Biophysical characterization of the association of intermediate filament proteins with translocon machineries

The intermediate filament protein vimentin is considered to be a cytoplasmic protein that forms 10nm filaments, and is a key component of the cytoskeleton. The surprise is that it has recently been found to be involved in host cell defenses, binding to several pattern recognition receptors of the innate immune system. Vimentin binds to cell membranes, just like its coassembly partners desmin and GFAP. GFAP stabilizes a multimeric translocation complex important in autophagy by binding to the membrane. Vimentin is involved in bacterial invasion, including pathogen binding on the cell surface as well as virulence through the activity of the translocon and escape of the phagosomal compartment after the initial infection. Cytosolic vimentin is therefore integral to intracellular human pathogenic bacteria and the resulting innate immune signaling. It appears that bacterial virulence factors target vimentin to subvert its function and seal host cell fate as the bacterial infection progresses. Therefore identifying those bacterial membrane proteins that bind vimentin or determining the binding regions on vimentin for bacterial virulence factors are important goals for human health. Type III secretion systems in Salmonella enterica, Shigella flexneri, Yersinia spp., Bordetella, Burkholderia pseudomallei, pathogenic Escherichia coli, Vibrio spp., Pseudomonas spp., Chlamydia spp. are attractive targets for the discovery or design of novel anti-infective agents and vaccine approaches for human pathogens. The E.coli virulence factor IbeA and M. Avium MBP-1 both target vimentin, but in both cases the precise domains/sequences involved in the interaction are unknown. Neither are the domains involved in targeting autophagosomes to vimentin.
We will use a combination of genetic tools to discover binding sequences. The targeted approach is to recombinantly express vimentin, IbeA and MBP-1 and determine the binding domains by a combination of biophysical techniques, including coassembly assays, SEC, ITC and SPR. The second approach is to fish for IbeA and MBP-1 binding sequences sequences. NGS has revitalised phage display approaches, allowing for the quantification of millions of sequences resulting from biopanning.
Aims:
• To identify the vimentin sequences that bind IbeA and MBP-1
• To identify peptides to block vimentin binding using a biopanning approach

Methodology: The student will use recombinant technologies to produce vimentin and selected virulence factors for biophysical studies to determine the vimetin binding sites. These studies will be complemented by the development of functional assays. The recombinant proteins will be characterized using the range of biophysical technologies available in Durham