Long-term feeder cell-free cat intestinal organoid cultures to study Toxoplasma gondii’s sexual development

TL;DR

Researchers developed a new lab model using feline intestinal organoids to study how Toxoplasma gondii develops sexually, which only happens in cats.

Contribution

A feeder cell-free feline intestinal organoid system was established to study T. gondii sexual development in vitro.

Findings

Feline intestinal organoids supported elevated levels of T. gondii sexual stage-specific transcripts.

The model allows systematic investigation of host factors influencing parasite sexual development.

Advanced sexual stages were not observed, but the system is reproducible and controlled.

Abstract

Toxoplasma gondii is a protozoan parasite able to infect and survive in diverse host environments. However, its sexual reproduction, culminating in infectious oocysts, occurs exclusively in feline intestines. Recent studies identified the transcription factors AP2XII-1 and AP2XI-2 as crucial for pre-sexual development. Their depletion enabled merozoite formation in human fibroblasts, but progression to sexual stages appeared to require additional cues. Host-specific factors governing this process are suspected but remain elusive. Here, we describe a robust continuous feline intestinal organoid culture system without feeder cells to investigate whether the feline cellular and metabolic environment promotes sexual development of in vitro-generated merozoites. Using ultrastructural and transcriptional analyses, we found elevated levels of sexual stage-specific transcripts. While advanced sexual stage formation could not yet be observed, our feline intestinal organoid model provides a controlled and reproducible experimental system to systematically uncover the feline host factors and molecular mechanisms of T. gondii’s sexual development.

Establishment of continuously expanding feline intestinal organoids in feeder cell-free cultures provides a robust in vitro model for the study of Toxoplasma gondi’s sexual stages and other intestinal pathogens.

Figures

Establishment and differentiation of 3D cat intestinal organoid cultures.a Microscopy images of fIO cultures on day 5 after isolation showing the effect of cultivation temperature at 38.5 °C and Wnt surrogate compared to 37 °C and Wnt-3a-containing conditioned media (CM) on organoid formation. b, c Organoids were cultivated in growth or differentiation medium for 5 days before harvesting, fixation, and processing for immunofluorescence microscopy or extraction of RNA for RT-qPCR. In (b), fluorescence and brightfield microscopy images are shown. F-actin was stained with phalloidin, and DNA with

Characterization of feline ODMs.Single cells obtained from 3D fIO cultures were seeded into cell culture inserts to generate organoid-derived monolayers (ODMs). a Confocal immunofluorescence microscopy images of an ODM stained for F-actin (phalloidin). Nuclei were stained with DAPI. Shown are maximum intensity projections of z-stacks for each channel and the corresponding merge image of a single xy slice. Orthogonal views show the signal along the *z*-axis. Scale bar, 20 µm. b Electron microscopy of a thin section through an ODM, which was fixed on day 8 after seeding. Left image shows several

Effect of IAA treatment on feline ODMs and *T. gondii* growth.a, b Uninfected feline ODMs were treated with varying concentrations of IAA for up to 96 h. a TEER was measured every 24 h. Data represent mean ± SD (*n* = 3 independent filters per IAA concentration), with individual values shown as scattered points (jittered along the x-axis to prevent overlap). b Representative fluorescence microscopy images of ODMs after 96 h of IAA treatment. Nuclei were stained with DAPI, F-actin with phalloidin, and the tight junction protein ZO-1 was visualized by antibody staining. c Effect of IAA treatment

Infection and growth of RH AP2XII-1 AP2XI-2 parasites in cat ODMs and detachment of infected ODM cells.a Immunofluorescence microscopy images of cat ODM infected with RH AP2XII-1 AP2XI-2 parasites, 1 day post infection. Maximum projection and orthogonal projections of a z-stack are shown. Parasites are visualized with the GAP45 antibody. Detail shows a vacuole with multiple parasites (single *z*-plane). b Quantification of parasite growth following IAA-induced degradation of AP2XII-1/AP2XI-2. Treatment with 500 µM IAA was started on day 1 post infection. The cells were fixed at the indicated t

Endopolygeny following AP2XII-1 and AP2XI-2 depletion.Electron micrographs of sections through cat ODMs infected with 2xAP2 KD tachyzoites, either untreated or treated with 500 µM IAA for 48 h. a In the untreated condition (left), a tachyzoite is prepared for symmetrical daughter cell formation (endodyogeny), which is indicated by the presence of the inner membrane complex (imc). Following IAA treatment (right), a developing schizont with profiles of three nuclei is seen, indicating an early stage of endopolygeny. b Serial sections through a single IAA-treated parasite, with profiles of five n

Gene expression of *T. gondii* life stage markers after depletion of AP2XII-1 and AP2XI-2.a Feline ODMs or b, c HFF cells were infected with 2xAP2 KD tachyzoites. The culture medium in (a, c) was supplemented with 200 µM linoleic acid (LA) starting 24 h before infection. One day after infection, AP2XII-1 and AP2XI-2 were depleted by the addition of 500 µM IAA. RNA was extracted at the indicated time points for RT-qPCR. Gene expression was normalized to the reference gene casein kinase 1 (*CK1*), and fold changes were determined relative to the 0 h untreated control. Data normalization and anal

Full text

Introduction

Toxoplasma gondii is an apicomplexan parasite and the causative agent of the zoonotic disease toxoplasmosis. It can infect most warm-blooded animals, including humans. However, its sexual reproduction and oocyst formation occur exclusively in the feline intestinal epithelium. Although this process is crucial in the parasite’s life cycle and key to the generation of its environmental transmission stages, the mechanisms underlying T. gondii’s sexual development and determinants of host specificity remain largely unexplored. Research in this area is limited by the difficult accessibility of sexual stages and oocysts. Only a small number of labs worldwide can work with infected cats, and those that do often encounter public criticism^1^. Intestinal cell cultures from cats and rats have thus been used to study different aspects of sexual differentiation^2–4^. While the fast-replicating tachyzoite and semi-dormant bradyzoite stages can be investigated in cell culture, the complete life cycle cannot be replicated in vitro^5^.

Recent advances have identified key molecular regulators of parasite development. Notably, several Apetala 2 (AP2) transcription factors have been individually implicated in repressing the merozoite program in tachyzoites^6–9^. However, only the combined depletion of AP2XII-1 and AP2XI-2 is sufficient to unlock the pre-sexual transcriptional program^10^. Yet, these in vitro merozoites failed to progress to gametocytes, suggesting additional factors may be required for sexual commitment.

The importance of host factors in parasite sexual development is well-established. For instance, in Plasmodium falciparum, host-derived lysophosphatidylcholine modulates PfAP2-G transcription factor expression and sexual commitment^11,12^. We and others^9,10^ hypothesized that feline intestinal epithelial cells might provide the necessary environmental cues to facilitate the complete sexual development of in vitro-generated merozoites.

In this context, stem cell-derived intestinal organoid cultures have emerged as valuable models for studying parasite infections, as they closely mimic the complex architecture and cell type diversity of the intestinal epithelium^13–15^. However, organoids derived from domestic cats (Felis catus) have been reported to be particularly challenging to maintain due to early growth arrest^16–18^. Fibroblast feeder cell lines have been used to prolong the lifespan of cat organoid cultures, though this approach adds complexity to the cultivation process^19^.

In this study, we developed a feline intestinal organoid (fIO) culture system that supports long-term growth without feeder cell lines. We provide, for the first time, comprehensive ultrastructural data of both uninfected and infected fIOs. Using this refined and robust system, we tested whether in vitro merozoites, produced by depleting AP2XII-1 and AP2XI-2 in T. gondii tachyzoites, would progress further toward sexual development. Our findings indicate that, under the limited conditions tested, fIO cell cultures are not sufficient to promote the formation of sexual stages when using in vitro-produced merozoites as starting material, even when additionally supplemented with some metabolites presumed to be important in this context. However, they provide a robust feline cell culture system for further experimentation.

