Campylobacter pathogenesis: the Unfolded Protein Response (UPR), inflammation and human disease

Infectious diarrhoea is a global problem with Campylobacter being the most common bacterial cause. Despite its importance, the mechanisms by which Campylobacter infection promotes inflammation and disease in humans remain unclear. Campylobacter infection is the most common bacterial cause of human diarrhoeal disease. The species Campylobacter jejuni is responsible for over 80% of human cases with symptoms typically including bloody diarrhoea, fever and abdominal pains. A key precursor of diarrhoea is inflammation triggered when C. jejuni invades human intestinal epithelial cells (IECs), leading to tissue damage and disease.
The cell has developed a system for maintaining transcriptional fidelity of the ER - the unfolded protein response (UPR). Although it mainly acts upon imbalances in the homeostasis of unfolded proteins, several bacterial species like Campylobacter have been shown to activate this pathway to increase intracellular survival. Unpublished data from Gong et al. demonstrate campylobacter mediated UPR activation through the PERK and IRE1 arms. The paper however stopped short of identifying the molecular drivers that activate the different arms of the UPR.
Reactive oxygen species (ROS) are a group of oxygen-based chemical intermediaries with an uneven number of electrons. Normally, ROS production is mediated by the activation of the nitrous oxide (NOX) pathway and is counteracted by several defence mechanisms, such as antioxidant release and a balance, the 'Redox' state, is maintained. Interestingly, several manuscripts have established an antimicrobial role of ROS.
Previous work demonstrate that Campylobacter can modulate ROS production pathway components to aid intracellular survival and proliferation: C. jejuni differentially regulates intracellular and extracellular ROS production in human T84 and Caco-2 cells. C. jejuni downregulates the transcription and translation of nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX1), a key ROS-generating enzyme in IECs and antioxidant defence genes CAT and SOD1. Furthermore, inhibition of NOX1 by diphenylene iodonium (DPI) and siRNA reduced C. jejuni ability to interact, invade, and survive within T84 and Caco-2 cells.
The UPR and NOX pathways are activated to aid intracellular bacteria clearance and form part of the autonomous cell response but are susceptible to bacterial influence. The project will aim to investigate the interplay between the UPR and ROS in Campylobacter mediated infection - before, during and after activation.
Campylobacter mutants lacking certain virulence factors will be developed and their ability to invade host cells, as well as to activate the UPR and NOX pathways will be assessed. Transcriptional analysis of both bacterial and host cells during invasion assays will provide a better understanding of the genomic changes of both agents during the invasion cycle, and bacterial mutants will be used will be used to determine changes to the transcription of virulence factors, as well as host cell defence mechanisms. Particular attention will be given to changes in the transcriptome corresponding to increased inflammatory molecules such as interleukins. Different strains of C jejuni and various animal cell lines will be used to investigate the discrepancies in disease states observed in literature.
The biochemical basis of both signalling pathways will be investigated, and techniques such as Mass-spec, ELISA and pulldown assays will be used to identify individual components of the pathways of UPR and ROS pathway activation.
Once the core aspects of the project have been completed, the project become more expansive; It will combine 'Omic technologies to gain a better systemic understanding of the UPR and ROS systems when reacting to C jejuni infection and will relate the data to inflammatory processes. Other aims of the project include the development of a 3D cell culture model to better recapitulate in vivo environments.