Nutrient Uptake Portals in Toxoplasma gondii Tachyzoites

Abstract

The process of nutrient acquisition by Toxoplasma gondii tachyzoites is an attractive target for developing and designing drugs against toxoplasmosis, however, just recently it was revealed to be an important process to be understood. The present work helps address the lack of information about the exact sites where nutrient uptake in T. gondii. The endocytosis of proteins by tachyzoites of T. gondii was measured using both fluid-phase and receptor-mediated endocytic tracers. Quantitative analysis by flow cytometry revealed important differences in the percentage of labeled parasites, incubated with BSA, dextran, or transferrin. The analysis by confocal microscopy showed that the anterior portion of the conoid is one preferential site for binding BSA and transferrin to the tachyzoite, later localized within elongated structures present in the anterior region of the parasite. The ultrastructural analysis of multiple ultrathin sections displayed the endocytic markers at the following: (i) conoid, within rhoptries, (ii) in cup-shaped invagination of the parasite membrane (micropore) and, (iii) posterior pore. The present study brings data revealing three possible nutrient uptake portals in Toxoplasma tachyzoites that may contribute in the future to a therapeutic design with a view to treatment of toxoplasmosis.

1. Introduction

Toxoplasmosis is a disease that results from the infection caused by the coccidian parasite Toxoplasma gondii. It is a significant public health problem worldwide. About half of the world’s population is infected with Toxoplasma, but most people are asymptomatic [1]. One of the most severe manifestations of toxoplasmosis is when the acquisition of the infection occurs in the first trimester of pregnancy. The parasite can cross the placenta and reach the fetus causing congenital toxoplasmosis [2]. This infection can be systemic and result in fetal death, preterm delivery, intrauterine growth retardation, fever, pneumonia, hepatosplenomegaly, thrombocytopenia, or affect the eyes and brain [3, 4]. Considering toxoplasmosis in the current COVID-19 scenario, recently a study showed that Toxoplasma-infected patients are a greater risk of having a more severe course of the disease [5]. Despite extensive research on Toxoplasma since it’s discovery in 1908, some aspects of T. gondii nutrient acquisition have just recently received special attention and many remain poorly understood [6]. The initial discovery on nutrient acquisition by Apicomplexa (including Toxoplasma and it’s close relative Plasmodium falciparum) was from 1961, when Garnham discovered the micropore by in P. falciparum. Since, it has been considered a mechanism for nutrition in Apicomplexa [7]. With regard, specifically to Toxoplasma, only one article analyzed ultrastructurally the endocytosis of the parasite. These data have served for years as a reference, describing the role of the micropore as responsible for the incorporation of nutrients by tachyzoites and bradyzoites [8]. This structure is a continuous cup-shaped invagination of the plasma membrane located in the anterior region of the parasite. Associated with this membrane is the internal membrane complex, forming a concentric electron-dense ring that surrounds the “neck” of the micropore invagination [7]. The micropore, coated or uncoated by clathrin, is present in all three infective forms of the parasite (bradyzoites, tachyzoites, and sporozoites) [8, 9]. The rhoptries deserve to be mentioned too. They are in the number of 8–12 per cell and are the only known acidified organelles in T. gondii (pH of immature rhoptries is 3.5–5.5 and of mature rhoptries is 5.0–7.0). Recent studies indicate that they are most analogous to secretory lysosomal granules [10].

Despite the importance of Apicomplexa parasites, including Toxoplasma gondii, in public health and the fact that the nutrient acquisition process are in general attractive targets for antimicrobial drugs, knowledge of the mechanisms involved in this process in Toxoplasma is still scarce [11]. Thus, nutrient incorporation pathways and intracellular traffic in T. gondii are fields yet to be explored in depth. These studies could potentially contribute to a direct and specific therapy for this parasite, for the benefit of patients with disseminated toxoplasmosis.