Results

Generation, expansion, and long-term maintenance of the cat small intestinal organoids

Previous reports indicated that cat intestinal organoids are particularly difficult to expand and to maintain continuously^16,17^, prompting us to optimize media and cultivation conditions. Wnt-3a, essential for sustaining the stemness and proliferative capacity of intestinal cells, is typically supplied as part of a conditioned medium (CM) due to the water-insoluble and unstable nature of recombinant Wnt-3a protein^20^. However, studies suggest that serum in Wnt-3a CM may inhibit long-term organoid growth^20,21^. To overcome these limitations, we compared replacing Wnt-3a CM with Wnt surrogate, an engineered fusion protein that activates Wnt signaling and supports organoid growth at much lower concentrations^22^. Additionally, the physiological temperature of domestic cats (including the rectum) ranges from 37.8-39.8 °C^23,24^, which is higher than the conventional 37 °C used for human and murine cultures, leading us to test two different incubation temperatures.

Using our previously established protocols^15,25^, we isolated intestinal organoids from duodenal crypts of two adult domestic cats. Notably, intestinal crypts incubated at 38.5 °C in Wnt surrogate medium exhibited improved organoid formation compared to those cultured in Wnt-3a CM and/or at 37 °C, yielding more and larger organoids by day 5 post-isolation (Fig. 1a). With continued cultivation, the organoids showed remarkable self-renewal capacity, which did not require feeder cells, and were successfully propagated without any signs of growth arrest for 54 passages (316 days) up to now. No further optimizations were attempted, but these results suggest an additive beneficial effect of higher temperature and Wnt surrogate and were thus used in further experiments.Fig. 1. Establishment and differentiation of 3D cat intestinal organoid cultures.a Microscopy images of fIO cultures on day 5 after isolation showing the effect of cultivation temperature at 38.5 °C and Wnt surrogate compared to 37 °C and Wnt-3a-containing conditioned media (CM) on organoid formation. b, c Organoids were cultivated in growth or differentiation medium for 5 days before harvesting, fixation, and processing for immunofluorescence microscopy or extraction of RNA for RT-qPCR. In (b), fluorescence and brightfield microscopy images are shown. F-actin was stained with phalloidin, and DNA with DAPI. In (c), mRNA levels of intestinal cell type markers determined by RT-qPCR are shown. Data are presented as ΔCt values, calculated by subtracting the Ct values of the reference gene encoding ribosomal protein S7 (RPS7) from the genes of interest (GOI). This representation was chosen because no Ct values could be determined for SI and KRT20 in growth medium (consistent with expected very few transcripts in medium not supporting differentiation), preventing proper ΔΔCt analysis to deduce the fold-change of expression between groups. Note that lower ΔCt values correspond to higher expression levels. Mean of n = 2 technical replicates is shown. *P ≤ 0.05; ns not significant (P > 0.05) by unpaired two-tailed t-test. SC stem cell, EE enteroendocrine cell, E enterocytes, LGR5 leucine-rich repeat-containing G-protein coupled receptor 5, OLFM4 olfactomedin 4, CHGA chromogranin A, SI sucrase-isomaltase, ALDOB aldolase B, KRT20 keratin 20.

To assess the differentiation potential of cat organoids, they were cultured in differentiation medium devoid of stem cell-promoting and differentiation-inhibiting factors (Wnt-3a, nicotinamide, p38 inhibitor SB-202190, and transforming growth factor beta (TGF-β) receptor signaling inhibitor A83-01). After 5 days, organoids exhibited typical morphological features of differentiation, including budding structures and more columnar-shaped cells (Fig. 1b). Both undifferentiated and differentiated organoids showed the characteristic “apical-in” phenotype, in which the apical surface of the polarized epithelium faces the organoid lumen. This orientation was confirmed by phalloidin staining of F-actin in the apical brush border (see Fig. 1b).

To further evaluate organoid differentiation, cell type-specific marker gene expression was analyzed by RT-qPCR (Fig. 1c). Culturing in differentiation medium led to a reduction in stem cell marker gene expression, as indicated by higher Ct values for leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) and olfactomedin 4 (OLFM4). Conversely, the expression level of the enterocyte marker aldolase B (ALDOB) was increased in differentiation medium-cultured organoids, while the enteroendocrine cell marker chromogranin A (CHGA) remained unchanged. Additionally, two differentiation markers for enterocytes (sucrase-isomaltase (SI) and keratin 20 (KRT20)) were detected exclusively in organoids incubated in differentiation medium but not in growth medium.

Generation and characterization of cat organoid-derived monolayers

While 3D intestinal organoid cultures can be infected with T. gondii^26^, organoid-derived monolayers (ODMs) have been developed to overcome several limitations of the 3D model, such as low or poorly controllable infection efficiency or reduced cell viability^15^. We thus generated cat ODMs from 3D cultures using single cells, obtained through enzymatic digestion and mechanical disruption, that were then seeded onto cell culture inserts^25^.

Fluorescence microscopy confirmed the development of a polarized monolayer with apical F-actin (Fig. 2a), measured to be ~16 µm in height (Fig. S1a, b). This is comparable to the height of human ODMs (~20 µm), which we have generated using the same protocol^15^, and greater than those derived from mouse, pig, and chicken (<10 µm). The transepithelial electrical resistance (TEER), an indicator of monolayer integrity, was measured at 1827 ± 75 Ω cm² (mean ± SD, N = 3 ODMs) on day 4 post-seeding, and remained stable through at least day 10 (Fig. S1c). This value is relatively high compared to the values that we have observed for human, murine, porcine, and chicken intestinal ODMs (~100–500 Ω cm²)^15^, but still within the range of TEER values reported in the literature^27^.Fig. 2. Characterization of feline ODMs.Single cells obtained from 3D fIO cultures were seeded into cell culture inserts to generate organoid-derived monolayers (ODMs). a Confocal immunofluorescence microscopy images of an ODM stained for F-actin (phalloidin). Nuclei were stained with DAPI. Shown are maximum intensity projections of z-stacks for each channel and the corresponding merge image of a single xy slice. Orthogonal views show the signal along the z-axis. Scale bar, 20 µm. b Electron microscopy of a thin section through an ODM, which was fixed on day 8 after seeding. Left image shows several cuboidal cells arranged as a polarized epithelium with apical microvilli (mv) and typical cell-cell contacts (arrowheads). Right image shows a magnified view of the cell-cell contacts established between two of the cells (highlighted in the left image by a rectangle). tj tight junctions, ld lipid droplet, n nucleus, d desmosome, F filter; *, basal inclusion is likely residue of extracellular matrix used for filter coating. c Cell type marker gene expression in ODMs was analyzed by RT-qPCR. RNA was extracted from the harvested 3D spheroids or from ODMs on day 7 post-seeding. Data are presented as ΔCt values, calculated by subtracting the Ct values of the reference gene encoding ribosomal protein S7 (RPS7) from the Ct values of the genes of interest (GOI). Lower ΔCt values indicate higher expression levels. The mean of n = 2 technical replicates is shown. *P ≤ 0.05; ns not significant (P > 0.05) by unpaired two-tailed t-test. SC stem cell, EE enteroendocrine cell, E enterocytes, LGR5 leucine-rich repeat-containing G-protein coupled receptor 5, OLFM4 olfactomedin 4, CHGA chromogranin A, SI sucrase-isomaltase, ALDOB aldolase B, KRT20 keratin 20.

