A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni

The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens. Author summary Enteric pathogens have evolved numerous strategies to successfully colonize and persist in the human gastrointestinal tract. However, especially for the research of virulence mechanisms of human pathogens, often only limited infection models are available. Here, we have applied and further advanced a tissue-engineered human intestinal tissue model based on an extracellular matrix scaffold reseeded with human cells that can faithfully mimic pathogenesis-determining processes of the zoonotic pathogen Campylobacter jejuni. Our three-dimensional (3D) intestinal infection model allows for the assessment of epithelial barrier function during infection as well as for the quantification of bacterial adherence, internalization, and transmigration. Investigation of C. jejuni mutant strains in our 3D tissue model revealed isolate-specific infection phenotypes, in-vivo relevant infection outcomes, and uncovered the involvement of a small RNA pair during C. jejuni pathogenesis. Overall, our results demonstrate the power of tissue-engineered models for studying host-pathogen interactions, and our model will also be helpful to investigate other gastrointestinal pathogens.