2. Experimental design

2.1 T. Gondii tachyzoites isolation

Tachyzoites of Toxoplasma gondii (RH strain) were maintained in Swiss mice, weighing about approximately 21 g, through intraperitoneal passages with inoculum of 2 x 10⁶ parasites/animal. Mice were obtained from Science and Technology for Biomodels Institute (ICTB-Fiocruz). After 48 to 72 hours of infection, the parasites were collected from the peritoneal exudate and collected in phosphate buffered saline (PBS) solution, pH 7.2. The cell suspension was centrifuged at 200 g for 10 min and the supernatant containing the parasites was centrifuged at 1000 g for 10 min and the sediment rich in T. gondii tachyzoites, was washed 2 or 3 times in PBS solution, pH 7.2, and quantified in a Neubauer chamber [12]. All procedures to obtain the parasites from infected mice were performed according to the Safety Standards established by the Ethical Committee for Animal Use of Fiocruz, license L-042/2018 A2.

2.2 Endocytosis assays

The endocytic capacity of T. gondii extracellular tachyzoites was analyzed using the following endocytic tracers:

1. fluid phase markers: (i) bovine serum albumin (BSA), a 66 kDa protein, conjugated to fluorescein (BSA-FITC) or to colloidal gold particles (BSA-Au); (ii) peroxidase, 40 kDa glycoprotein, conjugated to colloidal gold (HRP-Au) and (iii) dextran, hydrophilic polysaccharide synthesized by Leuconostoc bacteria of 4.4 kDa conjugated to TRITC (Dextran-TRITC);

2. receptor-mediated endocytic markers: transferrin, an 80 kDa protein, conjugated to fluorescein (Tf-FITC) or to colloidal gold particles (Tf-Au).

2.3 Processing for analysis by flow cytometry

Flow cytometry analysis was performed after washing the tachyzoites in PBS pH 7.2 and incubating for 15 min, 30 min, 2 hr. and 4 hr. at 37°C with 0,2 mg/ml BSA-FITC, 5 mg/ml Dextran-TRITC or 1 mg/ml Tf-FITC diluted in PBS. After three washes in PBS, the parasites were fixed for 20 min at 4°C with 4% PFA, washed 3 times for 10 min each with PBS and analyzed by flow cytometry on the same day. Non-incubated tachyzoites with the fluorochrome-labeled tracers were used to calibrate the system for morphology and granularity. Data acquisition was performed using the FACSCalibur flow cytometer (Becton & Dickinson, San Jose, USA) equipped with Cell Quest software (Joseph Trotter, Scripps Research Institute, San Diego, USA). The analyses were performed using the WinMDI2.8 program on 10,000 events acquired in a pre-established region corresponding to the parasites at the Cytometry Flux Platform at Oswaldo Cruz Institute.

2.4 Processing for analysis by laser scanning confocal microscopy

T. gondii tachyzoites freshly isolated from Swiss mice and purified from peritoneal lavage as described in 1.1 were incubated with 0,2 mg/ml BSA-FITC or Tf-FITC, for periods of 10 min, 30 min, 1 hr. or 2 hr. at 37°C, followed by washing in PBS. A drop containing the tachyzoites was incubated at 37°C for 5–15 min for parasite adhesion on a slide previously coated with poly-L-lysine. The parasites were then washed twice with PBS, followed by fixation for 5 min at room temperature with 2% paraformaldehyde (PFA). The parasites were then washed in PBS and distilled water and the coverslips mounted in DABCO (1,4 Diazabicyclo [2.2.2] octane - Triethylenediamine - “antifading”). The material was analyzed on an Olympus FV 300/BX51 laser scanning confocal microscope at the Biomanguinhos Applied Pharmacology Laboratory, Fiocruz. Fluorescence was stimulated by a 488 nm laser and 510 longpass filters combined with another 543 nm laser and 560/600 bandpass filters were applied. Differential interference contrast microscopy images were obtained simultaneously with fluorescence images in different focus planes.

2.5 Colloidal gold-protein complex

For ultrastructural analysis colloidal gold particles with an average diameter of 15 nm were obtained according to the Frens method [13]. For the formation of the colloidal gold-protein complex, the pH of the colloidal gold was adjusted to 5.5 for conjugation with albumin (BSA); 8.0 for peroxidase (HRP) and 5.0 for transferrin (Tf). The concentration of each protein required to stabilize 10 ml of colloidal gold was added and the protocol followed according to the method of Slot and Geuze (1985) [13]. Endocytic tracers were purchased by Sigma-Aldrich-St. Louis, MO, USA.