Further characterization of ODMs was conducted using thin-section electron microscopy (EM) (Figs. 2b and S2). The epithelial cells primarily formed a monolayer. Occasionally, the epithelial layer appeared pseudo-stratified or, more rarely, clearly stratified. The epithelial cells were cuboidal with short apical microvilli and exhibited typical cell-cell contacts, i.e., tight junctions and desmosomes. We regularly observed large lipid droplets in the epithelial cells. Lipid droplet formation is recognized as a mechanism to alleviate cellular lipotoxic stress^28^, which could indicate that ODMs were exposed to a lipid oversupply in the medium. Secretory activity was observed in many cells of the ODM at different levels and was characterized by the presence of secretory vesicles of variable size and slightly granular content in the apical region of the cells. The granules seem to release their content at the surface of the epithelium by bulging the apical plasma membrane and exocytotic fusion of membranes (Fig. S2).

Interestingly, at low frequency, but consistently, individual cells exhibited variations in electron density (Fig. S2d). One might speculate that these variations reflect distinct cell types, as higher electron densities have been reported for mucus-secreting intestinal cells compared to absorptive cells^29^, but other cellular or experimental factors may also contribute to this observation. Of note, to our knowledge, these are the first published electron micrographs of fIOs, and only a few ultrastructural studies of intestinal samples from cats for comparison have been published^29–32^.

RT-qPCR analysis was performed to evaluate ODM differentiation (Fig. 2c). Compared to undifferentiated 3D organoids, ODMs exhibited reduced expression of stem cell markers LGR5 and OLFM4, and only a trend towards lower expression of the enterocyte marker ALDOB. Differentiation markers for enteroendocrine cells (CHGA) and enterocytes (SI and KRT20) were not detected, suggesting altogether incomplete differentiation.

Overall, EM analysis and RT-qPCR analysis indicated that the degree of maturation and cell type diversity in our ODMs was limited, primarily reflecting the epithelial mucosa near the crypt base, where most cells are undifferentiated epitheliocytes^33^. However, the cellular architecture of the generated cat ODMs resembled the microanatomy of the cat’s duodenal epithelium in relevant aspects (polarized single-layered/pseudo-stratified epithelium with secretory activity), though in vivo epithelial cells appear more columnar, possess larger microvilli^33^, and reveal a clear differentiation into enterocytes and goblet cells.

AP2 depletion causes upregulation of sexual stage markers but impedes parasite proliferation in cat ODMs

Simultaneous conditional depletion of the transcription factors AP2XII-1 and AP2XI-2 in tachyzoites using the auxin-inducible degron (AID) system has been shown to promote the transition to merozoites in HFF cells, a non-natural cellular environment for sexual development^10^. However, in those experiments, the formation of mature micro- or macrogametocytes was not observed, and knockdown tachyzoites exhibited deficiencies in invasion and reduced infectivity in those fibroblasts^10^. In the related apicomplexan parasite P. falciparum, the activity of the transcription factor AP2-G can be influenced by extra-parasitic metabolite concentrations, thereby regulating sexual differentiation^34^. These and other findings^35^ prompted us to investigate whether feline ODMs provide a more permissive environment for rapid merozoite expansion and re-invasion, as observed in vivo^36^. We hypothesized that ODMs could promote sexual development of the AP2XII-1 and AP2XI-2 double-knockdown strain (2xAP2 KD) by removing an apparent but yet unidentified metabolic or regulatory roadblock to differentiation, even in the RH strain background that is otherwise oocyst-incompetent^37^.

Because indole-3-acetic acid (IAA), the inducer used in the AID system, is known to be cytotoxic at high concentrations in yeast^38^, mammalian cells in vitro and in vivo^39,40^, and to some extent also affects the growth of the apicomplexan intestinal parasite Cryptosporidium parvum^41^, we evaluated the tolerability of IAA in feline ODMs. IAA was titrated over a range of 125–500 µM, and barrier integrity was monitored by TEER measurements (Fig. 3a). Only after 72 h of IAA treatment, a sharp reduction in TEER was observed, indicating delayed barrier compromise, while shorter incubations (≤48 h) had no effect. This reduction was not strictly dose-dependent within the tested range, since all tested IAA concentrations resulted in a similar TEER decrease. Despite this decline, monolayer architecture remained largely intact up to 96 h, even at 500 µM IAA (Fig. 3b). IFA analysis showed that while F-actin staining was reduced with higher IAA concentrations, the signal for the tight junction protein zonula occludens-1 (ZO-1) retained its intensity and belt-like distribution.Fig. 3. Effect of IAA treatment on feline ODMs and T. gondii growth.a, b Uninfected feline ODMs were treated with varying concentrations of IAA for up to 96 h. a TEER was measured every 24 h. Data represent mean ± SD (n = 3 independent filters per IAA concentration), with individual values shown as scattered points (jittered along the x-axis to prevent overlap). b Representative fluorescence microscopy images of ODMs after 96 h of IAA treatment. Nuclei were stained with DAPI, F-actin with phalloidin, and the tight junction protein ZO-1 was visualized by antibody staining. c Effect of IAA treatment on the growth of T. gondii RH-YFP parasites not carrying the auxin-inducible degron system in feline ODMs. Treatment with 500 µM IAA was started on day 1 post infection. Quantification of parasites per host nuclei based on microscopic images and GAP45 as a marker for T. gondii. GAP45-stained areas were masked and quantified to determine the total parasite area per host nucleus, normalized to t = 0. Small open symbols represent values from three random areas per filter, large closed symbols with error bars indicate their mean ± SD. (One filter with quantification of n = 3 areas each).

To establish a baseline for interpreting the effects of IAA treatment on parasite growth in subsequent infection experiments with the 2xAP2 KD, we first tested its impact on parasites not carrying the AID system (RH-YFP; Fig. 3c). Some attenuation of RH-YFP growth over 96 h was detected upon addition of 500 µM IAA, likely reflecting the above described negative impact of IAA on feline ODMs beyond 48 h incubation. Collectively, these data show that IAA concentrations up to 500 µM are tolerated by ODMs, at least within the timeframe relevant for the following infection assays.

