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Elucidating the molecular targets of bacterial nano-syringes

Primary Supervisor: Dr Alice Rothnie, Life & Health Sciences

Secondary supervisor: Dr Nick Waterfield (Warwick) & Dr Alan Goddard (Aston)

PhD project title: Elucidating the molecular targets of bacterial nano-syringes.

University of Registration: Aston University

Project outline:

The evolutionary ‘arms race’ between bacteria and their hosts has led to a wide range of sophisticated strategies and mechanisms which enable manipulation of host cells, often resulting in lethality. In recent years, particularly since the “Resolution Revolution” in the field of (cryo) Electron Microscopy, it has become apparent that these mechanisms can be extremely elaborate in the form of large macromolecule complexes, as well as more ‘conventional’ exotoxins etc.

Among the most elegant and well known of these bacterially-derived macromolecular complexes are the various “Secretion Systems”, particularly the Type 3 and Type 6, which effectively function as ‘harpoons’ embedded in the cell wall of the bacterium, which mediate cell-to-cell transfer of biologic material. More recently, structures which function as nano-scale ‘torpedos’ or syringes have been discovered, which are released by the bacteria into the extracellular milieu and are capable of acting ‘at a distance’. First discovered in Serratia and Photorhabdus species of bacteria, these so-called “Virulence Cassettes” are short gene clusters of approximately 16 genes, which contain everything necessary to produce, spontaneously self-assemble, and load with (typically toxic) cargoes, these remarkable entities. These ‘nano-syringes’ are evolutionarily related to bacteriophages, and function in a very similar way: Once free in the extracellular environment, they will diffuse and ‘seek out’ a target cell. One of the genes within the cassette gives rise to a so-called ‘tail fibre’ protein which, similar to bacteriophage, is responsible for binding to the relevant cell surface marker/receptor. Once bound, the nanosyringe vehicle contracts and an inner sheath of protein is propelled out to puncture the cell barrier. With the cell barrier pierced, the payloads contained within the nanosyringe vehicle are released into the cell cytosol.

One of the key features remaining to be elucidated is how the ‘nano-syringes’ interact with the target cell – what in the target membrane do their tail fibres bind to precisely? Work to date suggests possible candidates include cell surface (glyco)proteins, specific glycolipid components, or potentially some combination of the two. This collaborative project will combine expertise from the University of Warwick in producing and assaying the ‘nano-syringe’ tail proteins, with membrane expertise at Aston University. In particular polymer lipid particle approaches such as SMALPs (styrene maleic acid lipid particles) will be utilised in order to identify membrane components that bind to the purified tail proteins. These lipid particles are important as they will present membrane proteins within a native lipid bilayer environment, representing all possible molecular targets of the nano-syringes. Components that bind to the tail proteins will then be identified and investigated in more detail to study the affinity and specificity of the interaction.

These PVCs are being exploited by the new spinout company Nanosyrinx as potential delivery vehicles for biotechnological and healthcare applications. Advances made in understanding the targeting, and capacity to engineer/retarget this system will have immediate translational impact.

References:

  1. Vlisidou, Isabella, et al. "The Photorhabdus asymbiotica virulence cassettes deliver protein effectors directly into target eukaryotic cells." Elife 8 (2019): e46259.
  2. Jiang, Feng, et al. "Cryo-EM structure and assembly of an extracellular contractile injection system." Cell 177.2 (2019): 370-383.
  3. Kube, Sebastian, and Petra Wendler. "Structural comparison of contractile nanomachines." AIMS Biophysics 2.2 (2015): 88.
  4. Yang, G., et al. "Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth." Journal of bacteriology 188.6 (2006): 2254-2261.
  5. Pollock NL, Lee SC, Patel JH, Gulamhussein AA, Rothnie AJ* (2018) “Structure and function of membrane proteins encapsulated in a polymer-bound lipid bilayer.” BBA Biomembranes 1860(4); 809-817.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Microbiology & Structural Biology

Techniques that will be undertaken during the project:

  • Protein expression, solubilisation and purification
  • Molecular biology/cloning
  • Binding assays
  • Chromatography
  • Mass spectrometry
  • Lipid analysis
  • Protein biochemistry
  • Fluorescence spectroscopy
  • Tissue culture

Contact: Dr Alice Rothnie, Aston University