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. 2025 Feb 4;19(2):e0012855.
doi: 10.1371/journal.pntd.0012855. eCollection 2025 Feb.

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

Affiliations

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

Carlos J Ramírez-Flores et al. PLoS Negl Trop Dis. .

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.

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Conflict of interest statement

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.

Figures

Fig 1
Fig 1. T. gondii localization in mouse jejunal villi after ingesting cysts-containing brains.
Jejunal localization of T. gondii after the natural route of infection. (A) Schematic representation of (i) the villus and its structures and (ii) the experimental setup. Images represent single events captured in independent infected mice after 3 days post-ingestion. Single mouse ingested brains containing ~4 ×ばつ 102 cysts. (B) Confocal images of infected villus with T. gondii after 3 days post-ingestion of cysts-containing brains. Each image represents single and isolated events captured in independent mice. T. gondii is localized in the intestinal lumen (i) or in the jejunal stroma (ii). In (B) the villi are stained for actin (red, rhodamine phalloidin), T. gondii (green, polyclonal antibodies), and nuclei (blue, DAPI). (Ci) Schematic representation of the experimental setup for detecting T. gondii infection by IVIS in the small intestine of mice fed cysts-containing brains. Single mouse ingested brains containing ~4 ×ばつ 102–~5 ×ばつ 102 cysts. (Cii) Representative image of the distribution of T. gondii infection by IVIS in the small intestine of mice fed cysts-containing brains after 5 days post-ingestion. (D) Grouped scatter plot showing the number of infection spots detected in the small intestine of seven mice in two independent experiments. (E) Grouped scatter plot of luminescence flux measured in the small intestine of infected mice. Data in (D) and (E) represent the observation of the events registered in seven infected mice in a combination of two independent experiments, with values presented as means ± SD (*p ≤ 0.05). (F) Representative epifluorescent images of T. gondii infection in the jejunum of mice fed brain containing cysts at 3- and 5-days post-feeding. SAG1-positive parasites (tachyzoites) in the stroma. (G) Confocal 3D projection of tachyzoites replicating in the stroma. (H) Epifluorescent image showing the absence of BAG1 in a villus of the infected jejunum. Lines in yellow define the villus edges. (I) Grouped scatter plots showing the fluorescence intensity of SAG1 in infection spots (left plots) or the number of SAG1-positive PVs detected in either the stroma or enterocytes within the jejunum (right plots). (J) Quantification of the observed infected villi positive for SAG-1 parasites per section of intestine. (K) Representative of an epifluorescent image showing tachyzoites replicating within regions of excessive cell infiltration at 5 days post-feeding with brains. Intestines are stained for WGA (green), SAG1 (red), BAG1 (red), and nuclei (blue, DAPI). Data in (I) represents a combination of at least fifteen different intestinal structures, with values presented as means ± SD (***p ≤ 0.001, ****p ≤ 0.0001). Data in (J) represents a combination of at least 12 independent observations in different sections of the same or different infected mouse. Schematic representations in (A and C) were as created with BioRender.com.
Fig 2
Fig 2. In vitro bradyzoite differentiation of type I and II strains at 33°C.
(A) Plot represents the number of intact vacuoles of ME49 parasites in the presence of HFF media or differentiation media. Quantification was done in at least ten different fields after 6- and 8-days post infection. Images to the right show representative fluorescent imaging of infected HFF cells. (B) Plots to the left shows the quantification of positive vacuoles to SAG1, BAG1 or both in ME49 parasites after incubation for 6 days in differentiation media at 37°C or 33°C. Plots to the right show positive vacuoles to SAG1, BAG1 or both in type II and type I parasites after incubation for 12 days in differentiation media at 33°C. Images to the right show representative fluorescent imaging of infected HFF cells. (C) Plots to the left show the quantification of PVs positive to DBA after 7 days in differentiation media in ME49 and RH parasites. Images to the right show representative fluorescent images of DBA parasites (D) Plots to the left show the quantification of PVs positive to DBA after 8- and 12-days in differentiation media in ME49 and RH parasites. Images to the right show representative fluorescent images of DBA- or BAG1-positive type I parasites after 8- or 12-days in differentiation media. (E) Plots show the in vitro differentiation of EGS strain at 33°C and at 37°C. Plots show live fluorescent imaging of EGS parasites detecting mCherry-expressing tachyzoites or GFP-expressing bradyzoites. Quantifications were performed by quantifying the number of vacuoles (top plots) or by measuring the fluorescent area (bottom plots). (G) Survival curve of mice fed bread with a range of 1 ×ばつ 102 – 1 ×ばつ 105 in vitro-generated bradyzoites. Three Swiss Webster mice were tested for each experimental condition.