Next, the growth of 2xAP2 KD tachyzoites in feline ODMs was analyzed by IFA using antibodies against GAP45, which decorates the inner membrane complex of the parasite. Some vacuoles contained multiple parasites, confirming successful infection and parasite proliferation (Fig. 4a). We then induced the degradation of AP2XII-1 and AP2XI-2 with 500 µM IAA to induce merogony^10^ and monitored parasite growth for 4 days (Fig. 4b) by quantifying the GAP45^+^ area as a proxy for parasite numbers. Parasite numbers perpetually increased in -IAA samples, suggesting continuous reinfections. In contrast, parasite numbers plateaued after IAA treatment, mirroring the phenotype of reduced infectivity previously reported in fibroblasts^10^. Interestingly, parasite numbers even declined at later time points. This may be caused by the detachment of infected host cells from the filter membrane, a phenomenon that was observed by microscopy (both EM and IFA) occasionally and with both type I and type II strains (Fig. 4c, d). Shedding of intestinal epithelial cells is a physiological process and part of the rapid turnover of the epithelial lining that occurs independently of infection^42^. Enterovirus-infected colonic organoid cells, on the other hand, have been shown to be specifically extruded from the surface of apical-out organoids via a force-sensing mechanism dependent on mechanosensitive ion channels^43^. Whether T. gondii infection may similarly trigger host cell detachment requires further experiments.Fig. 4. Infection and growth of RH AP2XII-1 AP2XI-2 parasites in cat ODMs and detachment of infected ODM cells.a Immunofluorescence microscopy images of cat ODM infected with RH AP2XII-1 AP2XI-2 parasites, 1 day post infection. Maximum projection and orthogonal projections of a z-stack are shown. Parasites are visualized with the GAP45 antibody. Detail shows a vacuole with multiple parasites (single z-plane). b Quantification of parasite growth following IAA-induced degradation of AP2XII-1/AP2XI-2. Treatment with 500 µM IAA was started on day 1 post infection. The cells were fixed at the indicated time points for immunofluorescence microscopy using anti-GAP45 to stain parasites and DAPI for nuclei. GAP45-stained areas were masked and quantified to determine the total parasite area per host nucleus, normalized to t = 0. Small open symbols represent individual data points from three random microscopic fields per filter, while large closed symbols indicate the mean of these values across n = 2 independent experiments (denoted by different symbol shapes). c Electron microscopy image of cat ODMs on day 7 after infection with the ME49 strain. The parasitophorous vacuole (PV) fills almost the entire host cell (HC). Cell contacts to neighboring cells seem mostly intact, but there is only limited basal contact to the filter (F) support, which could suggest an early stage of detachment. The right image is a magnification of the boxed region. PVM parasitophorous vacuole membrane. d Advanced stage of cell detachment, captured by confocal microscopy. Images are of 2xAP2 KD-infected ODMs following 48 h treatment with 500 µM IAA. Orthogonal projections of z-stacks are shown, and detaching cells (labeled 1 and 2) are enlarged on the right. Note the host cell nuclei (asterisk) and the faint F-actin signal (arrow) indicating the host cell boundary. Scale bars, 20 µm unless stated otherwise.

We then sought to analyze the presence of sexual stages by IFA using antibodies raised against the macrogametocyte marker amine oxidase 2 (AO2, TGME49_286778^44^). However, we observed non-specific antibody binding to uninfected host cells, leading us to use EM instead, as it does not rely on specific markers and provides superior morphological resolution. Ultrastructural analysis after 48 h IAA treatment showed that in untreated cultures the parasites maintained tachyzoite morphology, and some could be seen undergoing endodyogeny, characterized by the symmetrical formation of two daughter cells (Fig. 5a). In contrast, in IAA-treated 2xAP2 KD the formation of multinucleated schizonts was occasionally observed (Fig. 5a). To rule out the vague possibility that a lobulated nucleus might appear as separate profiles in single thin sections, we performed serial-sectioning EM and traced multiple distinct nuclei through consecutive planes of a single parasite (Figs. 5b and S3). These findings confirm previous observations in HFF cells, demonstrating that AP2XII-1- and AP2XI-2-depletion induces merogony^10^. Within the collected sets of images, we did not observe larger schizonts corresponding to later stages of endopolygeny (which have been found in HFFs), nor could we detect gametocytes.Fig. 5. Endopolygeny following AP2XII-1 and AP2XI-2 depletion.Electron micrographs of sections through cat ODMs infected with 2xAP2 KD tachyzoites, either untreated or treated with 500 µM IAA for 48 h. a In the untreated condition (left), a tachyzoite is prepared for symmetrical daughter cell formation (endodyogeny), which is indicated by the presence of the inner membrane complex (imc). Following IAA treatment (right), a developing schizont with profiles of three nuclei is seen, indicating an early stage of endopolygeny. b Serial sections through a single IAA-treated parasite, with profiles of five nuclei traced and color-coded across consecutive planes. Three sections (numbers 6, 26, and 58) are shown; the full series is provided in Fig. S3. R rhoptry, N nucleus, NM nuclear membrane.

We also examined the effect of IAA on the ultrastructural morphology of host cells in infected ODMs at 48 h and observed some signs of degradation, including enlarged intercellular spaces, compared to mock-treated controls (Fig. S4). However, tight junctions and overall epithelial architecture remained mostly intact, consistent with the uninfected control experiments (Fig. 3a, b), and this did not impair the assessment of parasite phenotypes.

Despite the lack of mature gametocyte detection in our EM analysis, we further assessed the parasite’s stage progression by RT-qPCR (Fig. 6). Importantly, T. gondii casein kinase 1 (CK1, TGME49_240640) was used as a reference gene for its stable expression level across all developmental stages and the parasite cell cycle, in contrast to other frequently used reference transcripts (Fig. S5a, c). CK1 expression is also not altered upon AP2 knockdown, as confirmed by RNA sequencing data^10^ (Fig. S5b). Expression of the merozoite marker GRA11B (TGME49_237800) was considerably upregulated upon depletion of AP2XII-1 and AP2XI-2, while SAG1 levels, a tachyzoite marker, decreased. This further confirms the tachyzoite-to-merozoite transition. Intriguingly, we observed an increased expression of several sexual stage markers, namely the amine oxidase 2 (AO2) (TGME49_286778), expressed in macrogametes and involved in dityrosine crosslinking in oocyst wall formation^44,45^, the predicted microgamete flagellar dynein motor protein (TGME49_500062)^18^, and the hypothetical oocyst wall protein 1 (HOWP1, TGME49_316890), expressed in late gamete stages and oocysts^44,45^. These three markers were detected after 24 h of IAA treatment, with their expression further increasing after 48 h. However, the decline in vacuole numbers following IAA treatment, and hence low amounts of parasite RNA, prevented analysis beyond this time point. Notably, gametocytes have been observed in the intestines of kittens fed with tissue cysts as early as 20 h after type D merozoite formation^46^.Fig. 6. Gene expression of T. gondii life stage markers after depletion of AP2XII-1 and AP2XI-2.a Feline ODMs or b, c HFF cells were infected with 2xAP2 KD tachyzoites. The culture medium in (a, c) was supplemented with 200 µM linoleic acid (LA) starting 24 h before infection. One day after infection, AP2XII-1 and AP2XI-2 were depleted by the addition of 500 µM IAA. RNA was extracted at the indicated time points for RT-qPCR. Gene expression was normalized to the reference gene casein kinase 1 (CK1), and fold changes were determined relative to the 0 h untreated control. Data normalization and analysis were performed as described by Taylor et al.^95^. Bar charts show the geometric mean, along with individual data points from n = 2 independent experiments (represented by different symbol shapes), each including two technical replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-way ANOVA followed by Tukey’s multiple comparison test.

Sexual stage marker expression in infected HFFs

To further investigate whether the observed increase in sexual stage gene expression was specific to cat ODMs, we also infected HFF cells and performed RT-qPCR (Fig. 6b). As expected, depletion of AP2XII-1 and AP2XI-2 led to a considerable increase in the expression of the merozoite marker GRA11B. Surprisingly, a rise in mRNA levels was also observed for the sexual stage markers AO2, TGME49_500062, and HOWP1, though this increase was not statistically significant (P > 0.05), which is in line with previously reported transcriptomic analyses (refs. ^6,9,10^, see “Discussion”). Importantly, HFF cells did not survive in the ODM infection medium, necessitating different culture conditions and limiting direct quantitative comparisons between the experiments. Nevertheless, the results suggest that feline ODM host cells enhance sexual stage gene expression following 2xAP2-depletion compared to HFF cells.

Given a previous report identifying linoleic acid (LA) as a potential trigger of sexual development^18^, we tested the effect of 200 µM LA on the 2xAP2 KD strain in HFFs. Gene expression patterns remained largely comparable to those observed in media conditions without additional LA, with no further increase in sexual stage marker expression (Fig. 6c). Notably, 200 µM LA was also included in the ODM infection medium, as it has been reported to be essential for sexual development of bradyzoites in cat cell culture^18^.

