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Modeling Toxoplasma gondii-gut early interactions using a human microphysiological system [1]

['Carlos J. Ramírez-Flores', 'Department Of Medical Microbiology', 'Immunology', 'University Of Wisconsin-Madison', 'Madison', 'Wisconsin', 'United States Of America', 'Department Of Infectomics', 'Molecular Pathogenesis', 'Center For Research']

Date: 2025-05 

Abstract Oral transmission of parasites via environmentally resistant cyst stages in contaminated food or water is a common route of human infection, but there are no effective vaccines available for any enteric parasitic infection. Our knowledge of parasite cyst stage conversion and interaction with the intestinal tract is limited. Here, we investigate infection dynamics of Toxoplasma gondii cyst-stage in murine jejunum and human intestinal microphysiological systems. We focus on parasite ingress, replication, and conversion of the cyst stage to the rapidly replicating dissemination stage. In vivo bioluminescent imaging of mice fed cysts revealed spots of infection throughout the jejunum and ileum, which were selected for further analyses. Immunostaining showed parasite migration and replication predominantly in the stroma, with minimal replication in enterocytes. We recapitulated bradyzoite infection in human intestinal microphysiological systems and showed stage conversation and migration through collagen. This integrated approach elucidates complex host-parasite interactions, highlighting the value of microphysiological systems in advancing understanding and identifying potential therapeutics.

Author summary Toxoplasma gondii is a widely distributed parasite that infects many people around the world. Its infection is linked to behavioral and metabolic disorders and can lead to serious health complications, particularly in individuals with weakened immune systems. In this study, we investigate how the parasite infects the intestine in the early days of infection and begins to spread within the gut in mice. We focus on the dormant form of the parasite, known as bradyzoites, and examine when they transform into the actively replicating tachyzoite stage within mouse intestines. Our in vivo data reveal that T. gondii primarily replicates in the intestinal stroma during early infection. To further understand this process, we used microphysiological systems to model intestinal infection in humans. This innovative approach allowed us to track the conversion between parasite stages and their subsequent dissemination. Our findings highlight the complex interactions between the host and the parasite, demonstrating the potential of microphysiological systems to uncover critical aspects of T. gondii infection. This research enhances our understanding of how this parasite spreads in humans and may inform future strategies for prevention and treatment.

Citation: Ramírez-Flores CJ, Hryckowian ND, Gale AN, Babatunde KA, Lares M, Beebe DJ, et al. (2025) Modeling Toxoplasma gondii-gut early interactions using a human microphysiological system. PLoS Negl Trop Dis 19(2): e0012855. https://doi.org/10.1371/journal.pntd.0012855 Editor: Sarah Ewald, University of Virginia, UNITED STATES OF AMERICA Received: October 2, 2024; Accepted: January 20, 2025; Published: February 4, 2025 Copyright: © 2025 Ramírez-Flores et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All data are available in the main text or the supplementary materials. Funding: This work was supported by a Food Research Institute (LJK), National Institutes of Health National Institute of Allergy and Infectious Diseases 1R01AI172874 (LJK, SCK, and DJB), and a Ruth L. Kirschstein Postdoctoral Individual National Research Service Award from the National Institutes of Health National Institute of Allergy and Infectious Diseases F32 AI172084 (NDH). University of Wisconsin Carbone Cancer Center Support Grant NIH P30CA014520. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: We have read the journal's policy, and the authors of this manuscript have the following competing interests: DJB holds equity in Bellbrook Labs LLC, Tasso Inc., Salus Discovery LLC, Lynx Biosciences Inc., Stacks to the Future LLC, Turba LLC, Flambeau Diagnostics LLC, and Onexio Biosystems LLC. DJB is also a consultant for Abbott Laboratories. The other authors declare that they have no competing interests.

