About the Project
Applications are invited for a 4-year PhD studentship within a multidisciplinary and integrative research team.
Background:
Periodontitis is a ubiquitous chronic inflammatory disease which affects approximately 50% of the UK adult population [1] and causes considerable patient morbidity. Periodontitis is initiated and propagated by a pathogenic plaque biofilm which drives an aberrant destructive host response that causes the local tissue damage which is characteristic of this disease. Current treatments are labour intensive, lengthy and costly which is in part due to oral bacteria living in communities known as biofilms, which can be extremely resistant to standard antibacterial interventions and subsequently hard to eradicate. Bacteria use a plethora of means to coordinate their lifestyle, one being the use of di-nucleotide second messengers, such as c-di-AMP and c-di-GMP. Previous studies have identified the production of these second messengers by key periodontal pathogens P. gingivalis [2] and Treponema denticola [3] and shown their involvement in biofilm formation, adhesion and invasion. Despite these encouraging results, no further research on this topic has been reported.
Significant research has however been undertaken on other pathogens (for example Staphylococcus aureus [4] and Mycobacterium tuberculosis [5]) with data showing that these second messengers are not only important regulators of virulence, but also have a secondary role in activating the interferon regulatory factor (IRF) pathway [5, 6]. These studies demonstrated that cyclic-di-nucleotides (CDN) act as pathogen associated molecular patterns (PAMPs) that can modulate the IFN production during infection.
Fully understanding the bacterial host interaction offers the potential to develop new therapeutic targets to aid disease management. The human immune system has developed various ways to fight bacterial infections. Amongst these type I interferon (IFN) responses are pivotal for antimicrobial defences. Modulation of the level of bacterial second messengers can influence the interferon regulatory factor (IRF) pathway leading to altered levels of IFN-β. The activity of the immune system is controlled by these type-1 IFNs, and if deregulated, disease can be exacerbated.
Indeed an improved understanding of the role of CDNs could lead to a new mechanistic understanding of disease pathogenesis and associated development of novel immunotherapeutic approaches as is being undertaken in other infectious diseases.
Aims:
Understanding of the role of second messengers (ci-di-AMP and c-di-GMP) in the switch from healthy to disease-associated oral biofilms in the progression from health to gingivitis and periodontitis.
1. Assay the levels of second messengers in oral biofilms with a range of compositions (containing for example Streptococcus mitis, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in the disease-associated biofilm) to identify key individual bacterial producers (in vitro).
This part of the project will involve the establishment of a multispecies biofilm using the species mentioned above and initial characterization. [7] General microbiology techniques, anaerobic microbiology, cultivation of anaerobic biofilms on coverslips, DNA extraction, qPCR (to quantify the different species), data analysis, CDN extraction.
2. Assay the impact of exogenously added second messengers on biofilm formation, adhesion and invasion.
Measuring the impact of CDNs on biofilm formation (growth, biomass), adhesion to and invasion of host cells (using cell lines) using advanced microscopy (confocal) to visualize the biofilm, imaging analysis to quantify biofilms, crystal violet biomass quantification, adhesion assays [2] and an antibiotic protection assay to quantify invasion [2].
3. Compare the levels of second messengers present in plaque samples from periodontitis patients and healthy controls.
Plaque samples will be obtained from the biobank and/or patients and CDNs will be extracted and analysed as in part 1.
Funding Notes
We will consider applications from prospective students with:
- a good biomedical, microbiology, biology or similar degree (minimum of a 2:1)
- a source of funding to cover tuition fees and bench fees.
For more information regarding the project, please contact Dr S. Kuehne ([Email Address Removed]).
For more information about the eligibility, programme, or the application process please contact Viktorija Ziabliceva ([Email Address Removed]).
References
1. Adult Dental Health Survey 2009. The Health and Social Care Information Centre, UK. 2011
2. Identification of a Diguanylate Cyclase and Its Role in Porphyromonas gingivalis Virulence. Chaudhuri S, Pratap S, Paromov V, Li Z, Mantri CK, Xie H. Infect Immun. 2014 Jul;82(7):2728-35.
3. Inactivation of Cyclic Di-GMP Binding Protein TDE0214 Affects the Motility, Biofilm Formation, and Virulence of Treponema denticola. Bian J, Liu X, Cheng YQ, Li C. J Bacteriol. 2013 Sep;195(17):3897-905.
4. Cross-talk between Two Nucleotide-signaling Pathways in Staphylococcus aureus. Corrigan RM, Bowman L, Willis AR, Kaever V, Gründling A. J Biol Chem. 2015 Feb 27;290(9):5826-39.
5. A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis. Dey B, Dey RJ, Cheung LS, Pokkali S, Guo H, Lee JH, Bishai WR. Nat Med. 2015 Apr;21(4):401-6.
6. Group B Streptococcus Degrades Cyclic-di-AMP to Modulate STING-Dependent Type I Interferon Production. Andrade WA, Firon A, Schmidt T, Hornung V, Fitzgerald KA, Kurt-Jones EA, Trieu-Cuot P, Golenbock DT, Kaminski PA. Cell Host Microbe. 2016 Jul 13;20(1):49-59.
7. Development of an in vitro periodontal biofilm model for assessing antimicrobial and host modulatory effects of bioactive molecules. Millhouse E, Jose A, Sherry L, Lappin DF, Patel N, Middleton AM, Pratten J, Culshaw S, Ramage G. BMC Oral Health. 2014 Jun 28;14:80.
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