Development of primary cell-based infection models to study pathogenesis of Campylobacter jejuni

Campylobacter jejuni is currently the leading cause of bacterial foodborne gastroenteritis in humans. However, only a few C. jejuni virulence-determining factors involved in host cell interactions in-vivo have been identified, in part due to the lack of appropriate infection models that accurately reflect the human host environment. So far, infection studies have mainly employed animal or in-vitro cell culture models based on immortalised cell lines (e.g., Caco-2 cells), which are limited in their ability to model the complexity of intact three-dimensional (3D) tissue in the human host. To add physical characteristics of host intestinal tissue, like the structure of crypts and villi, we previously developed a 3D infection model based on tissue-engineering that incorporates a biological scaffold, the small intestinal submucosa, seeded with Caco-2 cells. This 3D infection model allowed for monitoring pathogenesis related phenotypes of C. jejuni mutant strains analogous to phenotypes previously observed in animal models, which could not be reflected in 2D monolayers. Nevertheless, the use of immortalized cell lines in an infection model limits the resemblance of specific host tissue characteristic, such as cell type diversity. These shortcomings hinder the identification of host cell types targeted by C. jejuni, as well as the determination of bacterial survival and replication within host cells. Therefore, we are now adapting primary cell-based in-vitro infection models of the small intestine by employing human intestinal organoids for C. jejuni infections. So far, we have successfully set up 3D and 2D monolayer cultures of human jejunum and ileum organoids in our lab. First infection experiments of organoid-derived monolayers demonstrate that independent of the multiplicity of infection (MOI) and patient origin of organoids, C. jejuni can infect primary jejunum cells and in several of these, clusters of multiple bacterial cells can be observed. Using these humanized infection models, we aim to get a deeper understanding of C. jejuni pathogenesis, including host target cell identification as well as epithelial and pathogen factors mediating infections.