Development of modified organoid culture protocols for interrogation of interactions between Toxoplasma gondii and the intestinal epithelium

Mon9  Apr12:00pm(15 mins)
Where:
Stream 2 - Llandinam A6

Authors

L Luu2; N Randle2; S D Armstrong2; J Wastling1J L Coombes2
1 Keele University, UK;  2 University of Liverpool, UK

Discussion

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction has the potential to shape the course of the systemic immune response, and may be an attractive target for prophylactic therapy in livestock. However, we understand surprisingly little about the molecular pathways governing the interactions between T. gondii and the intestinal epithelium.

The in vitro 3D culture of intestinal epithelium as organoids (or “mini-guts”) shows great promise for modelling infection. Organoids retain key architectural features of, and contain all of the major differentiated epithelial cell types found in, the intestinal epithelium. However, current protocols yield cultures with an enclosed luminal space that precludes the large-scale application of infectious agents to the apical surface of the epithelium.

Here, we have adapted organoid culture protocols to generate collagen-supported semi-monolayer cultures with an exposed lumen for practical application of pathogens. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations with host-defensive function, such as goblet and Paneth cells. They are susceptible to infection with, and support replication of, T. gondii.

Using quantitative label-free mass spectrometry, we show that infection of the intestinal epithelium in our model is associated with upregulation of host lipid biosynthesis pathways, including isoprenoid biosynthesis. While T. gondii can synthesise isoprenoid precursors in the apicoplast, optimal survival and growth relies on the use of host cell isoprenoids. Consequently, host isoprenoid biosynthesis may represent an attractive drug target.

In conclusion, our adapted semi-monolayer model offers a tractable tool for understanding how interactions between T. gondii and the host intestinal epithelium influence the course of infection.

This work was funded by a Biotechnology and Biological Sciences Research Council Tools and Resources Development Fund grant (BB/M019071/1)

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