killed mice at a comparable level to the WT strain (Fig. 3D). One plausible explanation for the reduction in the number of brain cysts is the differential requirement for AMAs for invasion by tachyzoites and bradyzoites. KO-AMA4 tachyzoites would successfully invade brain cells using AMA1–RONs, convert to bradyzoites, and form cysts. However, the bradyzoites that escape these cysts lack AMA4 and would become less efficient to reinvade neurons [the primary target cell for the brain-tropic form of T. gondii (40)]. Dzierszinski et al. (7) showed that bradyzoites are highly motile and are able to escape cysts without reverting to tachyzoites, disrupting the vacuole or killing the host cell, and eventually reinvade adjacent cells. This process was proposed to provide a mechanism to increase the number of cysts in the brain. Although it has not been visualized in vivo, this observation is in line with the observed increase in tissue cyst burden in the brains of chronically infected animals, despite the low frequency of cyst rupture (41), and the recent observation that conditional mutagenesis of motility/egress factors in bradyzoites decreases cyst burden (42). Considering these data, our work further illustrates the requirement of reinvasion of bradyzoites for the increase in cyst number during chronic toxoplasmosis. In summary, the present work shows that both bradyzoites and tachyzoites use MJ-dependent processes but with different protein complexes. The switch in AMA repertoire may help bradyzoites, after escaping brain cysts, to overcome the immune response mounted against AMA1 during the acute phase (43). An alternative, but not mutually exclusive hypothesis is that different complexes are used for invasion of different cell types. Since AMA4 is highly expressed in bradyzoites but also in sporozoites (16), and in merozoites (44), and that all three stages infect enterocytes, it is tempting to speculate that AMA4 is also a peculiar invasion factor of enterocytes.
In tachyzoites, the end of invasion is marked by a well-defined residual dot visible by IFA (17), corresponding to junction proteins at the posterior end of the parasite. In contrast, a trail of AMA2, AMA4, and RONs is visible at the posterior end of newly invaded bradyzoites. Interestingly, ROP1, a rhoptry protein translocated to the PVM at the early step of PVM formation (36), also decorated the trail, supporting the hypothesis that the trails correspond to the PVM which might be translocated away from the original site of invasion due to bradyzoite high motility before the separation of the PVM from the host cell membrane (7). This observation is reminiscent of the trails observed at the posterior end of invading sporozoites (35). These trails defined the routes followed by individual sporozoites during their intracellular or intercellular migration. Sporozoites are able to do cell traversal and cell-to-cell passage, a process that might contribute to reach a suitable replicative site in the intestine (45, 46). Indeed, at 2 h postinfection, most of the sporozoites were found in ileal enterocytes and occasionally in goblet cells, while later at 6 to 12 h postinfection, sporozoites were not detected in the intestinal epithelium, but found exclusively in cells of the underlying lamina propria, which support parasite migration (45, 46). Future work is needed to establish whether bradyzoites also use cell-to-cell traversal in the intestine or the brain as previously suggested (7).
Several studies used AMA1 to target toxoplasmosis by protein, multiepitope, or DNA vaccines (47), but the most promising AMA1 vaccination approach was obtained for malaria infection. It was based on the use of a recombinant PfAMA1 protein in complex with a short domain of PfRON2 (26, 27). Here, we showed that Toxoplasma AMA1 in complex with TgRON2 peptide also generated efficient protection against both acute and chronic toxoplasmosis. The immunization with rAMA1–RON2D3 elicited a strong immune response which was illustrated by specific high antibody titers that provided protection by inhibiting the invasion process. As proposed by Srinivasan et al. (26), a plausible scenario of this enhanced protection with the complex may be due to the exposure of TgAMA1 immunogenic epitopes upon binding of TgRON2D3 to TgAMA1 pocket and the generation of more potent invasion-inhibitory antibodies, that would eventually prevent the interaction of AMA1 with RON2 and the disruption of the MJ formation. This hypothesis equally applies for AMA4 when in complex with RON2L1D3. Indeed, the protection was improved upon immunization with the rAMA4–RON2L1D3 complex, compared to rAMA4 alone. This result opens the path to the inclusion of other complexes, like AMA2-RON2 and AMA3-RON2L2 in an attempt to target all T. gondii stages.
Materials and Methods
Parasite and Cell Culture.
Tachyzoites were serially passaged in human foreskin fibroblasts (HFFs) (American Type Culture Collection CRL 1634) cultured in Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO, Invitrogen) and supplemented with 10% of fetal bovine serum (FBS), 50 µg/mL penicillin–streptomycin, and 2 mM glutamine. T. gondii type I RH stains deleted for the ku80 gene (RH Δku80) and for ama1 gene (RH KO-AMA1), T. gondii type II strain 76 K, type II strain Pru, and type II strain ME49 hxgprt-ko ku80-ko strain (referred hereafter Me49Δku80) (48) were used throughout this study.