Introduction Toxoplasma gondii is a cosmopolitan parasite that infects nucleated cells in warm-blooded animals [1,2]. It is estimated that approximately one-third of the human population is infected by T. gondii [3]. In the United States, 20–30% of people are seropositive; in contrast, in Brazil and France, it is estimated that more than half of their population are seropositive [3–7]. This widespread distribution has led many to consider T. gondii to be one of the most successful parasites known in nature [8]. Despite high rates of infection, there are currently no vaccines available for humans, and treatment options are limited. A live attenuated vaccine called ToxoVax (MSD, New Zealand) effectively reduces losses in breeding sheep, highlighting the potential for future human vaccines [9]. Human infection typically occurs through ingestion of undercooked meat containing tissue cysts, or through water or food contaminated with mature oocysts released in feces from felines, such as the domestic cat [10–13]. T. gondii infection in healthy hosts typically manifests as an asymptomatic infection characterized by parasite encystment in muscular tissue and brain tissue [13–15]. However, in immunocompromised individuals, parasite reactivation can lead to chorioretinitis and blindness, or even fulminant encephalitis [16,17]. In pregnant individuals, T. gondii infects the fetus, causing congenital toxoplasmosis with severe outcomes like hydrocephalus, chorioretinitis, and intellectual disabilities [18,19]. Tissue cysts contain bradyzoites, a life stage characterized by the presence of amylopectin granules, and the expression of specific markers including BAG1, LDH2, ENO1, and BRP1 [20–24]. Bradyzoites are known for their slow-replicating nature, contributing to the parasite’s ability to persist chronically in infected hosts [15,22]. Upon consumption, cysts undergo digestion by pepsin in the host’s stomach [15,25–28]. A current well regarded theory states that upon reaching the small intestine, parasites promptly infect enterocytes, revert to the rapidly replicating tachyzoite stage, disseminate throughout the host, and eventually develop into the cyst stage, which is a hallmark of chronic infection [10]. While this infection progression has been studied in mouse models, the initial interactions in the small intestine following the ingestion of cysts is not yet well understood. Oocysts contain eight infectious sporozoite parasites, which have been shown to rapidly infect ileal enterocytes, migrate to the lamina propria, and convert to tachyzoites within 24 hours [29,30]. Subsequently, parasites replicate in the lamina propria, spread to various organs, and reach the brain by day 6 post-feeding [29,30]. While intestinal bradyzoite infection appears to share similarities with intestinal sporozoite infection, our understanding of early bradyzoite infection in the small intestine is more limited. In mice fed bradyzoites, most of the early infection occurred in the distal half of the small intestine. The transition from bradyzoites to tachyzoites is suggested to appear 12–18 hours after feeding, as evidenced by parasite morphological changes in micrographs [31]. Within 1–3 days post-feeding, parasites were found replicating in both enterocytes and the lamina propria, with an increased parasite number in the intestine after 2–3 days. Subsequently, parasites were detected in the brain 6–7 days post-feeding [31]. Studying the first 3 days of the bradyzoite infection process in mouse intestines has proven challenging, hindering a comprehensive understanding of this process. Studies have focused primarily on the host immune response within the intestine after 3 days post-infection [32,33]. Gregg et al observed infected villi in the proximal jejunum of mice fed cysts 3 days post-consumption with the presence of macrophages, neutrophils, and monocytes in the lamina propria [32]. Coombes et al described the infection in the distal third of the small intestine of mice fed cysts [33]. By 5–6 days post-feeding, single parasites were detected within villi, with scarce locations containing already replicating parasites, often concentrated at the villi tips [33]. Despite the various approaches aimed at understanding early bradyzoite infection in the mouse model, our comprehension of processes such as bradyzoite release, interaction with the intestine in the early phases of the infection, parasite motility through mucus, villi infection, parasite conversion, and dissemination through the intestine and the organism remains limited. This limitation is particularly pronounced in humans, where the infection dynamics and interactions have obviously been limited to cell lines. Addressing these gaps is critical for developing novel approaches to prevent infection and vaccine development. Here we examine the initial phases of bradyzoite infection in mice and microfluidic models to simulate human intestinal interactions. By integrating microphysiological systems (MPS) with mouse infection studies, we aim to deepen our understanding of these processes in both mice and humans [34,35]. This study tracks early bradyzoite dynamics in the small intestine of orally infected mice and their replication in the jejunal stroma. We then use human MPS to explore the initial interactions and infections of bradyzoites in the human gut. Together, the use of both mouse and MPS allows us to identify key aspects of bradyzoite infection, including the site of replication in the villi, stage conversion kinetics, bradyzoite activation by pepsin, and parasite migration patterns within collagen. Our findings demonstrate that human MPS can effectively replicate bradyzoite conversion into tachyzoites and their infection patterns observed in mice. This work builds upon existing research and highlights the potential of MPS to study host-parasite interactions.

