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 transmembrane β-barrels even though the number of strands in the barrel varies by more than 1 order of magnitude. On the contrary, ClyA is a PFT produced by both E. coli and Salmonella enterica strains and forms rings of 12-13 protomers with the membrane spanning domain composed of a-helices. Hence, the pores formed by these three PFTs vary greatly from one another and are representative for the diversity of PFTs. We measured cell permeabilisation at the single cell level using two independent live-cell imaging methods, in erythrocytes and in nucleated cells. Both assays revealed that the assembly mechanism of PFTs is stochastic in its nature and that lowering the initial monomer concentration increases the variability and average time to successfully build first functional pores. The measured stochasticity reflects the chemical kinetics behind the assembly reaction and therefore carries important information about the underlying mechanism. In fact, analysis of the stochasticity of the processes led to the robust determination for each toxin of the minimal number of independent rate-limiting steps required for pore formation. Furthermore, scaling relationships in our data upon changes in initial toxin concentration constrain the possible underlying mechanisms for assembly and reveal that the steps leading to the aerolysin heptamer are composed of at least seven independent reactions whose rates scale with toxin concentration. Taken together, our findings suggest that that what is limiting for aerolysin is the conversion for each monomer to an assembly competent state, while for PFO a single limiting step is observed, likely corresponding to membrane insertion. A second aim of this thesis was to clarify the cellular consequences that occur upon pore formation by different PFTs and to find out how PFT-affected cells sense this event. This was done in collaboration with other members of the lab. Our results suggest that the cells monitor the breaches in the plasma membrane by assessing the correct homeostasis of the potassium ion inside the cell through a yet unknown mechanism. Interestingly, the cells seem to "monitor" the integrity of the membrane through the most upstream effect possible after pore formation, namely the flux of ions. Incidentally, the assays we developed in the first part of this thesis to "monitor" initial pore formation events are based on the same principle, namely the immediate loss of gradients that occur upon pore formation.

The role of the inflammasome in cellular responses to toxins and bacterial effectors

Françoise Gisou van der Goot Grunberg

Invading pathogens are recognized by mammalian cells through dedicated receptors found either at the cell surface or in the cytoplasm. These receptors, like the trans-membrane Toll-like Receptors (TLR) or the cytosolic Nod-like Receptors (NLR), initiate innate immunity after recognition of molecular patterns found in bacteria or viruses, such as LPS, flagellin, or double-stranded RNA. Recognition of molecules produced only by a specific pathogen, such as a viral envelop protein or a bacterial adhesin does not appear to occur. Bacterial protein toxins, however, might compose an intermediate class. Considering the diversity of toxins in terms of structure, it is unlikely that cells respond to them via specific molecular recognition. It rather appears that different classes of toxins trigger cellular changes that are sensed by the cells as danger signals, such as changes in cellular ion composition after membrane perforation by pore-forming toxins or type III secretion systems. The signaling pathways triggered through toxin-induced cell alterations will likely play a role in modulating host responses to virulent bacteria. We will here describe the few studied cases in which detection of the toxin by the host cell was addressed. The review will include not only toxins but also bacteria effectors secreted by the bacterium in to the host cell cytoplasm.