Generation of DNA Constructs, Transfection, and Transformant Selection.
To generate AMA4 KO in Me49 Δku80 background, we used a CRISPR/Cas9 strategy (18). The parasites were transfected with a DNA donor sequence and two pU6-Cas9 vectors containing a protospacer targeting respectively 20 bp before the start codon and 20 bp downstream the stop codon of the ama4 locus. The DNA donor sequence was amplified with KOD polymerase (Novagen) from pLIC-DHFR-HA3 using primers ML3234-ML3235 to generate a dihydrofolate reductase (DHFR) resistance cassette flanked by 30-bp homology arms corresponding to ama4.
To generate the complemented AMA4 line (Compl.), the full cDNA sequence of ama4 followed by a sequence encoding for 3 Ty tag and flanked by its own promotor (976 pb) and its 3′ UTRs (773 pb) was synthesized by GenScript and cloned into a modified pUPRT-TUB-G13-Ty plasmid that allows vector integration at the UPRT locus (49). The resulting pUPRT-AMA4-Ty3 plasmid was digested with NdeI, and the resulting fragment of 6,500 bp containing ama4 flanked by 700 and 500 bp of the 5′ and 3′UTR of the uprt locus respectively, was cotransfected with two sgRNAs targeting the uprt locus. These guides were constructed by annealing primers ML3445 and ML3446 (5′uprt) and ML2087 and ML2088 (3′uprt) in the pU6-Universal CRISPR/Cas9 vector. The integrations at the uprt locus were verified with primers ML5387-ML3403 and ML5386-ML5147.
Tagging of RON4L1 at the N terminus was done in Pru strain using the CRISPR/Cas9 strategy as described by Guerin et al in RHΔku80 strain (50).
Tagging of AMA2 at the C-terminus was done in Pru strain using the CRISPR/Cas9 strategy as described (29).
Primers used to generate the KO-AMA4 and its complemented version, or tagged lines, are listed in SI Appendix, Tables S1 and S2, respectively.
Transfections were done as described previously (51), and transfected parasites were selected with 20 µM of chloramphenicol for CAT integration and 1 µM pyrimethamine for DHFR integration. For Pru HA3-RON4L1, parasites transiently expressing cas9-YFP were sorted by FACS two days posttransfection. Clones were then isolated by limiting dilution in a 96-well plate, and integration was verified by PCR.
Plaque Assay.
Confluent HFF cells in 24-well plates were infected with ≈50 synchronized Me49Δku80, Me49Δku80 KO-AMA4, and Me49Δku80 KO-AMA4–complemented (Compl.) tachyzoites per well and incubated for 14 d at 37 °C. Infected cells were then fixed with cold methanol for 10 min and stained with Giemsa. Images were obtained using Olympus MVX10 macro-zoom microscope equipped with an Olympus XC50 camera. Plaque area measurements were analyzed using Zeiss Zen software (29).
In Vitro Bradyzoite Differentiation Assay.
HFF cells were cultured on coverslips in a 24-well plate and infected with 1,000 Pru tachyzoites in DMEM complete media. After 24 h incubation at 37 °C under 5% CO2 conditions, infected cells were cultured at 37 °C in induction medium (RPMI 1640 without NaHCO3, 3% FBS, 50 mM HEPES, adjusted at pH 8.2) and under ambient CO2 conditions (52). In order to maintain the basic pH, the medium was changed every other day. After 10 d, coverslips were fixed in 4% paraformaldehyde for IFA and cells infected with cysts were lysed with 1× Laemmli buffer for western blot analysis.
IFA and Parasite Immunoblots.
For IFA, infected HFF cells were fixed with 4% paraformaldehyde in PBS for 30 min followed by permeabilization with 0.2% Triton for 15 min and stained with the appropriate primary antibody listed below, followed by secondary antibody conjugated to Alexa Fluor (SI Appendix, Table S3). Images were acquired using a Zeiss Axiocam MRm CCD camera driven at the “Montpellier Ressources Imagerie” facility and Zeiss LSM 710 confocal microscope (Zeiss). All images were analyzed using Zeiss Zen software.
For western blotting, pellets of freshly egressed tachyzoites or HFF cells containing cysts following in vitro switch were lysed in 1× Laemmli buffer, and immunoblots were done as previously described (SI Appendix, Table S4) (53).
Generation of Individual Proteins.