ODM infection with ME49 tachyzoites

The 2xAP2 KD was generated in the lab-adapted RH strain, which is known to fail producing oocysts when its bradyzoites were fed to cats^37^. Thus, it is conceivable that this strain inherently lacks the ability to undergo sexual commitment, even after AP2XII-1 and AP2XI-2 depletion. Additionally, sustained depletion of the two transcription factors may cause a dysregulation in a broader regulatory network, preventing further stage transitions.

We recently reported the successful use of a type II strain (ME49 Δhpt luc^+^) for oocyst production in cats, confirming its capacity for sexual development despite being kept in culture for extended times^47^. It is unknown what natural cue is mimicked by the “unnatural” 2xAP2 knockdown via IAA treatment. Intended as an initial exploratory assessment, we therefore tested whether the feline environment itself, together with the metabolic adjustments arising from the culture medium, might be sufficient to initiate sexual stage conversion in the ME49 strain. We monitored the formation of developmental stages by EM for up to 7 days; however, all observed parasites retained tachyzoite morphology, and no evidence for endopolygeny was found (Fig. S6). We conclude from these preliminary experiments that there is no simple cat host cell factor or factors that would at least mimic the genetic depletion of AP2XII-1 and AP2XI-2 in an oocyst-competent parasite strain.

Discussion

In nature, the sexual development of T. gondii is restricted to the feline intestinal epithelium, but pre-gamete formation has recently been achieved in vitro by simultaneous depletion of the transcription factors AP2XII-1 and AP2XI-2 in tachyzoites^10^. However, the progression to sexual stages and the completion of the parasite’s life cycle have not yet been reproducibly and fully replicated in cell culture. To this end, we established robust, long-term expanding feline small intestinal organoid cultures. In contrast to previous studies^16,17^, we could continuously expand our organoid cultures for months without observing any growth arrest, also after freezing/thawing and re-culture. This was achieved without the need for co-culturing with growth-inhibited fibroblast feeder cells^19^, reducing handling effort and minimizing variability. Compared to previous protocols, major changes we implemented were the establishment and growth of fIOs at the cat’s normal body temperature (38.5 °C) and the replacement of Wnt-3a with a bi-specific Wnt surrogate. A detailed discussion of why these changes might have been instrumental for our success can be found in the supplement (Supplementary Discussion 1).

The central hypothesis of this study was that host cell cues could promote the developmental progression of in vitro merozoites to sexual stages (gametocytes and eventually oocysts). However, the inability of AP2XII-1 and AP2XI-2-depleted parasites to form new vacuoles and the absence of sexual stages in EM suggest that our experimental model did not provide the necessary factors or did not clear the roadblocks for in vitro sexual development. AP2 depletion did, however, lead to the upregulation of gametocyte genes, indicating a partial initiation of the developmental program. Previous RNA-seq data from human fibroblasts infected with the knockdown strain showed no considerable or only a minor upregulation of gametocyte-specific genes, most notably the microgamete marker TGME49_500062, with detected transcript levels being very low (< 4 transcripts per million)^10^. Similar results have been reported for individual AP2XII-1 and AP2XI-2 KDs^6,9,10^. RT-qPCR is more sensitive than RNA-seq for detecting low-abundance transcripts and might therefore have been able to reveal the expression changes in this study.

Our initial attempt at cat organoid isolation was limited to the duodenum, and given its success, we continued using duodenal organoids throughout this study. Adult stem cells from different sections of the small intestine are intrinsically programmed with location-specific identities, which are also reflected in their derived organoid cultures^48^. While gametocytes have more commonly been observed in the ileum, merogony and sexual development of T. gondii occur along the entire feline small intestine, including the duodenum^46,49^. Direct inoculation of bradyzoites into the cat’s duodenum led to oocyst excretion with the same prepatent period as oral infection^50^. Oocyst shedding has also been reported in cats following intrajejunal^51^ or intraduodenal^52,53^ tachyzoite deposition, albeit only with high inoculation numbers and longer prepatent periods. Another study could not observe oocysts in cat feces upon intraduodenal infection with tachyzoites^50^. Thus, our duodenal fIO could be useful to investigate this aspect in more detail.

Long before AP2s were known, it had already been proposed that “sexual differentiation (…) is a phenotypic change induced by some final host factor(s)”^54^. To better mimic the feline intestinal environment and to specifically cater to the parasite’s metabolic requirements during sexual stage conversion, our ODM infection medium included the following factors abundant in the intestine or unique to felids.

Butyrate is the predominant short-chain fatty acid in the intestine^55^, naturally produced by bacteria as a byproduct of dietary fibers. Butyrate was reported to influence the energy homeostasis and the degree of differentiation of small intestinal epithelial cells^56^. Interestingly, bacteria concentrations in the proximal small intestine of cats were found to be higher compared to other species, presumably an adaptation to the carnivorous diet^57^. Notably, however, germ-free cats have been reported to shed oocysts after infection^58^, indicating that the gut microbiota may only have a limited role in T. gondii development.

We further included two sulfur-containing amino acids: taurine, an essential amino acid for cats due to their very limited ability to synthesize it^59^, and felinine, an intriguing compound, because this amino acid is exclusively found in felids^60^. Excreted in large amounts in the urine of cats, and also found in their feces^61^, felinine is thought of as a territorial marker and putative pheromone precursor^60,62^. Strikingly, high quantities of the N-acetylated derivative of felinine have been detected in the small intestine^61^. This invites speculation about some hypothetical contribution of felinine, or related felid-specific compounds (isovalthine, isobuteine) that follow a similar metabolic route^59^, to T. gondii development. We also supplemented L-cysteine to support the synthesis of the exceptionally cysteine-rich proteins that the parasite expresses during the sexual stages and in oocysts^63,64^.

Since T. gondii undergoes rapid DNA synthesis in its enteroepithelial stages (EES), purine and pyrimidine demand rise, leading to increased gene expression related to pyrimidine biosynthesis, purine transporters, and salvage pathways (Fig. S7). We therefore also added adenosine as a key precursor for nucleotide synthesis, particularly for the ME49 ∆hpt strain, which cannot directly convert hypoxanthine, xanthine, or guanine to their respective nucleotides and depends on the interconversion from adenosine. Notably, intestinal cells have limited purine synthesis^65^ and also rely on dietary and microbiota-derived purines^66^, creating competition between parasite and host. The exclusive meat diet of felids is purine-rich, but degradation of purines to uric acid by anaerobic gut bacteria^67^ could further reduce purine availability for T. gondii.

Linoleic acid, a fatty acid abundant in cats and presumably accumulating in the intestine due to limited delta-6-desaturase activity, has been reported to be critical for T. gondii sexual development^18^. However, we observed no difference when comparing +/- LA in HFF cells (Fig. 6). Taken together, while we have not yet tested the effect of the absence of any of the additional factors in ODMs, their inclusion is based on several nutritional peculiarities of the intestinal feline environment and the metabolic demands of EES and oocysts of T. gondii. The fIO system invites the testing of more such metabolites.

Since prolonged cell culture cultivation can alter the parasite’s transcriptional network^37,68^, using recently acquired, non-lab-adapted strains may be advantageous. Obtaining tissue cysts from oocyst-infected mice would help mitigate these concerns and minimize confounding variables in the organoid model. A previous study used oocyst-derived tissue cysts to infect feline organoid cells and documented T. gondii development advancing beyond pre-sexual stages, leading to the formation of oocyst-like structures^18^. However, these oocysts were limited in number, unable to sporulate, and non-infectious to mice.