Springer-Verlag2007

Folding and Structure of the Pore Forming Toxin Aerolysin

Mircea Ioan Iacovache

The first obstacle encountered by a bacterial pathogen once inside the host is the plasma membrane surrounding the target cells. Throughout evolution bacteria has acquired and maintained genes that upon stimulation express proteins capable of damaging the membrane of other cells. Among these proteins pore forming toxins (PFTs) are a major class of bacterial effectors that are upregulated and secreted during bacterial infections. As their name suggests, pore forming toxins are proteins capable of inserting transmembrane pores in the membranes of the target cells which in turn leads to the lysis of the cell and release of nutrients. The mechanism by which the PFTs function during a bacterial attack has been the subject of extensive research over the years. In most cases PFTs are produced by the bacteria as soluble proteins that require the help of specialized secretion mechanisms to arrive as functional proteins in the external milieu. Once secreted by the producing bacteria these proteins diffuse towards the target cell and bind to the target membrane. Once bound to the plasma membrane of target cells they are capable of initiating a series of structural changes that will eventually lead to the conversion of the water-soluble PFT to a membrane inserted channel. The series of events and the characterization of the different structural changes required for a PFT to convert from a water-soluble protein to a membrane inserted channel is the subject of this thesis. Aerolysin, a PFT produced by Aeromonas hydrophilla, is one of the best candidates for a research into the details of the mode of action of bacterial PFTs. This particular PFT is produced by the bacterium as a soluble periplasmic protein and then secreted outside of the bacterium as a fully folded protein with the help of a type II secretion system. Binding to the target cell is achieved through two high affinity binding sites that recognize sugar modifications which are absent in A. hydrophila, a mechanism that insures that the producing cell is not damaged by its own PFT. Once bound to the target cell aerolysin requires proteolytic activation, a step which cleaves a C-terminal peptide (CTP). Activation is achieved using proteases present on the target cell and the removal of the CTP is thought to initiate the sequence of events leading to pore formation. Following activation aerolysin is able to oligomerize forming heptameric ring-like structures which spontaneously rearrange forming a transmembrane beta-barrel through the membrane. My thesis project, focused on the structural changes required in the mode of action of aerolysin, set off trying to identify the aminoacid sequence involved in the formation of the transmembrane beta-barrel. It was long thought that aerolysin would cross the membrane in a porin like fashin, forming a beta-barrel through the plasma membrane, primarily due to the lack of a hydrophobic patch of aminoacids in its sequence. An initial model proposed in the early '90s postulated that the only region that could form the transmembrane beta-barrel was the Domain 4 of the protein. In this model the removal of the CTP in the activation process would unravel the hydrophobic residues required for the beta-barrel formation and insertion. We and others were able to show however that the fourth domain of the protein is not involved directly in the formation of the pore and we identified a conserved loop in the third domain of the protein which is responsible for the formation of the beta-barrel. This loop presents an alternating pattern of hydrophobic and hydrophilic residues, a requirement for the formation of a transmembrane pore with a hydrophobic exterior and a hydrophilic cavity. Our research led us to propose a sequence of events upon insertion of the aerolysin pore in which a rearrangement of the DIII-loops of the seven monomers in the oligomer forms the initial beta-barrel and generates a hydrophobic tip which drives insertion of the structure through the membrane. Once the bilayer has been crossed the hydrophobic tips folds back on the membrane in a rivet like fashion, anchoring the pore. Following the identification of the DIII-loop as the region that forms the transmembrane pore my researched focused on the structural changes leading to the conversion of a water-soluble protein to a membrane inserted oligomer. While removal of the CTP is the key requirement for this conversion, the role of the CTP in aerolysin mode of action and the sequence of events triggered by its removal is not fully understood. Using a combination of in vivo, in vitro and in silico approaches we were able to show that the CTP plays a wider role in the aerolysin mode of action than previously thought. Indeed our research shows that the CTP is initially required for the correct folding of the soluble protein inside the bacterium, acting as a intramolecular chaperone during the folding of aerolysin. Following folding the CTP binds tightly to a hydrophobic pocket in the fourth domain of the protein locking the PFT in its soluble conformation, a role resembling C-terminal intramolecular chaperones previously described for tail spikes of bacteriophages or fiber forming collagen. This research will be continued with a study on the structural changes triggered by the removal of the CTP and their role in oligomerization and pore formation. The main focus of my thesis project has been however the determination of the structure of the oligomeric form of aerolysin. This part of the project is still ongoing and will be discussed in the final chapter of my thesis. Using 2D and 3D crystallography, AFM and modeling we hope to be able to improve our current understanding of the aerolysin heptameric form and the structural changes required in its formation.

EPFL2011

Folding and Structure of the Pore Forming Toxin Aerolysin

Mircea Ioan Iacovache

EPFL2011