The sequences encoding the mature ectoplasmic region of AMA1 (amino acids 64 to 484, TGME49_255260) and AMA4 (amino acids 58 to 553, TGME49_294330) were synthesized and subcloned into a modified pAcGP67b vector (Pharmingen) with a C-terminal Tobacco Etch Virus (TEV) cleavage site and a hexa-histidine tag. AMA1- and AMA4-encoding virus for insect cell protein production was generated and amplified as described (16). Then, 65 h after infection, the supernatant was harvested, concentrated, and proteins were purified by nickel–nitrilotriacetic acid (Ni-NTA) chromatography. The hexa-histidine tag was removed by TEV cleavage, and the protein was further purified by size exclusion chromatography (SEC) (Superdex 16/60 75) in HEPES buffered saline (HBS: 20 mM HEPES pH 7.5, 150 mM NaCl).
Sequences encoding a portion of RON2D3 (TGME49_300100; amino acids 1,297 to 1,333) and RON2D3L1 (TGME49_294400; amino acids 1,292 to Ser1324) were synthesized and subcloned into a modified pET32a vector (Novagen) with an N-terminal hexa-histidine, a thioredoxin (TRX) tag, and a TEV cleavage site. The fusion proteins were produced in Escherichia coli BL21 cells and purified by Ni-NTA and SEC as described above.
Purification of the Complexes.
Escherichia coli pellets expressing RON2D3 and RON2L1D3 TRX were thawed on ice and lysed in a French Press, and the insoluble material was removed by centrifugation. Purified, cleaved rAMA1 and rAMA4 were added directly to RON2D3 and RON2L1D3 clarified cell lysate, respectively, and incubated at 4 °C for 30 min. AMA1–RON2D3 and AMA4–RON2D3L1 complexes were then purified using Ni-NTA pull-down. Last, the TRX tag was removed after an overnight TEV cleavage, and the complexes were purified by SEC and analyzed by sodium dodecylsulfate (SDS)-PAGE.
In Vivo Studies: Ethics Statement.
All mice protocols were approved by the Institutional Animal Care and Utilization Committee (IACUC) of the American University of Beirut (AUB) (Permit Number IACUC#19-02-RN464) and by the French Council in Animal Care guidelines and following the protocols approved by the Institut Pasteur de Lille’s ethical committee (No. 11082-2017072816548341 v2). Mice were housed in specific pathogen-free facilities and monitored on a daily basis. Humane endpoints were used according to AUB IACUC which follows the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International and guide of animal care use book (Guide, NRC 2011). Mice were sacrificed if any abnormal phenotype is noted (18).
Immunization study.
Five- to six-wk-old female C57BL/6 mice were divided into two groups, a control group receiving only Freund’s adjuvant and an immunized group receiving the adjuvant along with one of the complexes (rAMA1–RON2D3 or rAMA4–RON2L1D3) or one of the peptides (rAMA1 or rAMA4). Subsequently, 40 µg of the complexes or peptides was added to 50 µL of PBS and then emulsified with an equal volume of adjuvant. The control mice group received 50 μL of PBS emulsified with 50 μL adjuvant. For mice receiving the two complexes together, mice were injected in a separate site with 40 µg rAMA1–RON2D3 and 40 µg rAMA4–RON2L1D3.
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.
Mice Survival and Evaluation of Brain Cysts Burden.
To assess virulence during the acute phase, 6- to 7-wk-old female BALB/c mice (Janvier Labs France) were intraperitoneally infected with 1,000 tachyzoites of type II Me49 ∆ku80, Me49∆ku80 KOAMA4 strains (N = 2, five mice per group). Mice were monitored daily throughout the course of infection.
To assess cyst burden of KO-AMA4 parasite during chronic toxoplasmosis, 7- to 9-wk-old female Swiss Mice (Janvier Labs France) were intraperitoneally injected with 200 tachyzoites of type II Me49∆ku80 strain (N = 3, 10 mice), Me49∆ku80 KO-AMA4 strain (N = 3, 10 mice), or complemented strain (Compl.) (N = 2, 10 mice) (N = 3, 10 mice per group).
To assess protection against the acute phase postimmunization, C57BL/6 immunized mice were challenged with 1,000 tachyzoites of type Pru and monitored daily throughout the course of infection.
To assess cyst burden after immunization, C57BL/6 immunized mice were challenged with 15 cysts of 76 K strain by oral gavage. One month after infection, the brains of two mice were pooled, and brain cysts were harvested as previously described (54) and enumerated using a hemocytometer or after labeling using Dolichos biflorus lectin.
Bradyzoite Isolation from Brains.
Bradyzoites were released from brain cysts by incubating the cyst solution with pepsin /HCl (pH = 2) in a final concentration of 0.05% at 37 °C for 15 min. Pepsin was then neutralized by adding complete DMEM (Gibco) supplemented with 10% FBS, 1 mM glutamine, and 1 mM penicillin/streptomycin and centrifuged for 15 min, at room temperature, 2,200 rpm.
Invasion and Invasion Inhibition Assay.