This study underscores the complexity of T. gondii sexual development and highlights the limitations of current in vitro models in fully recapitulating the parasite’s life cycle. Given T. gondii’s host promiscuity in other aspects of its life cycle (assumed to infect all nucleated cells in all warm-blooded animals), the difficulty in reproducing the felid niche, which allows initiation of EES development, is surprising. Looking for unique genetic, metabolomic, or nutritional differences compared to other possible hosts will eventually lead to the identification of physiological cue(s) that turn AP2 factors involved in this process on or off and set aside felids from all the others. Further studies are warranted to identify the precise cues that drive sexual commitment and oocyst formation, ultimately advancing our ability to model the complete life cycle of T. gondii outside of the feline host. In this respect, the generation of long-term expandable fIOs and therefore derived ODMs represents a valuable and important technical advance.

The fIO system will also be of value for studies of other enteric pathogens, where it could help to address open questions of their biology, difficult to study in animals. These include other coccidia besides T. gondii^69^, other protozoa such as Giardia sp. and Cryptosporidium sp.^70,71^ but also feline viruses, including feline coronaviruses^17,72,73^.

Current challenges of the culture model with regard to T. gondii’s sexual development

To our knowledge, this study presents the first description of cat duodenal organoid cultures, whereas previous reports have been limited to ileal and jejunal organoids^16,17,19^. While we also generated organoids from a second animal and successfully established organoids from the jejunum and ileum as well, these were not investigated further. Thus, whether our protocol is particularly effective for duodenal organoids versus other intestinal sections needs to be evaluated.

The ODMs resembled the intestinal epithelium, but showed a limited degree of differentiation and cell type variety. Although gametocytes have only been reported in feline enterocytes^74^, other cell types may secrete factors required for parasite development. Further differentiation may be achieved using air-liquid interface (ALI) conditions, in which the top medium layer is removed. The life cycle of C. parvum has been recapitulated using intestinal organoids, and ALI conditions drove monolayer differentiation and improved C. parvum growth compared to submerged cultures^14^. The small intestinal epithelium experiences relatively low oxygen levels (~2–3%) compared to those present in standard culture systems (~21%), and such hypoxic conditions can influence cellular physiology^75^. While we did not investigate the effects of varying oxygen levels, incorporating hypoxic culture conditions or oxygen gradients represents important avenues for future research. In addition, they could also constitute physiological cues that are involved in sexual differentiation.

We assessed the developmental progression of in vitro-generated merozoites using the 2xAP2 KD strain. As such, the findings are limited to this strain and may not be generalizable to other engineered strains that induce the expression of sexual stages^6–9,76^ or in vivo merozoites. Continuous degradation of AP2XII-1 and AP2XI-2, while crucial to reach the merozoite stage, may disrupt the regulatory cascade required for further maturation. A potential approach to assess this would be transient AP2 depletion, allowing their presence at later stages. However, accurately timing the expression of transcription factors in individual parasites throughout their developmental progression – which may not be synchronous across the culture – could be important but not easy to achieve.

Another challenge that needs future consideration was the unexpected sensitivity of cat ODMs upon longer incubation periods with IAA (>48 h), consistent with recent results with mouse intestinal organoids^77^. Alternative inducers like 1-naphthaleneacetic acid^78^ or 5-Ph-IAA together with the new Auxin 2 system^79^ might be more suitable for long-term cultivation but require prior evaluation or implementation in T. gondii.

Our infection experiment with ME49 tachyzoites showed that even a parasite strain with known stage conversion potential does not readily enter the sexual nor the pre-sexual developmental trajectory in feline ODMs. However, the use of tachyzoites as the starting point for infection experiments without prior genetic modifications seemed unsuccessful. Tachyzoites are likely required to first form bradyzoites in the feline intestinal epithelium before transitioning to the EES^51^, but this intermediate step was not observed in our cultures. ODM infections with bradyzoites could therefore be the next step. Mature bradyzoites can be readily produced from tachyzoites in vitro^80,81^ and could be used as starting material on the way to a complete in vitro life cycle of T. gondii^5^. However, preliminary experiments have shown that successfully synchronizing both culture systems requires further adjustments.

A key challenge in studying T. gondii sexual development is the requirement for a high initial number of merozoites to reliably detect merozoite-to-gametocyte conversion, a process inherently occurring at low frequencies, even in vivo^82^. Since quantifying formed EES by microscopy proved difficult due to technical challenges, EM imaging of multinucleated schizonts present in the samples was used to illustrate potential stage progression. We further mitigated this limitation using sensitive RT-qPCR. While experiments with increased parasite and host cell numbers would likely improve the number and quantification of detectable stage conversion events, a caveat is the poor scalability of current ODM cultures. It is constrained mainly by the resource-intensive nature of the organoid cultivation, particularly compared to conventional in vitro cultures like HFF cells. A further challenge of this study was the difficulty in visualizing late developmental stages by antibodies, which in our hands proved to be unreliable in some cases. This could be addressed by generating and utilizing fluorescent reporter strains in future studies. Despite these limitations and challenges, however, optimizing an in vitro culture system as close as possible to the in vivo situation will be worth the effort.

Methods

Isolation of cat intestinal organoids

Cat intestinal organoids were isolated as described by refs. ^15,83^ and which is based on^84^. Briefly, cat intestinal tissue pieces were obtained from the University of Perugia (Italy), Department of Veterinary Medicine. Animal 1 was an adult female stray cat of unknown exact age with breast cancer and metastasis to the lung. Animal 2 was an adult female cat, 13 years old, of the European breed, with severe kidney failure. At necropsy, the lungs appeared hyperemic with hemorrhagic spots. The cat exhibited purulent and catarrhal rhinitis. Both cats came from a cattery and had no owners. They were under the responsibilities of the National Public Health Service and were euthanized to prevent avoidable suffering unrelated to this specific research. Therefore, no informed consent was required. Sections of the duodenum were excised from the cats’ small intestines. The samples were shipped on ice by air and were processed after their arrival the next day. All pipettes and tubes used during isolation were pre-coated using 0.1% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Small duodenal tissue pieces (approx. 5 mm^2^) were washed in 30 ml ice-cold PBS by gentle pipetting with a 10 ml pipette. PBS was replaced 5–10 times until the supernatant was almost clear. The tissue pieces were then incubated on ice in 30 ml PBS containing 2 mM EDTA, using a rocking platform shaker. The solution was decanted, and 20 ml ice-cold PBS was added to the samples. The tissue pieces were vigorously pipetted up and down 10 times using a 10 ml pipette to isolate intestinal crypts. The supernatant was collected, and this step was repeated 4 times. The crypt-containing fractions were combined and passed through a 70 µm cell strainer. The flow-through was then centrifuged at 150 × g for 5 min at 4 °C. The collected crypts were washed once with Advanced DMEM/F12 (Thermo Fisher Scientific, Cat. No. 12634028) and then resuspended in Advanced DMEM/F12 and mixed 1:1 with Cultrex basement membrane extract (R&D Systems, Cat. No. 3533-010-02). 50 µl droplets were placed into wells of a 24-well plate and hardened at 37 °C before adding cat organoid growth medium (0.5 nM Wnt surrogate Fc fusion protein (Wnt surrogate; ImmunoPrecise Antibodies, Cat. No. N001), 20% R-Spondin 1-conditioned media (CM)^85^, 10% Noggin-CM^86,87^, 50 ng/ml mouse recombinant epidermal growth factor (rec. EGF; Preprotech), 50 ng/ml human rec. EGF (Preprotech), 10 mM HEPES, 2 mM stable glutamine (Capricorn Scientific), 1×P/S (100 U/ml penicillin and 100 µg/ml streptomycin; Capricorn Scientific), 1×N2 (Thermo Fisher Scientific), 1×NCS21 (Capricorn Scientific), 2.5 µg/ml biotin (Santa Cruz Biotechnology), 1 mM N-acetyl-L-cysteine (Sigma), 10 mM nicotinamide (Sigma), 500 nM A83-01 (Sigma), 10 μM SB-202190 (Cayman), 10 µM ROCK inhibitor Y-27632 (Tocris) in Advanced DMEM/F12). For the first 5 days after isolation only, culture medium was supplemented with gentamicin (100 µg/ml, Capricorn Scientific), fungin (10 µg/ml, Invivogen), gastrin I (10 µM, Sigma), prostaglandin E2 (10 nM, Tocris), Lipid Mixture 1 (1:50, Sigma). Alongside the Wnt surrogate-containing medium, a second medium was tested that contained 50% Wnt-3a, R-Spondin 3, and Noggin-conditioned medium (L-WRN-CM)^88^ instead of Wnt surrogate. Medium was exchanged every 2–3 days. After their establishment, organoids were cultivated exclusively at 38.5 °C with Wnt surrogate medium.