Fig 3
Fig 3. Bradyzoite activation for infection of human intestinal microphysiological systems to study bradyzoite interactions.
(A) Schematic representation of bradyzoite infection niche in human intestinal MPS. (i) Live staining of actin showing the coverage of the lumen by Caco-2 cells. (ii) FITC-dextran live imaging assessing the polarity of the Caco-2 cell line in the MPS. (iii) Parasite replication at 48 hours post-infection with 1 ×ばつ 103 RH tachyzoites as a control for host cell viability. (B) Representative fluorescent images of parasite replication in HFF cells infected with brain bradyzoites, either activated by pepsin or subjected to mechanical lysis, at 9 days post-infection. Graph shows parasite kinetics of growth in HFF for pepsin-activated versus inactivated bradyzoites. (C) Representative fluorescent images of replication in HFF, Caco-2, and HIEC-6 infected with activated bradyzoites at 7 days post-infection. Graph shows parasite kinetics of growth in the three different cell lines. Schematic representation in (A) was created with BioRender.com.
Fig 4
Fig 4. Bradyzoite differentiation to tachyzoite in human intestinal microphysiological systems.
(A) Fluorescent images showing replication of alive ME49 mCherry bradyzoites in the Caco-2 lumen. (i)(ii), and (iii) show immunofluorescent images of the parasite conversion of ME49 mCherry bradyzoites to tachyzoites, with markers for tachyzoites (SAG1, red) and bradyzoites (BAG1, green) at 3 days post-infection. (B) Fluorescent images of alive EGS LDH2p-GFP/SAG1p-mCherry parasites undergoing differentiation in MPS, with bradyzoites shown in green and tachyzoites in red. Punctuated lines represent the edges of the lumen and its boundary with the matrix. Lumens in (A) and (B) were infected with a total of 3 ×ばつ 104 bradyzoites ME49 mCherry or EGS LDH2p-GFP/SAG1p-mCherry parasites. Plots represent the quantification of mCherry positive PVs (expressing SAG1) from day 2- to 5-post infection as bradyzoites. Plots are graphed as the average of positive PVs per lumen. A total of seven plates were analyzed, each one with at least three infected lumens.
Fig 5
Fig 5. Bradyzoite transmigration of Caco-2 cells in human intestinal microphysiological systemsand intestinal jejunal villi explants.
(A) Representative fluorescent images showing bradyzoite transmigration through the Caco-2 cells after 20 h post-infection of lumens. Punctuated lines in white represent the edges of the lumen and its boundary with the matrix. (B) Confocal images of explants of villi infected for 6 h (i) or 24 h (ii-iv) and then fixed. (i) Shows EGS LDH2p-GFP/SAG1p-mCherry bradyzoites localizing in the jejunal stroma. (ii a) shows a 3D projection of a bradyzoite localizing in the stroma of villus. (ii b) represents a Z-section of (ii a). (iii) Shows a 3D projection of bradyzoites transmigrating through the enterocytes. (iv) Confocal image of a bradyzoite localizing in the stroma of villus. (C) Confocal images of explants of villi fixed and then infected for 6 h (i) or 24 h (ii-iv). (i) Shows EGS LDH2p-GFP/SAG1p-mCherry bradyzoites localizing underneath the enterocytes. (ii a) shows a 3D projection of a bradyzoite localizing in the stroma of the infected fixed villus. (ii b) represents a Z-section of (ii a). (iii) 3D projection of bradyzoites interacting with the enterocytes of the fixed villus. (iv) Confocal image of a bradyzoite localizing in the stroma of fixed villus.
Fig 6
Fig 6. Active tachyzoite migration through 3D collagen I/fibronectin matrix and expansion of its infection in human intestinal MPS generated with Caco-2 double-lumens.
(A) Schematic representation of bradyzoite/tachyzoite infection niche in human intestinal MPS. (B) Fluorescent images showing the coexistence of EGS LDH2p-GFP/SAG1p-mCherry bradyzoites and tachyzoites in the infected intestinal lumen at 3 days post-infection, with insets highlighting the localization of bradyzoites in the 3D collagen I/fibronectin matrix. Fluorescent images tracking the migration in collagen and Caco-2 infection by EGS LDH2p-GFP/SAG1p-mCherry parasites after exteriorization from Caco-2 cells in the bottom Caco-2 lumen shown in (C) and (D). (C) Micrographs show tachyzoites localizing in the 3D collagen I/fibronectin matrix and actively migrating towards the top Caco-2 lumen by day 5 post-infection. (D) Right micrographs show tachyzoites expanding the infection to the adjacent top Caco-2 lumen. Punctuated circles show parasitic infection to the top Caco-2 lumen. Punctuated lines in white represent the edges of the lumen and its boundary with the matrix. Lumens were infected with 5 ×ばつ 104 bradyzoites of the EGS LDH2p-GFP/SAG1p-mCherry strain. Magnified photograph in (B) shows different parasites captured in different planes than those in unmagnified photograph. Prominent areas in (C) and (D) are the result from foci of infection. Schematic representation in (A) was created with BioRender.com.

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