Primary Supervisor: Dr Sarah Kuehne, School of Dentistry

Secondary supervisor: Professor Mike Millward (UoB)

PhD project title: The role of cyclic di-nucleotides in the pathogenesis of Fusobacterium nucleatum

University of Registration: University of Birmingham

Project outline:

Background: Fusobacterium nucleatum is an anaerobic bacterium primarily found in multi-species dental plaque biofilm, where it is seen as a key player in the emergence of a dysbiotic microflora in periodontitis (gum disease). However, F. nucleatum has also been associated with non-oral diseases such as atherogenic cardiovascular disease, rheumatoid arthritis, inflammatory bowel disease and in particular colorectal cancer. [1, 2]

Bacteria use a plethora of means to coordinate their lifestyle and virulence, one being the use of di-nucleotide second messengers, such as c-di-AMP and c-di-GMP. These molecules have been studied extensively in human pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa and Mycobacterium tuberculosis, but are still under-researched in dental pathogens. [3] Only recently, we have shown the presents of c-di-AMP in cultures of F. nucleatum and we have been able to identify genes encoding a putative cyclase (producing c-di-AMP) and a putative phospho-di-esterase (degrading c-di-AMP). [unpublished data]

Aim: This study aims to understand the role of ci-di-AMP in the pathogenesis of F. nucleatum.

Methodology: Levels of c-di-AMP will be measured under different physiological conditions, in co-aggregation experiments and multispecies biofilms. Mutant strains lacking the putative enzymes detailed above, will be generated and used alongside the parental (wildtype) strain to study the role of c-di-AMP. In addition to the growth experiments this will also include cell invasion assays using oral and colon epithelial cells as well as 3-D organotypic cell-cultures to investigate interactions with the immune system. [4, 5]

Scientific outcomes: It is expected that this project will contribute significantly to a greater understanding of the impact and the role(s) of c-di-nucleotides in the lifestyle and pathogenesis of Fusobacterium nucleatum.

References:

1. Han YW et al; Current Opinion in Microbiology. 2015; p.141-147.
2. Edwards AM et al; Infection and Immunity. 2006; 74(1).
3. Andrade WA et al; Cell Host Microbe. 2016; Jul 13;20(1):49-59.
4. PARK J et al; International Journal of Oral Science. 2016; 8(3). p.138-144.
5. Millhouse E et al; BMC Oral Health. 2014; Jun 28;14:80.

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

Techniques that will be undertaken during the project:

• Microbiology:
• Anaerobic bacterial culture, generation of gene knock outs, biofilm and planktonic growth, imaging (confocal, electron microscopy), nucleotide extraction
• Cell biology:
• Culture of mammalian cells (oral and colon) and 3D-cultures,
• Biological assays to assess cell growth, proliferation, adhesion, bacterial-cell interactions.
• qPCR, Cytokine assays to study inflammatory response

In addition, to the wet-lab skills the student will be trained in necessary in silico skills, including basic bioinformatics for sequence analysis.

Contact: Dr Sarah Kuehne, University of Birmingham