Organoid maintenance

Passaging was performed as described previously^25^. Briefly, Cultrex-embedded organoids were harvested by scraping the ECM drops off the plate with a pipette tip. Organoids were collected and centrifuged at 500 × g for 5 min at 4 °C, washed with cold Advanced DMEM/F12. Cells were dissociated by enzymatic digestion with TrypLE Express for 5 min at 37 °C and then mechanically disrupted by forcing the suspension through an 18 G needle. After another washing step, the organoid cells were resuspended in Advanced DMEM/F12 and mixed with Cultrex in a 3:7 ratio. Drops were seeded and hardened as described for the organoid isolation. Wnt surrogate-containing growth medium was added, and organoids were incubated at 38.5 °C. Medium was exchanged every 2–3 days. The passaging interval was 5–7 days.

Differentiation of 3D organoids

For the differentiation of 3D organoids, the growth medium of 5-day-old cultures was replaced by Wnt-3a-free differentiation medium (based on ERN medium described in ref. ^15^, consisting of 5% R-Spondin 1-CM, 5% Noggin-CM, 1 mM HEPES, 2 mM stable glutamine, 1×P/S, 1×N2, 1×NCS21, 2.5 µg/ml biotin, 1 mM N-acetyl-L-cysteine, 50 ng/ml human rec. EGF, 50 ng/ml mouse rec. EGF and 10 µM Y-27632 in Advanced DMEM/F12). Organoids were kept in differentiation medium for 5 days. Cultures kept in growth medium served as a control.

Organoid cryopreservation and thawing

Organoids from 4 to 6 wells were harvested as described above, but after the first centrifugation step, the pellet was resuspended in 1 ml freezing medium (10% DMSO and 10% fatty acid-free BSA (Sigma) in Advanced DMEM/F12) and transferred to cryovials. Vials were placed into a freezing container (CoolCell LX) that was transferred to −70 °C. After 24 h, cryovials were transferred to liquid nitrogen for long-term storage. To thaw cryopreserved organoids, the cryovials were quickly placed in a 37 °C water bath. The thawed vial content was immediately transferred to a centrifuge tube with 9 ml Advanced DMEM/F12. After centrifugation at 500 × g for 5 min at 4 °C, the pellet was resuspended in Advanced DMEM/F12, mixed with Cultrex 1:1. Seeding and subsequent cultivation was performed as described above.

Establishment of organoid-derived monolayers (ODMs) on cell culture inserts

ODMs were created as described in ref. ^25^. Briefly, 0.6 cm^2^ polycarbonate cell culture filter inserts with 0.4 µm pore size (MilliCell) were placed in cell culture plates and pre-coated with 150 µl Cultrex (1:20 in Advanced DMEM/F12). After an incubation step at 4 °C for 3 h or overnight, the Cultrex mix was removed, and filters were incubated at 37 °C for 30 min. Organoids were harvested, and single cells were prepared as described above. The obtained cells were resuspended in ODM seeding medium (same as organoid growth medium (Table S1) but without A83-01 and SB-431542) and seeded into the filter insert. The cultures were kept at 38.5 °C. The next day and the day after, the medium was replaced with regular ODM medium (same as ODM seeding medium but without Wnt surrogate and Y-27632). After that, the medium was replaced every 2–3 days.

Transepithelial electrical resistance (TEER) measurements

TEER was measured to determine the formation of electrophysiological tight ODMs. Well plates with cell culture inserts were placed on a heating block set to 38.5 °C, and TEER was measured using a Millicell ERS-2 Voltohmmeter (Merck Millipore) with an Ag/AgCl chopstick electrode (STX01). TEER values were normalized to blank resistance (cell culture insert with no cells) and 1 cm^2^ surface area.

Fibroblast and parasite cultures

Human foreskin fibroblasts (HFF; BJ-5ta, ATCC CRL-4001) were grown in DMEM High Glucose (4.5 g/l) with Stable Glutamine (Capricorn Scientific, Cat. No. DMEM-HPSTA) supplemented with 10% fetal bovine serum (FBS low endotoxin; Capricorn), 1×P/S (100 U/ml penicillin and 100 µg/ml streptomycin) at 37 °C in 5% CO_2_ atmosphere. For passaging, cells were washed with PBS and incubated at 37 °C with Trypsin-EDTA (0.05%) in PBS until detached. Medium was added, and the cell suspension was used at a subcultivation ratio of 1:5 to 1:10. Toxoplasma gondii tachyzoites (strains are listed in Table S2) were maintained by continuous passage in confluent monolayers of HFF cells in tachyzoite medium (DMEM High Glucose (4.5 g/l) with Stable Glutamine, 10% FBS, 1×P/S).

Infection, auxin-induced degradation, and apicidin treatment

T. gondii-infected host cells were scraped from cell culture flasks, and the parasites were released by repeated passes through a 23 G tubing. Cell debris was removed by centrifugation at 100 × g for 5 min. The supernatant was collected and centrifuged at 300 × g for 10 min to pellet the parasites. The parasites were then resuspended in the respective medium for infection.

For the infection of HFFs, the medium consisted of DMEM High Glucose (4.5 g/l) with Stable Glutamine, 10% FBS, and 1×P/S. Confluent HFF cell monolayers in 6-well plates were infected with 6 × 10^5^ parasites per well. Two hours post infection, the medium was replaced with fresh infection medium with or without 200 µM linoleic acid (LA) supplementation.

For the infection of cat ODMs, the regular ODM medium described above was additionally supplemented with 5 mM adenosine (TCI, cat. no. A0152), 1 mM butyric acid sodium salt (Acros, cat. no. 10474715), 0.8 mM taurine, 12.7 mM L-cysteine, 0.5 g/l bile from bovine and ovine (all Sigma-Aldrich), and 120 nM L-felinine (Toronto Research Chemicals, cat. no. F231250). In the knockdown experiment, the medium was additionally supplemented with 200 µM LA (Sigma, cat. no. L1012). One day prior to infection, ODMs were pre-incubated with this medium in the apical compartment. For infection, 400 µl of parasite suspension with 6 × 10^5^ parasites were placed into the apical compartment of each well. The basal compartment was filled with regular ODM medium supplemented with only 120 nM L-felinine. To initiate the degradation of AP2XII-1 and AP2XI-2, the medium was changed after 24 h, and fresh medium with 500 µM 3-indoleacetic acid (IAA; Sigma cat. no. I2886) was added. The medium was prepared using a stock solution of 500 mM IAA in 100% EtOH. For the untreated controls, medium with the equivalent amount of EtOH was used. For apicidin treatment^89^, fresh medium with 75 nM apicidin (Adipogen, cat. no. AG-CN2) was added.

