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Dissecting host-pathogen interactions of Clostridium difficile

Principal Supervisor: Dr Meera Unnikrishnan - WMS

Co-supervisor: Dr Andrew Millard - WMS

PhD project title: Dissecting host-pathogen interactions of Clostridium difficile

University of Registration: University of Warwick

Project outline:

Clostridium difficile is a leading cause of hospital-acquired diarrhoea. C. difficile has been isolated from multiple animal hosts and is a leading cause of diarrhoea in piglets in several parts of the world. The strains that cause disease in pigs were found identical to human isolates, indicating an animal reservoir of C. difficile. Community-acquired CDI has been on the rise in recent years, and studies have suggested that livestock via the environment and/or food.

C. difficile colonisation of the gut by is an important part of CDI pathogenesis. Bacterial interactions with the gut mucosa are crucial for colonisation and establishment of infection, yet, key events including bacterial attachment and gut penetration, are poorly defined. The C. difficile virulence factors, toxin A and toxin B have been extensively studied and are known to induce cell death, disruption of the intestinal epithelial barrier and severe inflammatory responses. On the other hand, few C. difficile surface associated and extracellular or secreted proteins that affect host interactions have been examined. A better understanding of the host-bacterial interface will lead to new strategies against CDI.

Although murine and hamster models of CDI have contributed to the identification of bacterial determinants of colonisation there is a lack of information regarding precise host cell pathways triggered by specific bacterial factors. A major hurdle in investigating host-C. difficile interactions has been the strict requirement of an anaerobic environment for C. difficile growth. We have recently established innovative co-culture systems for Clostridium difficile using vertical diffusion chamber (VDC) systems for extended C. difficile-cell co-culture that allow simultaneous co-incubation of bacteria and cells under different gaseous environments. Further ongoing enhancements of this system include three-dimensional (3D) colon cultures. These novel systems provide an opportunity to perform in-depth molecular and cellular analyses on C. difficile interactions with gut epithelial cells.

The major goal of this project is to identify proteins and pathways that are crucial to C. difficile colonisation. In order to identify genes required at different stages on the colonisation process, we will employ a novel method which combines high-throughput sequencing technology and high-density transposon-mutant libraries-Transposon Directed Insertion Site Sequencing (TraDIS, also known as Tn-seq), where thousands of mutants were sequenced in parallel. TraDis allows for comprehensive identification of gene requirements under a selected condition. This method has been employed in the identification of genes essential bacterial growth in conditions relevant to infection, including biofilms and different infection models. Roles of individual components important for infection will be further investigated using bacterial mutants and knockout cell lines.

BBSRC Strategic Research Priority: Food Security

Techniques that will be undertaken during the project:

The student will be trained in a range of basic microbiology, molecular biology, cellular and bioinformatics techniques. Specific techniques include cDNA library preparation, genome sequencing, data analyses, bacterial cloning and immunofluorescent staining. The student will receive training in mammalian cell culture and advanced microscopy techniques.

Contact: Dr Meera Unnikrishnan, University of Warwick