Discussion T. gondii enters the host through ingestion, with the intestine as the primary site of infection initiation. Studying the bradyzoite’s first interaction within the intestine has been limited given the complexity of working with mouse intestine and the inadequacies of live imaging sensitivity [38]. While previous works have explored the interaction of tachyzoites with human intestinal cell lines in 2D models [46], studies on bradyzoite infections in these cell lines are notably scarce. To fill these gaps, we used MPS with human intestinal cells to complement our examination of bradyzoite infection in a mouse model. We examined the dynamics of host-parasite interactions such as ingress, site preferences for invasion and replication, and the kinetics of stage conversion from bradyzoites to tachyzoites within the gut. Although the initial site of the infection has been controversial between the jejunum [32,33] and the ileum [29,30,33], our findings support T. gondii localization within both sections during early infection with a preference for parasite replication in the jejunum. For that reason, we focused on analyzing the jejunum for our in vivo infections. Parasites were predominantly observed in the intestinal stroma, with fewer parasites in enterocytes (Fig 1). Particularly noteworthy was the early presence of parasites near cell-to-cell junctions, suggesting active transmigration from the lumen to facilitate dissemination [30,32]. Previous research has suggested that extracellular tachyzoites degrade tight junction proteins and affect the epithelial polarity in 2D and 3D-transwell models as a mechanism for parasite active transmigration of the biological barriers [47–49]. In contrast to previous reports where intestinal infection often concentrated at the villi tips [33], our study found limited replication within enterocytes (Figs 1, S1, S3 and S4). This result may be attributed to the rapid turnover of enterocytes as in humans and mice, they are typically replaced every 2 to 3 days [50]. For sustained replication and dissemination, T. gondii may target more stable environments such as stromal tissue. Previous work has shown sporozoites in the lamina propria after just 6 hours post-oral ingestion of oocysts [30], suggesting a rapid and transient migration through the enterocytes. Parasite avoidance of enterocytes may also be driven by unfavorable metabolic activity in enterocytes or exposure to digestive enzymes in enterocytes [51]. In previous reports, sporozoite or bradyzoite conversion to tachyzoites relies on qualitative assessments using transmission electron microscopy or embedded tissue sections, lacking stage-specific antibody staining [29–31]. In our study, we used SAG1 for tachyzoites and BAG1 for bradyzoites to follow their conversion process, as these stage specific markers are commonly used in the field [52]. Tachyzoites exhibited active replication within the stroma starting at day 3. Detection of parasites replicating in the jejunum before day 3 post-ingestion was not feasible in our samples using embedded tissue or in vivo imaging, likely due to the initially low number of parasites on 1- and 2-days post-feeding. This limitation prompted us to use intestine scrapes and subsequently transition to MPS for our investigations (Figs 4–6). The formation of slightly swollen intestinal areas by day 5 post-ingestion, possibly due to infiltration of immune cells, further underscores the complex interplay between T. gondii and the host immune response [32,33]. The observation that enlarged and infected intestinal areas were visible to the naked eye suggests the potential to identify foci of infection without the need for a fluorescent strain. However, this phenomenon requires further investigation and quantification, which will be addressed in a subsequent report. Parasites were found near endothelial tissues (S5D Fig), suggesting potential routes for hematogenous spread, aligning with observations of Trojan horse-like migration and their infiltration to the central nervous system [45]. In vitro differentiated bradyzoites have been used to progress the understanding of HFF cell infection dynamics, particularly the role of the moving junction [53]. In vitro bradyzoites were recently evaluated for their structural, metabolic, and functional similarities to in vivo cysts, resembling them in ultrastructure, temperature stress, and tolerance to antiparasitic agents. They also presented functional hallmarks to in vivo cysts infectivity in mice and their resistance to pepsin digestion for up to 60 minutes [54]. These findings provided a foundation for using in vitro bradyzoites as a model to investigate bradyzoite infections. Our revised protocol for cultivating bradyzoites in vitro yielded high quantities for type II strains and effectively induced bradyzoite markers in the poorly cystogenic RH strain (Fig 2). The resistance to pepsin digestion was crucial for activation, confirming their functionality as bradyzoites. Pepsin digestion has been extensively used for exteriorizing T. gondii cysts in a period of time between 2 min and 2 h [53–57]. Currently, there