RNA extraction and RT-qPCR

3D organoids were washed with PBS before the extracellular matrix was dissociated with ice-cold cell recovery solution (Corning, cat. no. 354253). Recovered organoids were transferred to 15 ml tubes pre-coated with 1% BSA in PBS. The tubes were filled to 12 ml with PBS and centrifuged at 70 × g for 5 min at 4 °C. Collected organoids were resuspended in TRI Reagent (Zymo, cat. no. R2050-1) and transferred into a 1.5 ml Eppendorf DNA LoBind tube (cat. no. 0030108051) for subsequent RNA extraction. For ODMs and HFFs, medium was removed, and TRI Reagent was added to the filter insert or well, and cells were detached by pipetting up and down. The suspension was collected in Eppendorf tubes. Total RNA was isolated using the Direct-zol RNA Microprep Kit (Zymo, cat. no. R2062), including on-column DNase I digestion to remove genomic DNA (gDNA). RNA was quantified spectrophotometrically at 260/280 nm with the Infinite M200 Pro plate reader (Tecan). RNA was reverse-transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, cat. no. RR047A).

Quantitative polymerase-chain reaction (qPCR) was performed using Luna Universal qPCR Master Mix (NEB, cat. no. M3003) with “No-RT” controls included for each sample. All qPCR primers (Table S3) were verified for amplification efficiency within 95–105% and specificity by melt curve analysis in CFX Maestro software. Where applicable, stage-specific mRNA expression was induced by apicidin treatment of tachyzoites as described^89^. qPCR was performed with a Bio-Rad C1000 cycler with CFX96 system using the following parameters: 10 min at 95 °C, followed by 35 cycles of 20 s at 95 °C, 30 s at 60 °C, and 20 s at 72 °C. Expression was normalized to an internal reference gene: RPS7 for cat cells^90^ and CK1 (TGME49_240640) for T. gondii. The comparative Ct (ΔΔCt) method was used to quantify fold expression relative to untreated samples.

Immunofluorescence microscopy

ODMs were washed with PBS, fixed (4% paraformaldehyde in PBS) for 20 min, quenched (50 mM NH_4_Cl in PBS) for 15 min, washed with PBS twice, permeabilized (100 mM glycine, 0.25% Triton X-100 in Tris-buffered saline (TBS)) for 20 min, and incubated in blocking buffer (3% BSA, 1% normal goat serum (Thermo Fisher, cat. no. 16210072), 0.02% Triton X-100 (Sigma), 0.05% Tween-20 (Bio-Rad), and 0.02% sodium azide (Sigma) in TBS) for 3 h at room temperature. Primary antibody incubation was performed at 4 °C overnight, and secondary antibody and dye incubation was performed for 1 h at room temperature. After each step, ODMs were washed three times with washing buffer (0.02% Triton X-100, 0.05% Tween-20 in TBS). After the final wash, the inserts were rinsed once with deionized water, and the filters were separated from the frame using a scalpel. The filters were mounted onto glass slides with Fluoromount-G (SouthernBiotech, cat. no. 0100-01).

3D organoids were recovered as described above (see “RNA extraction and RT-qPCR” section) and prepared for imaging following the protocol by Dekkers et al.^91^. Briefly, the organoids were fixed for 45 min, and the blocking and labeling steps were performed in 1.5 ml Eppendorf Protein LoBind tubes (cat. no. 0030108116). All steps were performed at 4 °C, and the staining step was performed overnight. After labeling, organoids were incubated with fructose-glycerol clearing solution (60% glycerol and 2.5 M fructose) for 20 min at room temperature. The organoids were then placed onto a slide and sealed with a coverslip using two layers of sticky tape. Confocal microscopy was performed using a Leica Stellaris 8 laser scanning microscope equipped with a 63× objective (HC PL APO CS2 63×/1.40 OIL). Images for parasite vacuole quantification were taken using a Leica Mica Widefield microscope with a 10× objective (PL FLUOTAR 10×/0.32). Images were processed with FIJI 2.3.0 and assembled with Affinity Designer 1.9.2.

Antibodies and stains

Antibodies and stains for immunofluorescence assays (IFA) were used in the following dilutions: rabbit anti-GAP45^92^, 1:5000; rabbit anti-ZO-1 (Invitrogen, cat. no. 61-7300) 1:25; anti-rabbit Alexa Fluor 568 (Invitrogen, cat. no. A10042) 1:250; Phalloidin-iFluor 488 (Abcam, cat. no. ab176753), 1:1000; DAPI (Sigma-Aldrich, cat. no. D9542), 0.2 μg/ml.

Quantification of parasite growth

Parasite growth was quantified from immunofluorescence microscopy images taken with a Leica Mica Widefield microscope equipped with a 10× objective. Parasites were stained with anti-GAP45 and nuclei with DAPI. For each condition and time point, three images were taken at random locations per filter. Each field contained >1300 host nuclei. GAP45-positive areas and DAPI-stained host nuclei were determined and quantified using Leica Application Suite X software and the StarDist plugin for FIJI^93^. The parasite area-to-host nuclei ratio was calculated and normalized to t = 0. Data were visualized using GraphPad Prism version 9.0.

Thin-section electron microscopy (EM) of ODM

ODMs were chemically fixed on their filter substrate by replacing the medium with fixative (1% paraformaldehyde, 2.5% glutaraldehyde, 0.05 M Hepes with Hanks balanced salt solution). Filters were post-fixed with osmium tetroxide, tannic acid, and uranyl acetate, dehydrated in ethanol, and embedded in Epon resin, with acetone as an intermedium, as described before (part “Thin section EM – Step-by-step protocol of machine-assisted epoxy-resin embedding”^94^). Thin sections were produced with an ultramicrotome at 60–100 nm section thickness and collected on naked copper grids or solid silicon substrates. On-section staining with uranyl acetate and lead citrate was used to further increase the contrast. Imaging was done with a transmission-electron microscope (Tecnai Spirit, Thermo Fisher) operated at 120 kV and a CMOS camera, or with a scanning-electron microscope (Teneo VS, Thermo Fisher) operated at 2 kV using the T1 detector to collect backscattered electrons. Serial sectioning was done at a mean section thickness of 90 nm. Sections were collected on silicon wafers and stained with lead citrate. Imaging of respective cells in all sections was performed by using the field-emission scanning-electron microscope. Profiles of nuclei were traced manually.

Amino acid sequence alignment

CLUSTAL multiple sequence alignment by MUSCLE (3.8) was performed on the EMBL-EBI website (www.ebi.ac.uk/jdispatcher/msa/muscle), visualized with Jalview 2.11.3.3, and arranged and accentuated with Affinity Designer. Sequences were retrieved from the Uniprot database (release 2025_01, 05-Feb-2025): human Wnt-3a (P56704), feline Wnt-3a (A0A2I2UTQ5), feline EGF (A0A5F5Y0J7), and sequences for recombinant human and mouse EGF were retrieved from the company website (Preprotech).

Statistics and reproducibility

Statistical analyses were performed using GraphPad Prism software (version 9.0). Specific statistical tests and the number of replicates are detailed in the “Methods” section and figure legends. For comparisons between two groups, an unpaired two-tailed t-test was used. For comparisons involving more than two groups, two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was applied. P values < 0.05 were considered statistically significant. Independent experiments refer to biological replicates from different organoid and parasite culture passages conducted on separate days, while technical replicates refer to replicates of the same sample.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information Description of Additional Supplementary Files Supplementary Data Reporting summary