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

Legionella pneumophila is a Gram-negative bacterium that causes Legionnaires'
disease, an often-fatal pneumonia. It secretes a unique protein onto its extracellular
surface; the Legionella collagen-like (Lcl) protein. This functions in both recognising
sulphated glycosaminoglycans (GAGs) on the host cell surface which mediates
adhesion, but also promotes biofilm formation through binding environmental divalent
cations. Lcl is formed of an N-terminal region (Lcl-NTR) responsible for anchoring it to
the bacterial surface after its secretion, an extensive collagen-like region which
extends it away from the surface, and a unique C-terminal domain (Lcl-CTD). We have
determined the crystal structure of Lcl-CTD and have used NMR and MD simulations to
understand how it binds GAGs (e.g. heparin, chondroitin). Using biochemical and
biophysical techniques we have shown how magnesium binds and causes changes
which enables Lcl-CTD to recognise lipopolysaccharides (LPS). LPS is a major
component of bacterial membranes, and this binding allows adjacent bacteria to then
aggregate. 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 they
behave within an artificial L. pneumophila membrane using MD simulations and
validate these findings using targeted point mutations of Lcl in L. pneumophila using
biofilm assays. The primary supervisor (Garnett) will be responsible for biochemistry,
structural biology and bacterial work, the secondary supervisor (Lorenz) will lead the
MD 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 how
these complexes behave within an artificial membrane using MD Aim4: Test theories
that arise with targeted lcl mutations in L. pneumophila