2.6 T. gondii tachyzoites endocytic assays by transmission electron microscopy

For ultrastructural analysis 10⁸ tachyzoites were centrifuged at 1000 g for 10 min and the sediment was resuspended in PBS containing BSA-Au, HRP-Au or Tf-Au at protein/PBS ratios of 1:10. Parasites were incubated at 4°C for 20 min and then at 37°C for periods of 5 min to 4 h. After this incorporation kinetics, the solution containing tachyzoites was centrifuged at 1000 g for 10 min and the pellet washed 2 times in PBS solution. Then, tachyzoites were fixed for 30 min at 4°C at 2.5% glutaraldehyde (GA) in cacodilate buffer with 2.5 CaCl₂ and 3.5% sucrose, pH 7.2, washed and centrifuged three times for 15 min in the same buffer. They were post-fixed for 30 min at room temperature, with 1%, osmium tetroxide and washed with the same buffer. The parasites were dehydrated in a graded acetone series and embedded in an epoxy resin (PolyBed 812). Thin sections were stained with uranyl acetate and lead citrate and then examined under a transmission electron microscope (Jeol JEM1011) at the Rudolf Barth Electron Microscopy Platform at Oswaldo Cruz Institute.

3. Results

3.1 Bovine serum albumin

For quantitative analysis, flow cytometry was used as a tool to check the association of the fluid phase endocytosis marker, BSA-FITC to tachyzoites. The region (R1) was previously established as corresponding to the parasites (Figure 1A). The negative control refers to the group of parasites not incubated with the fluorochrome (Figure 1B). The labelling of tachyzoites with BSA-FITC was time-dependent, with percentages of 16.5%, 17.5%, 27.5%, and 32%, of labeled parasites after incubation at 37°C for 10 min, 30 min, 1 h and 2 h, respectively (Figures 1C–F).

Figure 1.

Representative histograms of flow cytometry showing the kinetic of BSA-FITC internalization by T. gondii tachyzoites incubated at 37°C for different lengths of time. (A-B) Parasites incubated with PBS. Graphics showing the morphology of the parasites. (A) Size and granularity (FSC x SSC) and the region of analysis R1. (B) Negative control of the marker. (C-F) FACS analysis of parasites incubated with BSA-Au for periods of 10 min to 2h. The kinetic show that the labelling is time-dependent.

The confocal microscopy analysis after incubation of T. gondii tachyzoites with BSA-FITC for periods of 5 min to 2 h at 37°C revealed that a small population of parasites showed labelling (Figure 2A–D). After 5 min incubation the marker was strictly localized in the anterior region of the parasite body, corresponding to the apical complex (Figure 2A and B). After 30 min of incubation a higher concentration of the tracer was observed in the apical region of the parasite in addition to a fine granulation with symmetrical distribution along the first third of the tachyzoite body (Figure 2C). Few tachyzoites showed the tracer already internalized (Figure 2C). Parasites kept for 2 hours at 37°C in the presence of BSA-FITC revealed the fluorescent marker located at the tip of the apical region and in a possible invagination of the body (possibly micropore) and as well intracellular marker concentrated in the posterior region corresponding to basal complex (Figure 2D).

Figure 2.

Confocal microscopy analysis of T. gondii tachyzoites incubated with Bovine Serum Albumin conjugated with FITC (BSA-FITC). (A and B) At 37 º C for 5 min: (A) the tracer is observed in the apical region (arrow) and (B) a fine granulation extending symmetrically along the first anterior third of the parasite’s body. (C) At 37°C for 30 min: the marker is located in the first third of the parasite body (arrowhead). (D) After 2 h at 37°C: BSA-TRICT is internalized through its posterior region (arrowhead).

Transmission electron microscopy analysis showed that BSA-Au labelling was not homogeneous among tachyzoites. Incubation for 20 min at 4°C revealed discrete labelling in some parasites with one or two gold particles associated with their membrane, particularly in the vicinity of the apical region (not shown). Parasites analyzed after incubation for 30 min at 37°C showed tachyzoites contained gold particle in a depression of the plasma membrane, showing morphological features compatible with a microspore (Figure 3). The intracellular localization of the BSA-Au complex was observed in rhoptries of some parasites after incubation for 1 h at 37°C (data not shown).

Figure 3.

