Project Outline

The gastrointestinal tract is lined by a layer of mucus, which serves as a barrier separating the microbiota and the underlying epithelial cell layer (Paone and Cani, 2020). Bacterial-mediated mucin degradation provides the microbiota with an important nutrient source. However, the bidirectional interactions between GI-microbiota and the mucus layer are poorly understood, particularly during disease states in which the mucin composition undergoes significant changes. In the stomach, the mucins change dramatically during a pre-cancerous stage of gastric cancer (GC) known as intestinal metaplasia (IM). During IM, there is an imbalance of typical gastric mucins and an enrichment of intestinal mucins, such as Muc2. Concurrent with these changes, the gastric microbiota undergoes ‘dysbiosis’ where there is an enrichment of opportunistic oral bacteria and a reduction in the levels of Helicobacter pylori, the causative agent of GC. Although these two phenomena are known to play a role in the development of GC, there is a fundamental lack of understanding of the interplay between the mucins and the gastric microbiota; does the change in mucins during IM select for members of the gastric microbiota or does the microbiota drive changes to the expression of these mucins?

To answer this question, this project will use advanced cell culture techniques to generate human gastric organoids from healthy and IM gastric tissue. Gastric organoids are cultured in vitro from gastric endoscopic biopsies and mimic the multicellularity and three-dimensional architecture of the gastric environment observed in vivo. Mucosoid cultures are two-dimensional organoids that produce a thick layer of mucus and are more amenable to high-throughput infection studies than their three-dimensional counterparts (Boccellato et al in 2019). These mucosoid cultures have successfully been established in our laboratory from healthy gastric tissue and will provide an excellent tool to compare fundamental interactions between mucins and the autologous microbiota from either healthy or IM gastric tissue.

Key objectives for this project are as follows:

1. Generate healthy and IM gastric mucosoid cultures and characterise the composition of the mucus layer, using a suite of mucin-active enzymes.
2. Isolate and identify bacteria from healthy and IM human gastric tissue, using bacterial culture and 16S ribosomal RNA sequencing.
3. Co-infect healthy and IM organoids with up to 3 of the most dominant bacteria isolated in aim 2. Characterise polymicrobial interactions, mucin gene expression and bacterial-mediated mucin degradation, using RNAScope, confocal imaging and mucin-active enzymes.

References

Boccellato F, Woelffling S, et al. (2019). Polarised epithelial monolayers of the gastric mucosa reveal insights into mucosal homeostasis and defence against infection. Gut. 68(3):400-413.

Paone, P. and Cani, P.D. (2020) Mucus barrier, mucins and gut microbiota: the expected slimy partners? Gut. 69:2232-2243.

Techniques

The following techniques will be used for this project:

Bacterial culture, 16S ribosomal RNA sequencing, RNAScope fluorescence in situ hybridisation, confocal imaging, human organoid culture, mucin enzyme arrays, chronic models of co-infection