Topological analysis of type 3 secretion translocons in native membranes

PFPs (Pore-forming proteins) perforate cellular membranes to create an aqueous pore and allow the passage of ions and polar molecules. The molecular mechanisms for many of these PFPs have been elucidated by combining high resolution structural information of these proteins with biochemical and biophysical approaches. However, some PFPs do not adopt stable conformations and are difficult to study in vitro. An example of these proteins are the bacterial Type 3 Secretion (T3S) translocators. The translocators are secreted by the bacterium and insert into the target cell membrane to form a translocon pore providing a portal for the passage of T3S toxins into eukaryotic cells. Given the important role that the T3S systems play in pathogenesis, methods to study these translocon pores in cellular membranes are needed. Using a combination of protein modifications and methods to selectively permeate and solubilized eukaryotic membranes, we have established an experimental procedure to analyze the topology of the Pseudomonas aeruginosa T3S translocon using P. aeruginosa strain variants and HeLa cell lines.