Tight junctions in chicken gut and Campylobacter infection

A high number of broiler chickens are colonized with Campylobacter and poultry products represent the main source of C. jejuni infections in humans. The number of human cases of Campylobacter is continuously rising throughout the world, with severe consequences on human health and the economy. In contrast to the general perception that C. jejuni is only a commensal in chickens, recent studies indicated that the colonization of the gastrointestinal tract with some Campylobacter isolates has a negative impact on the birds health and well- being. Although Campylobacter largely accumulate in the caeca, the infection also influences certain functions within the small intestine. We were able to demonstrate that an experimental Campylobacter infection could have many consequences on gut health. Of importance is the disruption of the intestinal barrier by C. jejuni, resulting in a higher intestinal permeability. As a consequence, translocation of bacteria to internal organs is promoted, an effect not solely restricted to Campylobacter. The studies need to be extended i) to resolve the functional mechanisms of a Campylobacter infection on the gut barrier and ii) to extend existing knowledge of Campylobacter strains with different infection profile in chickens. The main hypothesis of the project is that Campylobacter colonization has an important influence on the tight junction proteins in the chicken gut, whereby the bacterium translocates across the host intestinal epithelium and spreads to extra-intestinal tissues. Our work will be focused on tight junction proteins, as the tight junction is influenced by Campylobacter infection. In addition, the difference in effects on the intestinal barrier between disseminating and non-disseminating Campylobacter strains will be evaluated. In vivo studies will be combined with a range of laboratory technologies, e.g. immunohistochemistry, freeze- fracture, two-photon microscopy, western blot and mRNA expression, to determine the composition and structure of the paracellular barrier in the chicken intestine at different times post infection. Elucidating the mechanisms by which C. jejuni strains disseminate into extraintestinal organs is crucial to understand the infection biology of this microorganism. The project would serve as a model of how gram-negative pathogens induce alterations of the intestinal function and gut health. The results will clarify the mechanisms used by C. jejuni to persist in the chicken gut and to invade internal organs. The proposed project offers a novel approach to investigate the interactions between Campylobacter and its primary host.

So far, the TJ protein tricellulin (MD2) has not yet been recognized in chickens. However, studies in other species have shown that MD2 plays an important role in sealing the tricellular junctions of three neighbouring cells and is somehow crucial for the TJ barriers against macromolecular passage. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), as an accurate, rapid, sensitive and affordable method, remains one of the genomic methods of choice for the quantification of specific mRNA sequences. RT-qPCR can either be performed with a non-specific DNA binding dye such as SYBR-Green or by using a fluorescence labeled internal DNA probe. Internal DNA probes detect only a target specific DNA sequence, which increases the specificity in the presence of other unspecific PCR products or primer-dimers. In this project, the development of a probe based Rt-PCR methodology to characterize the chicken tight junction proteins (TJ) in context of a Campylobacter infection was established for the first time. Sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2) were designed and probe-based RT-qPCRs were developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RTqPCRs to minimize the number of separate reactions and enable robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Campylobacter is the leading cause of bacterial foodborne illness worldwide. Our results imply that C. jejuni has harmful consequences on chicken gut health and we demonstrated that C. jejuni infection in chickens is associated with significant changes in the intestinal barrier, leading to intestinal dysfunction. Furthermore, the results indicated that C. jejuni exacerbates the intestinal paracellular permeability with consequenceson the translocation of bacteria across the mucosa and towards internal organs. We also analysed the structure of tight junctions by freeze-fracture electron microscopy, immunohistochemically by confocal laser scanning microscopy and quantify in immunoblots. Freeze-fracture replica labeling technique was used to study the dimensional distribution of tight junction proteins between cells. Taken altogether, we have characterized the structure of tight junctions and the regulation of barrier formation under physiological and pathological conditions in chickens.