The adhesion protein Intimin is a central virulence factor of so-called attaching and effacing pathogens, which includes organisms such as enteropathogenic and enterohaemorrhagic Escherichia coli. Both are major causes of diarrhoea. Intimin mediates tight binding to the epithelial cells of the intestine, which leads to changes in the host cell cytoskeleton and formation of an actin pedestal around the bacteria.
Intimin consists of three functional regions (panel A in figure): a short N-terminal peptide located in the periplasm, a transmembrane β-barrel domain that anchors Intimin in the outer membrane and an extended, multidomain extracellular region at the C-terminus. This extracellular structure, called the passenger, contains the adhesive domain at the distal end. The rest of the passenger is composed of four immunoglobulin (Ig)-like domains. The Intimin passenger is exported to the cell surface through the pore of the β-barrel domain in a process termed inverse autotransport. The current model for inverse autotransport posits the formation of a hairpin structure within the pore of the β-barrel domain that initiates secretion of the passenger (panel B in figure). The free energy for secretion is provided by sequential folding of the Ig-like domains at the cell surface, proceeding until the entire passenger is located outside the bacterial cell.
In this project, the Masters student will investigate factors that influence the efficient secretion of the Intimin passenger. The project consists of three sub-projects:
- Investigating the (re)folding kinetics of the proximal Ig-like domains
- The role of secondary structure elements in the loops of the β-barrel domain on passenger secretion
- The role of specific amino acid residues in the linker, the structure connecting the β-barrel to the passenger that resides inside the pore of the β-barrel
The aim of this project is to gain detailed insights into the inverse autotransport process, with the long-term goal of being able to rationally inhibit passenger secretion and thus provide novel antimicrobial compounds to combat infections.
The project will be jointly supervised by Jack C. Leo (at IBV) and J. Preben Morth (at NCMM). The project is suited to Masters students in Biochemistry or Molecular Biology with an interest in protein structure and folding, structure-function relationships, and the molecular basis of bacterial pathogenesis. The student is expected to have a strong theoretical background in biochemistry, ideally also in molecular biology and microbiology.
The project offers the opportunity to work in two well-equipped laboratories and gain experience in a variety of methods, including
- molecular cloning and site-directed mutagenesis
- recombinant protein production and purification
- detection methods (Western blot, fluorescent labels and fluorescence microscopy)
- biophysical techniques (fluorometry, stopped-flow measurements)
- basic structural biology
For more information, contact Jack C. Leo (j.c.leo@ibv.uio.no; tel. 228 59027)
Relevant literature:
Leo JC, Oberhettinger P, Yoshimoto S, Udatha DBRKG, Morth JP, Schütz M, Hori K, Linke D (2016). Secretion of the Intimin passenger domain is driven by protein folding. J Biol Chem., 291(38): 20096-112.
Leo JC, Oberhettinger P, Schütz M, Linke D (2015): The inverse autotransporter family: Intimin, Invasin and related proteins. Int J Med Microbiol., 305(2): 276-82.
Oberhettinger P, Leo JC, Linke D, Autenrieth IB, Schütz M (2015): The inverse autotransporter Intimin exports its passenger domain via a hairpin intermediate. J Biol Chem., 290(3): 1837-49.
Oberhettinger P, Schütz M, Leo JC, Heinz N, Berger J, Autenrieth IB, Linke D (2012): Intimin and Invasin are members of a family of autotransporters that export their C-terminus to the bacterial cell surface. PLoS ONE, 7(10): e47069.