is a published thesis showing pepsin treatment activates bradyzoite invasion [41]. In our study, we found that pepsin digestion plays a crucial role in activating T. gondii in vitro cultured- and brain isolated-bradyzoites, leading to enhanced parasite replication (Fig 3). Pepsin digestion was also vital to remove any remaining tachyzoite in our in vitro bradyzoite cultures, which agrees with previous findings [58,59]. Using human fibroblasts infected with brain cyst bradyzoites, researchers have previously observed the coexistence of bradyzoite and tachyzoite markers initially, with exclusive SAG1 expression emerging after 48 hours post-infection [59]. However, understanding this process in biologically relevant tissues such as the intestine remains incomplete. To address the gaps in understanding bradyzoite-to-tachyzoite conversion, we used Caco-2 cells in MPS mimicking human intestines (Fig 4). Bradyzoite infection and replication were significantly lower in Caco-2 cells compared to HIEC-6 (Fig 3C). Both lines are derived from cancer patients, but Caco-2 cells are derived from the colon, and HIEC-6 cells are derived from the small intestine [60–62]. In mice, the small intestine has been recognized as the predominant site of T. gondii infection [32]. In the MPS, HIEC-6 cells displayed unusual behavior by both failing to adhere to the collagen to form a complete lumen and extravagating into the collagen I/fibronectin matrix (S6 Movie). These phenotypes may be due to HIEC-6 inability to form tight junctions [63]. Caco-2 cells excelled in forming complete circular lumens in our MPS and consistently showed tachyzoite conversion within 3 days post-infection aligning with our in vivo results (Fig 4). Future studies incorporating co-cultures with gut microorganisms or immune cells in the human intestinal MPS could enhance our understanding of bradyzoite switching, potentially accelerating the process. Recently, researchers have published the use of endothelial cell lines in MPS for T. gondii tachyzoites studies opening the avenue for studying a complete bradyzoite and tachyzoite dissemination in the presence of immune cells [35,64]. These models offer valuable insights into stage conversion and infection progression, complementing traditional in vivo approaches. Additionally, co-infections with parasites like Schistosoma, Giardia, or Entamoeba histolytica—common in high-parasitic regions—may influence T. gondii virulence. The complex interactions between these pathogens and the immune response could impact T. gondii motility, switching behavior, and disease outcomes, making co-infection studies in MPS models crucial for understanding these dynamics. Our results demonstrate that bradyzoites actively migrate through the enterocyte layer and localize within the stroma, reminiscent of earlier studies where tachyzoites were observed in the lamina propria following oral ingestion of cysts or oocysts [28–30]. In the absence of systemic immune cells in our villi explants, this migration indicates that bradyzoites can actively penetrate the lamina propria. The parasites’ movement through the epithelial layer underscores their capability to invade deeper in the intestine early in the infection process. These findings validate the hypothesis that parasites actively traverse epithelial layers for dissemination purposes, potentially facilitated by effectors secreted by the parasites as previously suggested [47,65]. Tachyzoites also exhibited active migration through the collagen I/fibronectin matrix, exceeding 200 µm in width, to reach and infect adjacent Caco-2 lumens (Fig 6). This phenomenon mimics potential host cell-independent routes of T. gondii dissemination within the host as a possible route for reaching distant intestinal areas. Using collagen I for the lumens was particularly beneficial for tracking parasite migration, aligning with earlier studies using 3D collagen matrix assays to study the motility of tachyzoite-infected dendritic cells [66]. Other studies using MPS have employed collagen I as a matrix to study endothelial parasite migration [35,64]. Exploring alternative substrates, such as other types of collagens, laminin, or Matrigel, all of which have been used as 3D models for studying tachyzoite motility [67–69], could provide deeper insights into T. gondii migration, motility, host cell invasion, and infection dynamics in MPS. MPS offer a promising platform to investigate interactions between bradyzoites and immune cells such as neutrophils and macrophages, which have traditionally been studied in 2D models. Our platform also provides the foundation for modeling human organs, such as the placenta for studying congenital toxoplasmosis, the eye for studying chorioretinitis, and the brain to study infection of the CNS and encephalitis. This study elucidates critical aspects of T. gondii early infection dynamics, including localization, differentiation, and migration within intestinal tissues. The combination of in vivo and in vitro approaches provided comprehensive insights into parasite-host interactions, shedding light on mechanisms that govern T. gondii pathogenesis and transmission.

