Structural studies into biofilm formation by the Legionella pneumophila collagen-like protein

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Legionella pneumophila is a Gram-negative bacterium that causes Legionnaires'disease, an often-fatal pneumonia. It secretes a unique protein onto its extracellularsurface; the Legionella collagen-like (Lcl) protein. This functions in both recognisingsulphated glycosaminoglycans (GAGs) on the host cell surface which mediatesadhesion, but also promotes biofilm formation through binding environmental divalentcations. Lcl is formed of an N-terminal region (Lcl-NTR) responsible for anchoring it tothe bacterial surface after its secretion, an extensive collagen-like region whichextends it away from the surface, and a unique C-terminal domain (Lcl-CTD). We havedetermined the crystal structure of Lcl-CTD and have used NMR and MD simulations tounderstand how it binds GAGs (e.g. heparin, chondroitin). Using biochemical andbiophysical techniques we have shown how magnesium binds and causes changeswhich enables Lcl-CTD to recognise lipopolysaccharides (LPS). LPS is a majorcomponent of bacterial membranes, and this binding allows adjacent bacteria to thenaggregate. We will now use NMR and cryo-EM to study the structures of the Lcl-NTR+/- detergent and Lcl-CTD +/- LPS, respectively. We then wish to understand how theybehave within an artificial L. pneumophila membrane using MD simulations andvalidate these findings using targeted point mutations of Lcl in L. pneumophila usingbiofilm assays. The primary supervisor (Garnett) will be responsible for biochemistry,structural biology and bacterial work, the secondary supervisor (Lorenz) will lead theMD aspects of the project. The specific aims of this PhD project are to: Aim1: Use cryo-EM to determine the structure of Mg2+/Lcl-CTD with/without LPS bound Aim2:Elucidate the structure of Lcl-NTR with/without detergent by NMR Aim3: Monitor howthese complexes behave within an artificial membrane using MD Aim4: Test theoriesthat arise with targeted lcl mutations in L. pneumophila