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

ORIGINAL RESEARCH published: 28 August 2019 doi: 10.3389/fcimb.2019.00300 An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium Edited by: Jeroen P. J. Saeij, University of California, Davis, United States Reviewed by: Laura Knoll, University of Wisconsin—Madison, United States Frank Seeber, Robert Koch Institute, Germany Michael S. Behnke, Louisiana State University, United States *Correspondence: Janine L. Coombes † These authors have contributed equally to this work as joint first authors ‡ Present address: Jonathan M. Wastling, Faculty of Natural Sciences, Keele University, Staffordshire, United Kingdom Specialty section: This article was submitted to Parasite and Host, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 29 March 2019 Accepted: 05 August 2019 Published: 28 August 2019 Citation: Luu L, Johnston LJ, Derricott H, Armstrong SD, Randle N, Hartley CS, Duckworth CA, Campbell BJ, Wastling JM and Coombes JL (2019) An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium. Front. Cell. Infect. Microbiol. 9:300. doi: 10.3389/fcimb.2019.00300 Lisa Luu 1† , Luke J. Johnston 1† , Hayley Derricott 1 , Stuart D. Armstrong 1 , Nadine Randle 1 , Catherine S. Hartley 1 , Carrie A. Duckworth 2 , Barry J. Campbell 2 , Jonathan M. Wastling 1‡ and Janine L. Coombes 1* 1 Department of Infection Biology, Faculty of Health and Life Sciences, School of Veterinary Science, Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom, 2 Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom 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. Keywords: enteroid, organoid, Toxoplasma gondii, intestine, cholesterol, statin, monolayer INTRODUCTION Toxoplasma gondii infection is commonly acquired following oral ingestion of cyst-containing meat, or oocyst-contaminated water and produce. As a result, the first encounter between parasite and host occurs in the small intestinal epithelium. Subsequently, the parasite travels from the intestine to the brain and other tissues, where it forms cysts that persist for the lifetime of the Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 August 2019 | Volume 9 | Article 300 Luu et al. Infection of Enteroids With T. gondii infected individual. This can have serious consequences to human health: reactivation of cysts in people whose immune systems are compromised can result in severe encephalitis and death. Furthermore, spontaneous abortion, stillbirth, and severe birth defects can occur if the infection is caught during pregnancy and transmitted to the fetus. Treatment for toxoplasmosis is available, but it can cause severe side effects and is ineffective against brain cysts. Thus, the development of novel vaccines and therapeutics remains an important research goal. An obvious target is the initial interaction between the parasite and intestinal epithelium, which is critical not only in controlling initial invasion and replication, but also in shaping the quality of the systemic immune response. Surprisingly little is known about how T. gondii interacts with the small intestinal epithelium of orally infected hosts. In vivo, it has been reported that dividing parasites can be observed in intestinal tissue as early as 1 day post infection (dpi), although our own work shows that parasites do not become readily detectable in the intestine until 5 dpi or later (Dubey, 1997; Kobayashi et al., 1999; Coombes et al., 2013). This makes it almost impossible to study the earliest interactions between parasite and host in a whole animal model. Experiments in cell line and explant culture models have shown that paracellular transmigration may play a significant role in early traversal of the epithelial barrier by T. gondii (Barragan et al., 2005). Tachyzoites cluster at intercellular junctions and transmigrate, often without significant disruption of the epithelial barrier (Barragan et al., 2005; Weight and Carding, 2012; Jones et al., 2016). This allows the parasite to rapidly disseminate, likely following invasion of motile immune cells (Barragan and Sibley, 2002; Courret et al., 2008; Weidner et al., 2016). Despite this rapid transmigration, there is clear evidence of communication between the parasite and the host epithelium. T. gondii uses host intercellular adhesion molecule 1 (ICAM-1), and the tight-junction protein, occludin, as receptors for transmigration, resulting in redistribution of occludin from tight junctions to an intracellular compartment (Barragan et al., 2005; Weight and Carding, 2012). Furthermore, a proportion of tachyzoites are seen to invade, rather than bypass, cultured epithelial cells. Nevertheless, we understand remarkably little about how epithelial cells respond to contact with the parasite, or if the parasite targets a specific cell type or location along the crypt-villus axis. To do this, we require sophisticated models (...truncated)