Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling

AbstractMany biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways

Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors

The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a β-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin

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

Exotoxin secretion: getting out to find the way in

During infection, most pathogenic bacteria deliver proteins to the host cell cytoplasm to manipulate host behavior. In this issue of Cell Host & Microbe, Spanò and colleagues describe a system where a bacterium produces an exotoxin while inside the host cell. Only after this exotoxin is transported to the mammalian cell surface and secreted into the extracellular milieu can it intoxicate the infected cell or noninfected distant cells

Pathogenic Pore-Forming Proteins: Function and Host Response

Organisms from all kingdoms produce pore-forming proteins, with the best-characterized being of bacterial origin. The last decade of research has revealed that the channels formed by these proteins can be very diverse, thus differentially affecting target cell-membrane permeability and consequent cellular outcome. The responses to these toxins are also extremely diverse due to multiple downstream effects of pore-induced changes in ion balance. Determining the secondary effects of pore-forming toxins is essential to understand their contribution to infection

Stabilizing patterning in the Drosophila segment polarity network by selecting models in silico

The segmentation of Drosophila is a prime model to study spatial patterning during embryogenesis. The spatial expression of segment polarity genes results from a complex network of interacting proteins whose expression products are maintained after successful segmentation. This prompted us to investigate the stability and robustness of this process using a dynamical model for the segmentation network based on Boolean states. The model consists of intra-cellular as well as inter-cellular interactions between adjacent cells in one spatial dimension. We quantify the robustness of the dynamical segmentation process by a systematic analysis of mutations. Our starting point consists in a previous Boolean model for Drosophila segmentation. We define mathematically the notion of dynamical robustness and show that the proposed model exhibits limited robustness in gene expression under perturbations. We applied in silico evolution (mutation and selection) and discover two classes of modified gene networks that have a more robust spatial expression pattern. We verified that the enhanced robustness of the two new models is maintained in differential equations models. By comparing the predicted model with experiments on mutated flies, we then discuss the two types of enhanced models. Drosophila patterning can be explained by modelling the underlying network of interacting genes. Here we demonstrate that simple dynamical considerations and in silico evolution can enhance the model to robustly express the expected pattern, helping to elucidate the role of further interactions

Structure and assembly of pore-forming proteins

Pore-forming proteins (PFPs), involved in host-pathogen interactions, are produced as soluble, generally monomeric, proteins. To convert from the soluble to the transmembrane form, PFPs assemble, in the vicinity of the target membrane, into ring-like structures, which expose sufficient hydrophobicity to drive spontaneous bilayer insertion. Recent findings have highlighted two interesting aspects: (1) that pores form via similar overall mechanisms even if originating from vastly different structures and (2) specific folds found in PFPs can be found in widely different organisms, as distant as prokaryotes and mammals, highlighting that pore formation is an ancient form of attack that has been remarkably conserved

Engaging the public or asking your friends? Analysing science-related crowdfunding using behavioural and survey data

Science-related crowdfunding enables public engagement with science. However, we know little about citizens engaging with science this way: Who are the people engaging with and donating to science through crowdfunding – and how do they decide how much to give? This study analyses behavioural and survey data from the Swiss crowdfunding platform wemakeit (N = 576). Results illustrate that a small, non-representative segment of the public engages with science through crowdfunding. Compared to the general public in Switzerland, these backers have an above-average education and income. Science-related crowdfunding mainly reaches citizens with an existing interest in science, personal ties to project initiators or the scientific community. The size of backers’ donations correlates with perceived personal appeals in campaigns or connections to initiators rather than projects’ scientific merit. While science-related crowdfunding thus opens up new avenues for public outreach by the scientific community, its potential for broader public engagement with science seems limited