IMIB - Institute for Molecular Infection Biology

Exploring virulence factors and regulators of the food-borne pathogen Campylobacter jejuni in a human 3D tissue infection model

Campylobacter jejuni is a highly prevalent food-borne pathogen of the human gastrointestinal tract and currently the leading bacterial cause of gastroenteritis worldwide. It has also been linked to inflammatory bowel disease and secondary neuropathies like the Guillain-Barré syndrome. So far it is still unclear which factors are important for its pathogenesis and what triggers the disease development in humans compared to the commensal lifestyle in chicken. C. jejuni lacks many classical virulence factors such as a type III or type VI secretion system known from other enteric pathogens. Therefore, it has been suggested that mainly the metabolic capabilities and motility are required for virulence. Current infection models for this pathogen are often limited in their ability to reflect the in-vivo situation in the human host. Thus, in cooperation with the Chair of Tissue Engineering and Regenerative Medicine in Würzburg (Prof. Heike Walles), we have been employing tissue engineering techniques to establish novel intestinal 3D tissue infection models that more closely recapitulate the micro-environment of the human intestine. These tissue models are based on acellularized extracellular matrix scaffolds, which are reseeded with human gastrointestinal cell lines [1, 2]. Dynamic cultivation conditions promote the development of a tight epithelial barrier. Infection of the 3D tissue model with diverse C. jejuni mutant strains showed adherence and internalization phenotypes that are not apparent in conventional in-vitro 2D cell culture systems. Based on dual RNA-seq analyses of host and pathogen transcriptomes in these infection models I aim at uncovering virulence and regulatory genes necessary for C. jejuni pathogenesis. Initial focus will be laid at bacterial sRNAs which might impact virulence. Upon construction of deletion mutants of selected, identified virulence regulator candidates, I aim at further studying their impact on infections and at identifying their cellular targets and the underlying molecular mechanisms. Furthermore, I will examine localization of novel virulence factors using super-resolution microscopy and perform 3D live-imaging of the infected tissue models using multi-photon laser-scanning and multiparameter light-sheet fluorescence microscopy. Moreover, I will employ mucus-producing cell lines and even human primary intestinal cells in the 3D tissue model and employ deep-sequencing techniques to study host-pathogen interactions in an environment more reminiscent of native host tissue.

References:

[1] Pusch J, Votteler M, Goehler S, Engel J, Hampel M, Walles H, Schenke-Layland K (2011). The physiological performance of a three-dimensional model that mimics the microenvironment of the small intestine. Biomaterials 2011; 32:7469-7478.

[2] Schweinlin M, Wilhelm S, Schwedhelm I, Hansmann J, Rietscher R, Jurowich C, Walles H, Metzger M (2016). Development of an advanced primary human in vitro model of the small intestine. Tissue Eng Part C Methods 2016; 22(9):873-83.