Nutrient Uptake Portals in *Toxoplasma gondii* Tachyzoites

*Marialice da F. Ferreira-da-Silva, Mauricio Magalhães de Paiva, Erick Vaz Guimarães and Helene S. Barbosa*

## **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 fluidphase 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.

**Keywords:** *toxoplasma gondii*, endocytosis, nutrient uptake, tachyzoites, ultrastructural analysis

#### **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:

a.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);

b.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**).

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**).

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 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.*

#### **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).*

#### **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.*
