MRC DiMeN Doctoral Training Partnership: Assembly of a bacterial killing machine

Staphylococcus aureus is a major antibiotic-resistant bacterial pathogen of humans. Although frequently found as a human commensal, it can cause life-threatening diseases including pneumonia and necrotising fasciitis. To colonise the host, it must compete with the resident microbiota. The Palmer group have demonstrated that S. aureus uses a Type VII protein secretion system (T7SS) to secrete toxins that target other bacteria, important for establishing colonisation. The T7SS comprises six components that homo- and hetero-multimerise to form a large secretion machine which spans the cell wall to deliver proteins to the surface. This studentship will elucidate how the T7SS is functionally assembled in vivo.

Objectives
Assess the temporal order of assembly of S. aureus T7SS components.
Determine the subcellular location of the T7SS and how it traverses the cell wall.
Develop a labelling strategy for in vivo structural analysis of the T7SS.

Experimental Approach
Fluorescence microscopy will be used to assess the temporal order of assembly of the T7SS in vivo. Fluorescent fusions will be constructed to the chromosomally-encoded components (EsaA, EsxA, EssA, EssB, EsaB, EssC) and their localisation assessed in strains where other components are present, and where they are systematically deleted. Pairs of fusions with complementary fluorophores will be used to determine whether components interact in vivo, using FRET. Two hybrid and co-purification approaches will be used to confirm interactions.

The Foster group have recently elucidated the architecture of the S. aureus cell wall at unprecedented detail, with large surface pores that traverse almost the entire thickness of the wall. Using correlative AFM and super-resolution fluorescence approaches we will map T7SS components within the topographical context of the cell wall and plot their dynamics as a facet of the cell cycle.

EsaA has a large extracellular loop that has been proposed to reach the cell surface. To probe this, cysteines will be engineered into the loop and assessed for surface exposure using thiol-reactive labels. These cysteine-engineered strains will used as tools to visualise the T7SS in situ, through nanogold labelling.

Further information on the research groups can be found at:

https://www.ncl.ac.uk/medical-sciences/people/profile/tracypalmer.html
https://www.sheffield.ac.uk/biosciences/people/mbb-staff/academic/simon-foster

And catch us on twitter! @proftracypalmer and @foster_lab

Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.

Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards

Further information on the programme and how to apply can be found on our website:
https://bit.ly/3lQXR8A