Primary Supervisor: Dr Meera Unnikrishnan, Warwick Medical School

Secondary supervisor: Professor Andrew McAinsh

PhD project title: Investigating C. difficile-host interactions in the gut

University of Registration: University of Warwick

Project outline:

Clostridium difficile, an anaerobic bacterial pathogen, causes C. difficile infection

(CDI), a major healthcare-associated challenge worldwide. It is a leading cause of hospital associated diarrhoea with high rates of infection in the elderly. C. difficile has also been isolated from multiple animal hosts and is a common 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.

Initial establishment of C. difficile in the gut is a key step in CDI pathogenesis. However, unlike other enteric pathogens, molecular interactions between C. difficile and the gut epithelium are poorly understood (Abt et al., 2016).The C. difficile toxins A and B are considered to be key virulence factors that break down the epithelial barrier. Surface-associated proteins including S-layer proteins, membrane and cell wall associated proteins (CWPs) have been associated with host cell adherence, extracellular matrix (ECM) degradation and biofilms. However, most research on C. difficile cell surface proteins has focused on their biochemical properties and roles in C. difficile physiology and underlying mechanisms during infection remain unclear.

From the host standpoint, few pathways activated during infection have been identified. Host cell receptors that the glycosylated toxins A and B bind to and inhibition of the Rho, Rac cellular pathways with the eventual disorganisation of actin filaments have been reported. Studies have begun to elucidate epithelial and innate responses to infection for e.g. IL-33 was recently shown to be a key driver of lymphoid cell-mediated responses to C. difficile. However, some aspects of infection, such as C. difficile interactions with the colonic mucus layer or the gut tissue ECM remain largely unexplored. On the whole, a better understanding of the gut-C. difficile interface is crucial for developing novel therapeutics, effective prophylactic strategies and improved diagnostic tools for CDI.

We have developed models in our laboratory to model the human gut bacterial interface (Anonye et al., 2019) and have recent performed transcriptomics (dual RNA-seq of the host and bacteria) during infection in this human gut model. Several interesting bacterial surface proteins and host pathways were identified using this approach. The goal of this study is to characterise C. difficile factors and host pathways activated by these during C. difficile infection.

This project will involve molecular and cellular microbiology, mammalian cell biology and advanced analytical approaches, involving techniques like mixed biofilm and infection assays, confocal microscopy, transcriptomics, mammalian gene knockout technologies (e.g. CRISPR/Cas) and in vitro human gut mimics. Elucidating specific pathways involved during initial infection will enhance our understanding of clostridial colonisation of the gut.

References:

BBSRC Strategic Research Priority: Understanding the Rules of Life: Microbiology

Techniques that will be undertaken during the project:

• Handling and culturing CL2 pathogens
• Anaerobic bacterial culture
• Biofilm assays
• Functional genomics (RNA-seq)
• Widefield and confocal microscopy
• Immunoblotting
• Mammalian tissue culture
• Mammalian gene knockout strategies (e.g. CRISPR/Cas, siRNA) in vitro infection/ coculture assays
• Human gut model studies

Contact: Dr Meera Unnikrishnan, University of Warwick