Ultrastructural analysis of tachyzoite of T. gondii incubated with BSA-Au. For 10 min. Image showing a gold particle (arrow) at a plasma membrane depression, which displays compatible characteristics such as a micropore.

3.2 Peroxidase

Experimental assays using HRP as a tracer of fluid phase endocytosis in tachyzoites were performed for transmission electron microscopy analysis only. No labeling was observed in the parasites after incubation at 4°C for 20–30 min (data not shown). The endocytic capacity of tachyzoites was tested during incubation for periods ranging from 5 min to 2 h at 37°C. During the course of the experiments, localization of HRP-Au on the surface of the parasites was rarely observed showing a low association of tracer. The labelling profile was altered when we increased the concentration of HRP-Au, revealing a greater association of colloidal gold particles on the surface of the tachyzoites (not shown). In all experimental assays performed at 37°C, the intracellular localization of HRP-Au in tachyzoites was exclusively into rhoptries (Figure 4A–C). Presence of HRP-Au particles was commonly observed in more than one rhoptry in the same parasite (Figure 4A–C).

Figure 4.

Ultrastructure of tachyzoites incubated for 5 min to 2 h at 37°C with HRP-Au. Longitudinal sections of parasites revealed particles of the HRP-Au complex (arrowhead) inside rhoptries (R). It was common to observed HRP-Au particles in more than one rhoptries (R) in the same tachyzoite.

3.3 Dextran

Quantitative analysis of tachyzoites labelling with dextran-TRITC was performed by flow cytometry. The negative control is parasites without prior incubation with the fluorescent tracer (Figure 5A and B). The results obtained from 10 min to 2 h of incubation at 37°C showed a slight increase in the labelling as a function of incubation time, however with very low values of 0.17% and 1.42% after 30 min and 2 hours, respectively (Figure 5C–F). We did not observe any parasite labeled by confocal microscopy.

Figure 5.

Analysis by flow cytometry of the incubation of tachyzoites with Dextran-TRITC. A: Tachyzoites incubated with PBS. Graphic show the morphology of the parasites, in terms of size and granularity (FSC x SSC) and the region of analysis R1. (B) Negative control. (C-F) The kinetics of Dextran-TRITC incorporation showed a low percentage of labelled parasites over time

3.4 Receptor-mediated endocytosis

Quantitative analysis of tachyzoites incubated with transferrin was performed by flow cytometry. Negative control is parasites maintained in PBS alone (Figure 6A and B). After incubation with Tf-FITC, we observed that the number of Tf-FITC-associated tachyzoites was relatively constant reaching levels of 12.74% and 15.05% after 10 min and 2 h, respectively (Figure 6C and F).

Figure 6.

Representative histograms of flow cytometry showing the kinetic of transferrin conjugated with FITC (Tf-FITC) internalization by T. gondii tachyzoites incubated at 37°C for different lengths of time. (A) Parasites incubated with PBS. Graphics showing the morphology of the parasites, in terms of size and granularity (FSC x SSC) and the region of analysis R1. (B) Negative control of the marking. (C-F) Kinetics of the incorporation of Tf-FITC by the tachyzoites. The percentage of marked parasites remained constant during incubation for 10, 30, 60 and 120 min.

By confocal microscopy analysis after 5 min incubation at 37°C with Tf-FITC we did not observe any tracer-associated parasites. However, 30 min or 2 h incubation at 37° C with Tf-FITC resulted in the uptake of the tracer by the tachyzoites. Tf-FITC was seen concentrated at one pole of the parasite’s body or distributed throughout its cytoplasm (data not shown). Ultrastructural analysis of tachyzoites incubated with Tf-Au showed a low association with the surface of the parasites. After incubation for 30 min at 37°C, tachyzoites were observed in transverse/oblique sections of the apical region to allow frontal visualization of the conoid. There was localization of the tracer at the tip of tachyzoites, positioned centrally (Figure 7B). These analyzes also revealed the presence of Tf-Au particles inside the rhoptries (Figure 7B and C).

Figure 7.

Ultrastructure of tachyzoites incubated for 5 min to 2 h at 37°C with transferrin-Au (Tf-Au). (A) Transverse/oblique section of the apical region of a tachyzoite with the frontal view of the conoid and the location of the Tf-Au particle at its tip, positioned centrally (arrow). (B and C) Longitudinal ultrafine cut of tachyzoite displays two Tf-Au complexes inside the same rhoptry (arrowhead).

