Dataset Information

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 host-pathogen intreaction shows great promise for modelling 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 modelling 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 dramatic reduction in 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Toxoplasma Gondii Mus Musculus (mouse)

TISSUE(S): Epithelial Cell

SUBMITTER: Stuart Armstrong

LAB HEAD: Janine Coombes

PROVIDER: PXD013306 | Pride | 2019-11-12

REPOSITORIES: Pride

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An Open-Format Enteroid Culture System for Interrogation of Interactions Between <i>Toxoplasma gondii</i> and the Intestinal Epithelium.

Luu Lisa L Johnston Luke J LJ Derricott Hayley H Armstrong Stuart D SD Randle Nadine N Hartley Catherine S CS Duckworth Carrie A CA Campbell Barry J BJ Wastling Jonathan M JM Coombes Janine L JL

Frontiers in cellular and infection microbiology 20190828

When transmitted through the oral route, <i>Toxoplasma gondii</i> 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 interact ...[more]

PMID: 31555604

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Project description:The innate immune response of mucosal epithelial cells during pathogen invasion plays a central role in immune regulation in the gut. Toxoplasma gondii (T. gondii) is a protozoan intracellular parasite that is usually transmitted through oral infection. Although much of the information on immunity to T. gondii has come from intra-peritoneal infection models, more recent studies have revealed the importance of studying immunity following infection through the natural per-oral route. Oral infection studies have identified many of the key players in the intestinal response; however, they have relied on responses detected days to weeks following infection. Much less is known about how the gut epithelial layer senses and reacts during initial contact with the pathogen. Given the importance of epithelial cells during pathogen invasion, this study uses an in vitro approach to isolate the key players and examine the early response of intestinal epithelial cells during infection by T. gondii. We show that human intestinal epithelial cells infected with T. gondii elicit rapid MAPK phosphorylation, NF-ÎºB nuclear translocation, and secretion of interleukin (IL)-8. Both ERK1/2 activation and IL-8 secretion responses were shown to be MyD88 dependent and TLR2 was identified to be involved in the recognition of the parasite regardless of the parasite genotype. Furthermore, we were able to identify additional T. gondii-regulated genes in the infected cells using a pathway-focused array. Together, our findings suggest that intestinal epithelial cells were able to recognize T. gondii during infection, and the outcome is important for modulating intestinal immune responses. Oral infection studies have demonstrated an increase in several cytokines and chemokines in response to T.gondii infection; however, the mixed population of the intestinal mucosa did not allow for the determination of the relative role that specific cell populations play in the production of these mediators. To address the role of intestinal epithelial cells to modulate the cytokine environment early following infection, we used specific pathway arrays to identify cytokines and chemokines induced 4 hours after exposure to T. gondii. At this time point most cells have become infected, but the parasites have not replicated.

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