Materials and methods Ethics statement Animals were treated in compliance with the guidelines set by the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin School of Medicine and Public Health (protocol #M005217), which adheres to the regulations and guidelines set by the National Research Council. The University of Wisconsin is accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care. Host cells lines cultivation All cultures were maintained in a 37°C humidified 5% CO 2 incubator unless specified. Briefly, Human Foreskin Fibroblasts (HFF, ATCC SCRC-1041) and Human Colorectal Adenocarcinoma Cells (Caco-2, ATCC HTB-37) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin-streptomycin. Human Intestinal Epithelial Cells (HIEC-6, ATCC CRL-3266) were cultured in OptiMEM 1 Reduced Serum Medium (Gibco) supplemented with 4% FBS, 10 mM GlutaMAX (Gibco), 20 mM HEPES (Gibco), and 10 ng/mL Epidermal Growth Factor (EGF). All cultures were periodically tested for mycoplasma and tested negative. Experimental mice Swiss Webster or C57BL/6 female and male mice, 8–12 weeks-old, were used for all the experiments. Infected or moribund mice were euthanized with CO 2 . Toxoplasma gondii tachyzoite cultivation T. gondii tachyzoites were cultured in HFF cells and passed every two or three days. To avoid the appearance of any tissue culture specific characteristics, all experimental procedures were done using low passage parasites and used no longer than passage twenty. T. gondii strains: a) type I: RH ΔKU80ΔHPT, RH mCherry and RH GFP; b) type II: PruΔHPT:luciferase, ME49 ΔKU80DHPT (donated by Dr. S. Lourido), ME49 mCherry, and M4; and type I/III: EGS LDH2p-GFP/SAG1p-mCherry [40] (donated by Dr. L. Weiss). Toxoplasma gondii in vitro bradyzoite cultivation Briefly, HFF cells were infected with 5 × 105 – 2 × 106 tachyzoites for 2 h. Then, extracellular parasites were removed and differentiation media (RPMI 1640 without sodium bicarbonate (Sigma-Aldrich), 1% FBS, and 40 mM HEPES, at pH 8.1.) was added. Infected cells in differentiation media were incubated at 33°C for up to 12 days in a thermostatic incubator under normoxic conditions with ambient CO 2 concentrations. The flask cap was tightly closed to avoid gas exchange. Bradyzoite and cysts formation were confirmed by staining SAG1, BAG1, or DBA. In EGS parasites, bradyzoite differentiation was monitored in vivo by the expression of GFP and loss of mCherry by using EVOS FL Auto system (Life Technologies) scope or a live cell incubator (Incucyte, Sartorius). Quantification of in vitro encystment was performed by counting at least 100 positive or negative vacuoles. The statistics were performed using one-way ANOVA in the GraphPad Prism software. Toxoplasma gondii in vivo encystment To develop chronic infection and collect brain cysts, Swiss Webster or C57BL/6 mice were inoculated intraperitoneally (i.p.) with 1 × 103 – 1 × 104 of freshly egressed T. gondii tachyzoites of M4, PruΔHPT:luciferase, ME49 mCherry, or EGS LDH2p-GFP/SAG1p-mCherry parasite strains. Mice were euthanized 28 days post-infection, and the brains were removed to feed other mice or to isolate brain cysts. Brain cysts isolation Cysts were purified from infected mouse brains infected for at least 28 days. Brains were harvested and homogenized in ice-cold PBS. Brain material was passed through 18-, 20-, and 22-gauge needles three times to desegregate the brain. To purify cysts, brain material was centrifuged and resuspended in 30% of Percoll-90 (90% Percoll and 10% 1X PBS) in PBS. Cysts were pelleted by centrifugation at 1,000 × g for 15 min, washed with PBS, counted in a hemocytometer. Survival curves of in vitro bradyzoite-infected mice Three male Swiss Webster mice per dose were naturally infected with in vitro cysts of EGS strain: doses were 1 × 102, 1 × 103, 1 × 104, or 1 × 105. For ME49 in vitro cysts, natural infection doses were 1 × 104 (one mouse) or 7 × 104 (three mice)”. Eight days in differentiation media for EGS and eleven days in differentiation media for ME49, monolayers containing in vitro cyst structures were scraped and lysed using a 27-gauge needle. In vitro cysts were quantified, adjusted to the corresponding burden, and centrifuged at 500 × g for 10 min. Pellets were resuspended in 100 µl of 2% sucrose and added into 0.5 cm3 fresh white bread cubes. Mice were fasted overnight [70] and consumed the bread containing in vitro cysts within a 1-hour window. ME49 mCherry in vitro cysts were used as a positive control of chronic infection. For both strains, mice health was monitored daily for health status. At 28 days post-infection, mice were euthanized, and their brains were collected and analyzed for the presence of cysts. Villi isolation from infected mice Swiss Webster mice were subcutaneously infected with oocysts of the M4 strain (type II, generously donated by Jeroen Saeji and David Arranz-Solís). Upon establishment of chronic infection, mice were euthanized, and their brains were isolated to release bradyzoites, following established protocols [55]. Bradyzoites were then propagated in HFF cells through one passage. Subsequently, 1 × 104 