4. Discussion

The study of nutrient uptake mechanisms by T. gondii represents a challenge, mainly because they are mandatorily intracellular parasites. They invade the host cell and replicate delimited within a parasitophorous vacuole (PV), delimited by a host-derived membrane that is extensively modified by the parasite to facilitate nutrient acquisition and minimize attacks from the host cell [14]. This compartmentalization represents physico-biochemical barriers, involving the plasma membrane of the host cell, the membrane of the PV and also the membrane of the parasite. Until recently, little has been explored regarding the pathways of incorporation of macromolecules by tachyzoites. The origin of this knowledge comes mainly from the very early studies on this topic written by: (i) Sénaud et al. (1976) who proposed the micropore as a nutritional organelle [7]; and (ii) Nichols et al. (1994) [8], who used the fluid phase tracer (HRP) to demonstrate the incorporation of cyst matrix material by bradyzoites through the micropore. This process involved the formation of vesicles coated or not by clathrin. Nevertheless, the proposal of the micropore’s role as a specialized endocytosis site is still under discussion [6]. Here we investigated the uptake capacity of tachyzoites by using macromolecules routinely employed for endocytosis assays, such as fluid phase and receptor-mediated endocytosis tracers..

The performance of the endocytosis assays, with single parasites through FACS enabled quantitative analysis of labeled parasites in a whole population using different endocytosis tracers. Our results showed that the incorporation or binding was tracer dependent. The highest values by FACS were achieved with BSA as endocytic tracer. Percent of parasites positive for labeling with BSA-FITC increased in time dependent manner from 16.5%, at 10 min to 32%, at 2 hours. In this case, it showed us a time-dependent labeling with the tachyzoites. On the other hand, by using the fluid phase endocytic marker dextran, it turned to be that their association was extremely low, independent of the time of incubation. The concentration of dextran used was not a determining factor of its association with tachyzoites, since using twice the concentration (5 mg/ml) we obtained similar association indexes (1.4% after 2 hours of incubation). They mentioned that only a minority of labeled parasites was found after incubation with transferrin, without having conducted a systematized study. However, results presented here show binding with. This tracer. In this case, the labeling was not time-dependent and reached levels of 15% of labeled parasites.

We tried to infer whether the size of the molecule would affect the ability of tachyzoites uptake. For example, the dextran tracer selected for this study was 4.4 kDA including fluorochrome. The BSA and transferrin tracers have mass approximately 66 kDa and 80 kDa, respectively. Our data demonstrate that the size of the molecule cannot be responsible for the level of incorporation of the marker, considering that the lowest indices obtained in the current work were observed during the assays with dextran, which was the smallest molecule employed by us and that due to its low association with tachyzoites, it was not possible to determine its intracellular location.

It was not possible in our experimental conditions to observe a large number of tachyzoites marked with the tracers used. This difficulty has been noted for fluid and receptor-mediated endocytic markers in the review article by Robibaro et al. (2001) [15]. Results showed that only with high concentrations (around 2 mg/ml) of dextran-FITC and lucifer yellow, allowed detection of uptake by the tachyzoites, allowed to detect uptake by the tachyzoites, reaching percentages of 1 and 8%, respectively, without, however, identifying the route of internalization or the location of the markers in the parasites. Prior data from incubation with BSA-FITC showed a diffuse labeling in some parasites, with no evidence of incorporation via vesicle-like structures.

We also showed the focal BSA-FITC labeling of the surface of the parasite was limited to the anterior surface of the parasite, limited to the anterior (apical) region, where the conoid is located. These data were later corroborated by ultrastructural analyses, where we showed BSA-gold nanoparticles marking the same region of the parasite. The endocytosis tracer was restricted to the two thirds of the anterior of the anterior region of the tachyzoite, contained in elongated vesicles located immediately below the tachyzoite membrane. Additionally, after 2 hours, we documented a diffuse distribution of the marker in the posterior region of some parasites. We demonstrated here that despite the low number of parasites capable of endocytosis with BSA, this kinetic study shows the adhesion of the marker to the surface of the parasite, restricted to the apical region, its subsequent incorporation into structures located in the first two thirds of the parasite body and a possible traffic of this tracer to the posterior region of the parasite.

