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. Gut commensals provide protection from pathogen colonization, known as colonization resistance, and several studies have demonstrated the strong link between C. difficile infection and the disruption of healthy gut microbiota due to antibiotic therapy. Although the initial stage of colonization is critical to infection, we do not understand the molecular basis of pathogen interactions with commensal bacteria that line the gut mucosa. Studies based on sequencing data from human or murine fecal samples have indicated association of certain bacterial species with protection from CDI. However, specific interbacterial interactions occurring at a single species level between C. difficile and gut commensals remain poorly understood.
C. difficile forms complex communities or biofilms, which are known to play a key role in enabling pathogens to persist during infection. Several bacterial factors such as cell wall associated proteins, flagella, quorum sensing proteins and signaling molecules like c-di-GMP are known to mediate formation of biofilms (Frost et al, 2021). Recent studies have shown that C. difficile forms mixed communities with other commensal bacteria both in vitro and in vivo, in animal models (Slater et al 2019). A comprehensive identification of genes that are involved in C. difficile biofilm formation, and in modulating pathogen-commensal interactions within mixed biofilms would be valuable for developing novel therapeutics and effective prophylactic strategies.
We have established assays and tools in the lab to study C. difficile biofilms. Recently we constructed a high-density transposon library in C. difficile. The goal of this project is to use this transposon library to understand the genes required for survival in mixed bacterial biofilms with selected commensals or a mixed commensal community. This project will involve molecular microbiology and bioinformatics, involving techniques like mixed biofilm assays, confocal microscopy, cDNA library preparation, DNA sequencing, gene manipulation, DNA sequence analysis and pathway modelling.
References:
- Frost, L.R., Cheng, J.K.J. and Unnikrishnan, M. (2021) Clostridioides difficilebiofilms: A mechanism of persistence in the gut? PLoS Pathogens 17 (3), e1009348
- Slater, R.T., Frost, L.R., Jossi, S.E., Millard, A.D. and Unnikrishnan, M. (2019) Clostridioides difficile LuxS mediates inter-bacterial interactions within biofilms. Scientific reports 9 (1), 9903
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 (TraDis)
- Sequence analysis
- Pathway modelling
- Gene knockouts (molecular biology methods)
- Widefield and confocal microscopy
- Immunoblotting
- Mammalian tissue culture
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