tachyzoites of the M4 strain were infected in mice to develop chronic infection. At least four brains containing cysts were collected and served as food for two healthy mice, following the natural route of infection. Briefly, two mice were fasted overnight [70] and consumed the brains containing in vitro cysts within a 1-hour window. Each mouse was fed with two brains, each containing ~2 × 102 cysts per brain. The number of cysts were determined isolating brains from two infected mice from the same population, as above described. After feeding, mice were euthanized 3 days post-feeding, and their intestines were isolated. Sections of 6–8 cm of jejunum of infected mice was gently washed with ice-cold PBS, opened longitudinally, and mucus layer was removed by scraping superficially with a glass slide as previously described [33]. Villi were then detached by gentle scraping, collected in tubes, and fixed in 4% paraformaldehyde for 30 min. The fixed villi were centrifuged at 100 × g for 5 min, washed twice with PBS, and processed for immunofluorescence assay. Floating villi were placed in slides and imaged in a confocal microscope (ZeissLSM 800 Laser Scanning Microscope). This experiment was repeated three times to find foci of infection. Bradyzoite excystation and pepsin activation Bradyzoite exteriorization was performed by incubating purified cysts with digestion buffer containing NaCl and pepsin (5 mg/ml pepsin, 100 mg/ml NaCl, 0.14 N HCl, pH 2.1) as described previously [54,56]. Excystation was carried out for 2 min to 2 h. Digestion solution containing free bradyzoites was neutralized by addition of 1% Na 2 CO 3 (v/v). Bradyzoites were counted using a hemocytometer, centrifuged at 2,000 × g for 10 min, and resuspended in the corresponding media. Mice in vivo imaging system To study T. gondii intestinal interactions followed by the natural route of infection, three males and four females Swiss Webster mice were infected by consuming brains containing cysts (refer to figure legends to see the mice number in each time point). Each mouse ingested two brains with a total approximated dose of 4 × 102–5 × 102 brain cysts. The number of brain cysts were determined by isolating brains from two infected mice from the same population, as above described. At 3 or 5 days post-infection, parasitemia in mice was detected by bioluminescence using the in vivo imaging system (IVIS, PerkinElmer) as previously reported [37]. Prior to imaging, mice were anesthetized with 4% isoflurane and then injected retroorbital and i.p. with 200 µL of D-Luciferin potassium salt (15.4 mg/mL in PBS). Immediately after the injection, mice were sacrificed, intestines were isolated and exposed to the IVIS detector, and images were collected. Background measurements were determined by injecting uninfected mice with D-Luciferin and collecting the images as previously described. Additional background controls included the imaging of food and pieces of muscle from infected mice. All conditions were repeated at least twice. For the analysis of infected areas, the small intestines was collected the duodenum, jejunum, and ileum which encompass 33 cm of length [71]. Duodenum was considered as the first 2 cm after the pyloric sphincter, jejunum constituted 15 cm after the end of the duodenum, and ileum constituted the last 15 cm before the cecum. Data quantification of the bioluminescent areas was performed by counting the number of events per intestinal region or by drawing a region of interest (ROI) box for each mouse and recording the total number of photons per second (Total Flux) [38]. Mice intestines immunohistochemistry Areas of infection in intestines were detected by IVIS. Three to four infected sections of intestines of 5 mm long were cut and immediately fixed in 4% formalin in PBS overnight and dehydrated in 75% ethanol. Paraffin-embedding and sectioning of intestines were performed by request in the Translational Research Initiatives in Pathology (TRIPath lab, University of Wisconsin-Madison). Paraffin-embedded intestines were serial cut in at least 4 sections per slice to facilitate antibody comparisons. Infected sections of the intestine were processed at least three times for detecting parasites in different planes. Giving a total of at least 36 sections of foci of infection per condition. Intestinal sections were incubated at 65°C in a water bath for 1 h, washed with xylene 3 times for 10 min to remove the paraffin, twice for 1 min with 100% ethanol, twice with 95% ethanol in water, once with 75% ethanol in water, and finally in water. The epitopes were retrieved by exposure to the low-pressure setting in a pressure cooker for 6 min in urea buffer (1.25 M Tris Base, 1 M Urea, pH 9.5). Sections were blocked with 3% goat serum in 0.05 M TBS for 1 h. Primary antibody was incubated at 4°C overnight in 0.05 M TBS. Primary antibodies against- SAG1 (donated by John C Boothroyd,1:40, rabbit polyclonal antibody), BAG1 (donated by Louis Weiss, 1:40, rabbit polyclonal antibody), T. gondii (Invitrogen, 1:40, rabbit polyclonal antibody). CD31 (BD Pharmingen, 1:40, rat polyclonal antibody), and CD45 (BD Pharmingen, 1:40, rat polyclonal antibody). Dyes included: wheat germ agglutinin Alexa 488 (Invitrogen, 1:100) and DAPI (Millipore Sigma, 1:1000). Secondary antibodies against- rabbit or rat Alexa 594 (Thermo Fisher Scientific, 1:100). Washed 3 times with 0.05 M TBS and mounted in VECTASHIELD antifade mounting medium (VectorLabs). Each antibody was tested in at least twice biological replicates and by duplicate. Wheat germ agglutinin was preferred over phalloidin to counterstain the embedded tissue due to punctuated and unclear patterns detected in our samples when staining actin. Samples were imaged on a Zeiss Axioplan III (Imager.M2, Carl Zeiss) or a confocal microscope (ZeissLSM 800 Laser Scanning Microscope). 