Aiming to study the receptor-mediated endocytosis pathway in T. gondii, we employed transferrin as ligand. Our data suggest that the mechanisms used by Toxoplasma tachyzoites, may be through: (i) the membrane in the posterior third of the parasite body, by the presence of vesicles containing HRP just below the plasma membrane, a region devoid of subpellicular microtubules, which would favor a greater endocytic activity of the membrane; (ii) the membrane in the first anterior third of the parasite body, due to the presence of vesicles and tubules containing HRP particles in this region. Transferrin-specific receptors have been demonstrated in some other protozoans [16, 17, 18, 19, 20, 21]. Regarding Plasmodium falciparum, the data are controversial [22, 23, 24, 25]. In the case of T. gondii, it has been described that lactoferrin, a protein of the transferrin family, binds to its surface [26]. Our quantitative results, through flow cytometry, showed that the association of transferrin with tachyzoites was stable over 2 hours (about 15%). The analysis of this association by confocal microscopy showed the localization of the protein in the interior of the parasites, initially concentrated in the apical region and after 2 hours, located in the median region of its body. T. gondii tachyzoites were able to incorporate transferrin suggesting the presence of receptors for transferrin. The presence of transferrin binding sites on the surface of a subgroup of parasites may be related to a certain stage of its cellular cycle, or be dependent on the induction of expression of these surface receptors in the presence of the ligand, as proposed by Botero-Klein et al. (2001) [27], during the characterization of heparin receptors in extracellular tachyzoites. Thus, these data indicate the need for the identification and characterization of this possible receptor for transferrin or an independent iron capture pathway in T. gondii. Since there is no excrement route of iron, nor in single - or multicellular organisms, its homeostasis is dependent on the regulation of the level of its uptake, essential for most eukaryotes and prokaryotes [28].

Nichols et al. [8] observed by ultrastructural analysis the internalization, of the fluid phase marker HRP by the micropore of T. gondii. Since the base of the micropore in both tachyzoites and bradyzoites is sometimes coated with clathrin, it is possible that receptor-mediated endocytosis via the micropore also occurs. The possible role of the micropore as a site of endocytosis is still unclear and under discussion. Endocytosis of fluid phase tracers via non-specific pinocytosis appears to occur at sites located below the apical region. Our studies by confocal microscopy on the internalization by tachyzoites of fluid phase markers and those of receptor-mediated endocytosis demonstrated the association occurred preferentially through the apical region of the tachyzoites, with their intracellular localization into elongated structures present in the first anterior third of the parasite body. Thus, our results suggest that the micropore is not the only single or preferred route of incorporation of macromolecule by the tachyzoites. The current ultrastructural analysis allowed us to accumulate numerous pieces of evidence of the intracellular presence of endocytic tracers in T. gondii.

The question is: How would the markers have access to the interior of the rhoptries? Possibly, by anterior region, via conoid, since it is a region of high exocytic activity allowing the secretion of compartmentalized molecules into the external milieu during the stages of binding (adhesive proteins of the micronemes) and invasion of host cells by parasites (proteins of the rhoptries, involved in the biogenesis of the PVM). It has been proposed that terminal part of the peduncle of the rhoptry fuses with the plasma membrane lining the conoid and the entire surface of the parasite [29] and enables movement of the rhoptry contents from the intracellular to the extracellular environment. It is possible that a reflux of extracellular material may occur into the rhoptries during the exocytosis of thier contents. Based on the evidence presented here, specifically colloidal gold particle presence in the anterior region of the conoid, and accumulation of fluorescent markers at the end of the anterior region of tachyzoites, seen by confocal microscopy (Figures 1–5) we hypothesize existence of an inverse pathway where molecules can transit into the rhoptry. Botero-Kleiven et al., (2001) [27], have described that heparin receptors, which we would expect to be located where there is access to host cell were localized in elongated structures, perpendicular to the longitudinal axis of the tachyzoite with a size ranging from 0.5–1.5 nm. This description is compatible with the morphology of the rhoptries, although the authors did not mention this hypothesis.