For invasion assay, 5 × 106 Me49∆ku80 and Me49∆ku80 KO-AMA4 tachyzoites were incubated on HFF cultures growing on coverslips in a 24-well plate on ice for 20 min. The plate was later placed in a water bath at 38.5 °C to allow the parasites to invade for 5 min. For invasion inhibition assay, total IgG was purified from sera of mice immunized with either rAMA1 or rAMA4 peptides alone, or with rAMA1–RON2D3 or rAMA4–RON2L1D3 complexes, using protein G agarose beads (GE Health Sciences), eluted in DMEM, and dialyzed against RPMI 1640 medium. A total of 5 × 106 tachyzoites of type I T. gondii (RHΔku80 or RHΔku80 KO-AMA1) were incubated with 0.5, 1, or 2 mg/mL of total IgG for 30 min at 37 °C, and invasion was performed in the presence of IgG. To quantify invasion, intracellular and extracellular parasites were differentially stained by IFA as described (29). Intracellular parasites were enumerated using the Zeiss Axioimager epifluorescence microscope. The experiment was done three independent times, and 20 fields were counted on each coverslip.
ELISA.
Total IgG was assessed using ELISA, and 96-well plates were coated with 100 µL/well of either complex (rAMA1–RON2D3 or rAMA4–RON2L1D3) or with single peptides (rAMA1 or rAMA4) at a concentration of 1 µg/mL, diluted in coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate). After overnight incubation at 4 °C, plates were blocked with 5% skimmed milk in Tris-buffered saline (TBS) for 2 h at room temperature (RT). Sera from respective mice were diluted at 1:5000 in dilution buffer (0.1% BSA, 0.05% Tween-20 in TBS), added (100 µL/well) to detect total IgG and incubated for 2 h at RT. Following extensive washing in TBS-0.1% Tween-20, plates were incubated with 100 µL/well of anti-mouse IgG antibody conjugated with peroxidase (Sigma) for 2 h at RT. After similar washing, peroxidase substrate 3, 3′,5,5′-tetramethylbenzidine (Sigma) was added for 20 min, and the reaction was stopped by 2 M HCl. The optical density was read at 450 nm using an ELISA reader (Multiskan EX).
Statistical Analysis.
All results are presented as mean value with SD. All in vivo experiments, ELISA, and invasion inhibition assay were analyzed using two-tailed Student’s t tests to determine statistical significance. For in vivo survival experiments, statistical significance was determined using the log-rank test using GraphPad. Statistical significance is indicated as * for P-value < 0.05, ** for P-value < 0.01, and *** for P-value < 0.001.
Supplementary Material
Appendix 01 (PDF)
Acknowledgments
We thank Sebastian Lourido for the pU6-Universal plasmid and Dominique Soldati-Favre for kind gift of the Me49∆ku80 cell line. This work was supported by the Laboratoire d’Excellence (LabEx) (ParaFrap ANR-11-LABX-0024) and the Fondation pour la Recherche Medicale (FRM) (Equipe FRM DEQ20170336725) to M.L. and by Canadian Institutes for Health Research grant 148596 to M.J.B. Dr. M.L. is an INSERM researcher. M.T.G.R. has been supported by ParaFrap (ParaFrap ANR-11-LABX-0024) and is a recipient of FRM (award no. FRM FDT201904008062). M.J.B. gratefully acknowledges the Canada Research Chair program for salary support. This work was made possible through core support from the Medical Practice Plan (Faculty of Medicine, AUB to Dr. H.E.H.) and the “Universite Montpellier-Centre National de Recherche Scientifique-Liban” fellowship to support R.N. under the direction of Dr. M.L. and codirection of Dr. H.E.H. Portions of the paper were developed from the thesis of R.N. We thank the AUB Core Facilities for providing access to their imaging, Animal Care, and core culture facilities. We also thank the Office of Grants and Contracts at the AUB.
Author contributions
R.N., M.T.G.R., D.M.P.-V., M.G., H.E.H., and M.L. designed research; R.N., M.T.G.R., D.M.P.-V., M.H., and T.M. performed research; M.J.B. contributed new reagents/analytic tools; R.N., M.T.G.R., D.M.P.-V., M.G., H.E.H., and M.L. analyzed data; and R.N., M.T.G.R., D.M.P.-V., H.E.H., and M.L. wrote the paper.
Competing interest
The authors declare no competing interest.
Footnotes
This article is a PNAS Direct Submission.
Contributor Information
Hiba El Hajj, Email: he21@aub.edu.lb.
Maryse Lebrun, Email: maryse.lebrun@umontpellier.fr.
Data, Materials, and Software Availability
All study data are included in the article and/or SI Appendix.
Supporting Information
References
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Appendix 01 (PDF)
Data Availability Statement
All study data are included in the article and/or SI Appendix.