Assembly Mechanisms and Cellular Effects of Bacterial Pore-Forming Toxins

The correct assembly of macromolecular protein complexes such as ribosomes or multisubunit membrane channels is essential for their function. Alterations in the process can lead to disease either by loss of function or by acquisition of toxic function. However, the precise mechanisms by which multimeric protein complexes assemble are generally poorly understood owing to the difficulty in reconstituting the process in-vitro. Thus, systems that undergo dynamic assembly and disassembly in solution, such as cytoskeletal proteins, have been particularly attractive. By contrast, the assembly of multi-subunit membrane protein complexes has been notably difficult to study. An interesting, somewhat in between, situation is provided by pore-forming proteins, the best-characterized subclass of which are bacterial pore-forming toxins (PFTs). These proteins can generally be produced recombinantly and in a highly stable soluble monomeric form, but assemble into homo-oligomeric ring-like structures upon addition to cells expressing the appropriate cell surface receptor. These rings concomitantly, or subsequently depending of on the PFT, undergo a conformational change that leads to exposure of hydrophobic surfaces and spontaneous membrane insertion. The built pores then perforate the membrane of the host cell. PFTs do not only occur in bacteria and have a very broad taxonomic distribution, occurring in organisms such as parasites, plants and mammals. However, for pathogenic organisms the PFTs are major virulence factors contributing to the disease caused by the organisms producing them. For this reason bacterial PFTs have been studied extensively during the last century and a lot of insight into their mode of action has been gained. However, despite the increasing knowledge on PFTs, the mechanisms and the kinetics of self-assembly of these complexes remain largely enigmatic. In particular it is unknown whether oligomerization occurs through the sequential addition of monomers or through interaction of multimeric intermediates, whether oligomerization is the rate-limiting step during the pore-formation process and whether the same rules apply to all toxins. This is mostly due to the fact that intermediates have not been visualized by either biochemical or structural methods. Also, with few exceptions, functional assays on the activity of PFTs have always been performed at the level of a population of cells. The aim of this thesis was to measure pore-formation at the single cell level to extract mechanistic information from the analysis of the stochasticity of the pore-formation process. We chose to study three PFTs produced by human pathogens: aerolysin, PFO and Cytolysin A (ClyA). Aerolysin is produced by Aeromonas hydrophila and forms pores of 1-2 nm in diameter, perfringolysin O (PFO) occurs in Clostridium perfringensis and is a cholesterol-dependent cytolysin (CDC) that builds channels of 25-30 nm. Both of these PFTs build structurally similar pores made of