Another route movement of proteins into rhoptries could be endocytosis via micropore, considering that: (i) previous results have been shown to be a site of endocytosis with vesicles formation at its base either coated or not by clathrin [8]; (ii) there is indirect evidence by confocal microscopy of the presence of heparin binding sites in the anterior lateral region of the parasite body that could correspond to the micropore [27]; (iii) our rare images (Figure 2A and 3C) implicate posterior pore endocytosis by demonstrating the location of markers, after short incubation times, in the posterior region of the parasite, in proximity to the pore (Figure 6). This pore has been described as a site of great exocytic activity of dense granules, as demonstrated by Sibley et al. (1995) [27]. Following the same line of reasoning proposed for endocytosis via the conoid, this could also be a site potentially capable of incorporating molecules. The involvement of the posterior pore in this process is supported by the review in Romano and colleagues attributing a multifunction ability to the basal complex (posterior pore) during its participation in host vesicle remodeling and/or lipid uptake [30, 31, 32].

Based on the results presented here and supported in literature we suggest a hypothetical model proposing 3 nutrient uptake portals of macromolecules by tachyzoites of T. gondii (Figure 8). The first portal (or site) proposes the endocytosis of macromolecules by conoid with its subsequent localization in the rhoptries interior. The second portal, by micropore, leads to vesicle-based transport to rhoptries or to the Golgi following to the rhoptries. Or yet, the third portal would be through the posterior pore with subsequent transport to the Golgi apparatus and/or to the rhoptries. Our hypothesis that the transit of molecules incorporated by T. gondii has the rhoptries as its final destination is based on evidence that they are the only acidified organelles of the parasite and that they would be analogous to lysosomal secretory structures which receive material from the endocytic pathways of the cell [33].

In the future, we hope there will be many strategies and approaches to control diseases, like Toxoplasmosis. One Health should bridge disciplines linking human health, animal health, and ecosystem health and that treating and managing Toxoplasmosis demands integrative approaches to breach disciplinary boundaries. Nevertheless, to characterize the mechanisms and portals involved in the acquisition of nutrients by tachyzoites of T. gondii and identifying its portals may be an important contribution to the understanding of the biology of the parasite and also be applied as a target for drug action.

5. Conclusions

The ubiquitous parasite Toxoplasma gondii has been the subject of intense investigation over the last years. Neverthless, there are still many open questions regarding the nutrient acquisition by this parasite. In this work, we tested the endocytic capacity of extracellular tachyzoites of T. gondii. Our results showed innumerous evidence of the intracellular presence of endocytic tracers in the anterior region of the conoid, inside the rhoptries, at the micropore and posterior pore of the parasite. As a result, we proposed a hypothetical scheme (Figure 8) aiming to summarize data presented here. These hypotheses should be confirmed through further studies using 3D reconstruction of serial ultrathin sections, for example, or through live cell imaging. The set of data presented here may contribute to new perspectives in this field, enabling in the future a better understanding of the biology of T. gondii, enabling application in therapeutic interventions in the treatment of toxoplasmosis.

Figure 8.

Schematic representation of three different nutrient absorption portals suggested for Toxoplasma tachyzoites: conoid reaching rhoptria, in red; micropore in blue and basal complex in green. The hypothesis is that the uptake of nutrients via the conoid, micropore or basal complex could be a intracellular transported route of nutrients to the rhoptries and be digested there, considering that the pH 5.0 of the rhoptries, is a favorable environment for the activation of lysosomal enzymes

Acknowledgments

We thank Sandra Maria de Oliveira Souza, Rômulo Custodio dos Santos and Genesio Lopes de Faria for technical assistance. We are grateful also for Carlos Bizarro from Confocal Platform of PDTIS-Fiocruz for help with an Olympus FV 300/BX51 laser scanning confocal microscope and Bruno Avila for the support with the images. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação Oswaldo Cruz (Programa Estratégico de Apoio à Pesquisa em Saúde - PAPES VI), Pronex - Programa de Apoio a Núcleos de Excelência - CNPq/FAPERJ, Edital FAPERJ N° 15/2019 Apoio a Redes de Pesquisa em Saúde no Estado do Rio de Janeiro and Instituto Oswaldo Cruz/Fiocruz.

Conflict of interest

The authors declare no conflict of interest.