**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 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.*

#### **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).*

#### **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**).

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**).

#### **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 hostderived 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 [16–21]. Regarding Plasmodium falciparum, the data are controversial [22–25]. 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].

#### *Nutrient Uptake Portals in* Toxoplasma gondii *Tachyzoites DOI: http://dx.doi.org/10.5772/intechopen.107853*

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 [30–32]**.**

#### **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*

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.