3D projections were reconstructed from z-stack sections using the Zen imaging software (Carl Zeiss). Quantification of infected areas was conducted by measuring fluorescence intensity. Regions-of-Interest corresponding to the fluorescence signal of SAG1 in the stroma or enterocytes in infected intestines were identified. The integrated pixel intensity of the entire region-of-interest was measured, and background fluorescence was subtracted. Quantitative analysis of images was performed using ImageJ software as described previously [72]. In addition, a quantification of the location of SAG1-positive vacuoles in the stroma or enterocytes was quantified and graphed. The statistics were performed using one-way ANOVA in the GraphPad Prism software. Ex-vivo infection of villi Healthy C57BL/6 mice were euthanized, and their intestines were isolated on an ice-cold Petri dish with PBS. Intestines were gently washed with 1× Pen/Strep and 25 μg/ml gentamicin in sterile PBS. Intestines were opened longitudinally, and villi detached as above described. Villi were washed once in antibiotics/PBS solution, centrifuged at 100 × g for 5 min, and resuspended in Leibovitz L-15 media (Thermo Scientific 31415029) supplemented with 10% FBS and 1× GlutaMAX (Gibco). Villi suspension was placed in chamber slides (Thermo Scientific 177372PK) covered by 3 mg/ml rat tail collagen I (Col-I, Corning), 0.5 N sodium hydroxide (Thermo Fisher Scientific), 50 ng/ μl human fibronectin (Millipore), mixed with 7.5 pH 5× PBS, and complete DMEM medium. Villi were challenged with ~5 × 105 of RH GFP tachyzoites or EGS LDH2p-GFP/SAG1p-mCherry in vitro bradyzoites for 6 h and 24 h. Samples were then fixed with 4% paraformaldehyde and processed for immunostaining. Viability assays were performed at the same time points by using SYTOX Green Nucleic Acid Stain (Thermo Scientific S7020). Samples were imaged on a Zeiss Axioplan III (Imager.M2, Carl Zeiss) or a confocal microscope (ZeissLSM 800 Laser Scanning Microscope). 3D projections were reconstructed from z-stack sections using the Zen imaging software (Carl Zeiss). Parasite growth assays To measure parasite growth, HFF, Caco-2, or HIEC-6 cells were seeded in 12- or 24- well plates. In vitro-differentiated or brain cysts-isolated bradyzoites of ME49 mCherry or in vitro-differentiated bradyzoites of EGS strain were isolated and excysted as above described using pepsin or by mechanical disruption using 30-gauge needle. Under-confluent cells were infected with 1 × 104 – 1 × 105 exteriorized bradyzoites. Plates were incubated in the Incucyte incubator for up to 9 days. For comparative assays, all cell types were seeded in the same plate, infected with the same parasites passage, and imaged and quantified under the same conditions as previously described [37,73]. Samples were quantified using the total mCherry area (µm2/image) as previously published [37,73]. The statistics were performed using one-way ANOVA in the GraphPad Prism software. Generation of human intestinal microphysiological systems MPS were fabricated using soft lithography techniques and filled with a collagen/fibronectin matrix to create a molded lumen that was seeded with a monolayer of Caco-2 cells to create an intestine mimic as previously described [35]. Briefly, the LumeNEXT device is created from two stacked PDMS layers, surrounding a removable PDMS rod, with separate inlet and outlet ports. After the main chamber is filled with extracellular matrix and polymerized, the PDMS rods are removed to form a molded lumen structure that can be seeded with cells. For specific features about MPS fabrication, please refer to Jimenez-Torres and collaborators [74]. Devices were UV-sterilized for 20 min. To promote matrix adhesion to PDMS, chambers were treated with 1% polyethylenimine (MilliporeSigma) in water solution for 10 min, followed by a treatment of 0.1% glutaraldehyde (MilliporeSigma) for 30 min, and then washed 5 times with water. ECM was prepared consisting of 3 mg/ml rat tail collagen I (Col-I, Corning), 0.5 N sodium hydroxide (Thermo Fisher Scientific), 50 µg/μl human fibronectin (Millipore), mixed with 7.5 pH 5× PBS, and complete DMEM medium. The pH of the ECM matrix was adjusted to pH 7.2 before loading the matrix into the central chamber of the device. ECM was polymerized at room temperature for 20 min and then moved to an incubator at 37°C for at least 1 hour. PDMS rods were then removed leaving molded lumen