An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium

Abstract

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii . Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.

Stem cell‐derived enteroid cultures as a tool for dissecting host‐parasite interactions in the small intestinal epithelium

“…While intestinal inflammation has been documented in other species, it is not a common feature of acute infection in humans or farm animals and caution is warranted when studying epithelial responses in these mice 44 . The ability to generate enteroid cultures easily from multiple species and from mice of multiple genetic backgrounds will be beneficial in this respect 14,17,18,45 …”

“…To reflect the physiologic route of infection via the apical epithelial surface, we have developed three enteroid‐based techniques for modelling T gondii infection: fragmentation, microinjection and open‐format enteroid cultures 14 . Fragmentation of enteroids increases the probability of T gondii infecting through the newly exposed apical epithelial surface.…”

“…Fragmentation of enteroids increases the probability of T gondii infecting through the newly exposed apical epithelial surface. However, although this method exposes the apical surface it does not limit infection to that site 14,16,74 . One way to restrict infection to the luminal surface is through the use of a microinjection system that delivers pathogens directly into the lumen of enteroids 4 .…”

“…Development of enteroid‐derived monolayers or ‘inside‐out’ enteroid models will be important in addressing this issue (Figure 1E,F). 10‐15 …”

“…Enteroid models have recently been applied to the study of T gondii and Cryptosporidium spp, yielding important biological insights 14,16‐25 . Since enteroids recreate many of the characteristics of the in vivo environment in a sustainable and cost‐effective way, they pave the way for a reduction in the use of animals in research into these important parasites.…”