My project

Novelty and timeliness

Many proteins resist or respond to mechanical deformation as part of their function. The importance of

understanding how mechanical force affects both the structure and the binding of proteins to each other and ligands / surfaces is exemplified by biofilm formation, which often occurs under hydrodynamic stress (i.e. in the vasculature). We have shown previously that a repetitive surface protein involved in host adhesion and bacterial-bacterial interactions (SasG) essential for biofilm formation displays extreme mechanical stability (Gruszka et al. Nature Commun (2015)). However, many questions remain unanswered that, if addressed, may lead to novel methods of preventing or removing biofilms. These include: why are these proteins so strong, how do they dimerise to form bacterial interactions, what happens if the strength of this interaction under force is weakened, what is the effect of destabilising these proteins (or their dimerization) on biofilm formation ‘in vivo’?

 Objectives

1. The effect on the mechanical strength of SasG of introducing β-strand-, hydrophobic- or charge-disrupting mutations will be assessed using force spectroscopy.
2. Variants with interesting force characteristics will be introduced into S. aureus to reveal their effect on biofilm formation. Custom microfluidic devices and three-dimensional confocal imaging will be used to quantify surface attached biomass and biofilm morphology.
3. Dimerization of SasG is necessary for biofilm formation but the mechanism, the role of metals and the effect of force are still unclear. We will perform ‘fly-fishing’ experiments to measure the formation and rupture force of the complex and the effects of metal ions and / or specific amino-acid substitutions.
4. We will select conditions / variants with modulated dimer properties and assess their effects in vivo. We will leverage microfluidic systems and automated image analyses, to understand how dimer destabilization affects the initialization of biofilms under physiologically relevant hydrodynamic conditions.

Resource and facilities available

The student will be in well-funded laboratories with all equipment needed. The School of Molecular and Cellular Biology / Faculty of Biological Science at Leeds has world-class facilities that include CD and fluorescence spectroscopy, state-of-the art mass spectrometry, NMR and electron microscopy. The project will make extensive use of two Atomic Force Microscopes that are optimised for force spectroscopy experiments (Asylum Research, MFP-3D).

Microfluidic experiments will be conducted at the University of Sheffield, where the necessary cell culturing, microfluidic device fabrication, and automated time lapse imaging facilities are available. This project will use image analysis algorithms capable of quantifying the behaviour of thousands of single cells simultaneously and as well as characterize the morphology of mature biofilms using 3D reconstructions.

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