structures within the ECM gel that can be lined with cells. To seed the lumens with epithelium, Caco-2 cells were resuspended at 20 × 106 cells/ml of supplemented DMEM. 3 μl of cell suspension was introduced into the lumens through the inlet port. The MPS was incubated upside down for 30 min, then flipped and incubated for 1 h. Nonadherent cells were removed by washing with culture medium, and additional medium was added to the inlet/outlet ports. Devices were incubated for 24–48 hours to permit the formation of a confluent monolayer of cells that mimic human intestinal lumens. Intestinal lumens were infected with 1 × 103 – 5 × 104 exteriorized bradyzoites. For the quantification of tachyzoite conversion, seven plates were infected with 3 × 104 in vitro bradyzoites. EGS tachyzoites were switched to the conditions described above and maintained for at least 7 days. Bradyzoite conversion to tachyzoites was monitored in the device using the EVOS FL Auto system and assessed by detecting PVs positive to mCherry/SAG1. Results are shown as a percentage of the average of red vacuoles in each lumen per plate, 36 lumens total were quantified. An additional staining for cell coverture in the lumens was tracked by actin staining using fluorescein phalloidin (Invitrogen). Barrier stability of lumens was tested using 2 mg/ml of 4 kDa FITC-Dextran (Sigma) and monitoring dextran diffusion. Immunofluorescence staining For immunoassays, infected monolayers were processed following modified protocol based on previous works [47,75]. DBA, SAG1, or BAG1 positive vacuoles, infected monolayers were fixed with 4% paraformaldehyde for 30 min or ice-cold methanol for 20 min. Methanol was also used to quench mCherry or GFP fluorescence in parasites. Fixed monolayers were permeabilized with 0.5% Triton X-100 for 5 min and blocked with 3% BSA and 0.2% Triton X-100 for 30 minutes. For stating the cysts structures, we used Dolichos biflorus Agglutinin (DBA) which specifically binds the mucin domain of N-acetyl-galactosamine of the CST1 glycoprotein in the cyst wall [76]. DBA was used coupled to rhodamine (Vector laboratories) or fluorescein (Vector laboratories) was diluted in 3% BSA and 0.2% Triton X-100 and incubated for at least 1 h. For immunostaining infected villi, fixed tissue was processed as previously described [37]. Fixed villi were permeabilized with 0.5% Triton X-100 for 5 min and blocked with 5% BSA for 30 min. Villi were incubated with polyclonal antibodies against- T. gondii (Invitrogen, 1:500, rabbit), overnight at 4˚C. Secondary antibody Alexa Fluor 488 (Thermo Scientific, 1:1,000, goat anti-rabbit) were incubated for 2 h at room temperature. For GFP or mCherry parasites no antibodies were used for detecting parasites. Villi were counterstained with rhodamine phalloidin (Cytoskeleton, Inc) or fluorescein phalloidin (Invitrogen), DAPI, and mounted using VECTASHIELD. Villi were imaged using a confocal microscope (ZeissLSM 800 Laser Scanning Microscope). Immunostaining of parasites in MPS followed a modified protocol based on a previous report [35]. Briefly, cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 (Fisher Scientific) for 10 min or ice-cold methanol for 20 min to quench mCherry fluorescence in parasites. Blocking was performed overnight at 4°C in a buffer solution (3% BSA and 0.1% Tween 20 (Thermo Fisher Scientific). Primary antibodies against- SAG1 (1:40, monoclonal mouse antibody DG52) or BAG1 (1:40, rabbit polyclonal antibodies) in buffer solution were added to the infected lumens and incubated at 4°C for 2 days. Secondary antibodies: rabbit Alexa Fluor 488 (Thermo Fisher Scientific, 1:100) and mouse Alexa Fluor 594 (Thermo Fisher Scientific, 1:100) were added to the buffer solution and incubated for 1 day at 4°C. Additionally, viability of cells in lumens were tracked by using CellMask Green Actin (Invitrogen), counterstained with DAPI, and imaged using a Nikon AXR Confocal Microscope. Statistical analysis Data were analyzed (Prism 9.0; GraphPad Software). Statistical significance was assessed using nonparametric Student’s t tests when comparing two conditions/groups, and when comparing more than two groups, significance was assessed using one-way analysis of variance (ANOVA).

Acknowledgments We thank Louis Weiss for the EGS strain, Jeroen Saeji and David Arranz-Solís for the M4 oocysts, and Sebastian Lourido for ME49ΔKU80ΔHPT strain. We thank members of the Knoll laboratory for their helpful discussions. We thank Michael Panas and John Boothroyd for providing the DG52 hybridoma for anti-SAG1 monoclonal antibody production. We thank the UW Small Animal Imaging and Radiotherapy Facility (SAIRF) for providing access to the IVIS imaging system, we thank the UW Optical Imaging Core (UWOIC) for use of the Nikon AXR confocal microscope (NIH grant number 1S10O34394-01), and we thank the Translational Research Initiatives in Pathology (TRIPath lab) for processing our intestinal samples. We thank members of the Beebe lab for their technical help and helpful discussions. We specially thank Imran Khan for his technical support.

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