Economou lab - Type III protein secretion


Type III protein translocase (T3S)
Several pathogenic bacteria have developed a fascinating tool to help them invade their animal or plant hosts. The Type III secretion (T3S) apparatus is a complex transmembrane machine with an elongated extracellular portion and comprises 17-25 subunits. Many T3S components are related to flagellar proteins. The T3S apparatus is thought to pierce the plasma membrane of the host cell and to inject toxins directly in the host cytoplasm! We are interested in the molecular structure, mechanism and nanobiotechnology potential of the T3S nanomachine. As model systems we focus on the T3S of the human enteropathogenic E.coli (EPEC).
Our T3S work has three foci:
1. T3S ATPase structure/assembly
The T3S ATPase was characterized from pathogenic Pseudomonas and EPEC. The T3S ATPase was purified to homogeneity with a decahistidine tag. The catalytically active form of the enzyme is a dodecameric particle of 12nm diameter as determined by biochemical analysis (cross-linking, gel filtration and blue native-PAGE), laser-light scattering, analytical ultracentrifugation (A.Lustig, Basel) and electron microscopy (collaboration with S.Mueller, Engel Lab, Basel and H.Stahlberg, UC Davis). The dodecameric form of HrcN is the prominent form at the membrane and is formed of teo hexameric rings (Mueller et al., 2006). Higher resolution structural analysis will involve electron microscopy studies of the dodecamer (collaboration with the Engel Lab, Basel) and X-ray crystallography(collaboration with W.Welte, Konstanz).
2. T3S ATPase catalysis
To understand how the T3S ATPase converts energy into mechanical work, we studied the ATPase activity of HrcN from Pseudomonas and more recently on EscN from EPEC. HrcN has a high level ATPase in vitro that is cooperatively stimulated upon dodecamerization (Pozidis et al., 2003). We anticipate that additional factors in the cell regulate this ATPase and we are trying to identify them. In addition, secretory toxins are expected to regulate this ATPase activity. We are currently studying these interactions using purified T3S substrate toxins and chaperones using biochemical and biophysical assays and are in the process of determining their structures using NMR (collaboration with B.Kalodimos, Rutgers U.).

3. T3S translocase assembly
Biochemical analyses and immunogold localization (collaboration with Ian Brown, Mansfield group, Wye College) showed that HrcN is a peripheral membrane protein. We wish to determine how HrcN binds to the membrane and in particular to determine whether contacts with the other T3S subunits are important. To this end we have purified 8 of the membrane subunits of the T3S machine and have prepared antibodies against them. Immunofluorescence, immunogold localization and biochemical analyses on isolated inner membrane vesicles are used to determine the intramolecular interactions between the T3S components.