Biochemical structure and immune mechanisms of action of novel immune modulatory factor secreted by Staphylococcus aureus

We have recent data which shows that Staphylococcus aureus (SA) secretes immune modulatory factors (IMF) that actively drive epidermal damage and cell death. Since SA is positively associated with eczema and chronic clinical outcome, we propose that these factors are key to future therapies and intervention. We are interested in both the identification and function of SA immune modulatory candidates, therefore this project will combine biochemical, molecular and immunological approaches.

The aim of this project is to characterise the biochemical nature and mechanism of action of novel factors secreted by SA which induce skin inflammation and death.

The methods used in this project will include:
• Readout assays Human keratinocyte release of cytokines will be assayed by ELISA; quantification of necrosis and apoptosis by flow cytometry; bacterial culture of Staphylococcus aureus (SA) and other staphylcoccocal strains (available in house).
• Identification of the novel IMF from SA Protein fractionation by HPLC, ion exchange and affinity chromatography; mass spectrometry for analysis of bioactive proteins.
• Confirmation of the IMF activity / Genetic manipulation to confirm IMF candidate Commercially available SA transposon mutant libraries will be screened for IMF activity. IMF candidates will be investigated by creation of deletion mutants of SA which can then be characterised using our keratinocyte cell model. Functional candidates will also be targeted using a CRISPR/Cas9 approach, taking advice from the Transgenic unit (TgU) within FBMH. The same candidate will then be knocked into S. epidermidis to show that bioactivity can be transferred into a normally bio-inactive staphylococcal strain.

The medium outcome is to identify a factor that drives the immune response in eczema, which will lead to much needed therapeutic agents. In the long term, understanding the pathogenesis of SA infection will allow for the development of inhibitors and possibly vaccines to prevent infection with this potentially devastating microbe.

The successful candidate will enter the PhD program within the School of Biological Sciences at the University of Manchester which provides high quality research skills and training through the Doctoral Academy. The supervisory team will provide the student with training in a number of laboratory techniques including: bacterial and mammalian cell culture, ELISA and flow cytometry, proteomics (protein fractionation and identification) and bacterial genetics (transposon mutagenesis, gene deletion and complementation, mutational analysis) as detailed in the project description above.

The student will be part of a team consisting of other PhD students, as well as a postdoc also supervised by Drs Arkwright, Pennock and Dr Xia. Additionally Dr Adamson of the core Trangenic Unit is an expert in CRISPR/Cas9 genome editing and will advise and train in CRISPR-based strategies and cutting edge genome editing technologies.

Candidates are expected to hold (or be about to obtain) a minimum upper second class honours degree (or equivalent) in the general area of microbiology or immunology. Candidates with experience or an interest in bacterial and cell culture, molecular biology and gene editing are encouraged to apply.