Folding and Structure of the Pore Forming Toxin Aerolysin

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

A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool

Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins.

Cells of the vascular system release spherical vesicles, called microparticles, in the size range of 0.1-1 μm induced by a variety of stress factors resulting in variable concentrations between health and disease. Furthermore, microparticles have intercellular communication/signaling properties and interfere with inflammation and coagulation pathways. Today's most used analytical technology for microparticle characterization, flow cytometry, is lacking sensitivity and specificity, which might have led to the publication of contradicting results in the past.We propose the use of nano-liquid chromatography two-stage mass spectrometry as a nonbiased tool for quantitative MP proteome analysis.For this, we developed an improved microparticle isolation protocol and quantified the microparticle protein composition of twelve healthy volunteers with a label-free, data-dependent and independent proteomics approach on a quadrupole orbitrap instrument.Using aliquots of 250 μl platelet-free plasma from one individual donor, we achieved excellent reproducibility with an interassay coefficient of variation of 2.7 ± 1.7% (mean ± 1 standard deviation) on individual peptide intensities across 27 acquisitions performed over a period of 3.5 months. We show that the microparticle proteome between twelve healthy volunteers were remarkably similar, and that it is clearly distinguishable from whole cell and platelet lysates. We propose the use of the proteome profile shown in this work as a quality criterion for microparticle purity in proteomics studies. Furthermore, one freeze thaw cycle damaged the microparticle integrity, articulated by a loss of cytoplasm proteins, encompassing a specific set of proteins involved in regulating dynamic structures of the cytoskeleton, and thrombin activation leading to MP clotting. On the other hand, plasma membrane protein composition was unaffected. Finally, we show that multiplexed data-independent acquisition can be used for relative quantification of target proteins using Skyline software. Mass spectrometry data are available via ProteomeXchange (identifier PXD003935) and panoramaweb.org (https://panoramaweb.org/labkey/N1OHMk.url)

Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry

Bacterial pore-forming toxin pneumolysin: Cell membrane structure and microvesicle shedding capacity determines differential survival of immune cell types

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore‐forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans‐membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T‐cell line Jurkat, and myeloid cell lines U937 and THP‐1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP‐1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first‐line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin

Ultrasensitive Label-Free Detection of Protein–Membrane Interaction Exemplified by Toxin-Liposome Insertion

Measuring the high-affinity binding of proteins to liposome membranes remains a challenge. Here, we show an ultrasensitive and direct detection of protein binding to liposome membranes using high throughput second harmonic scattering (SHS). Perfringolysin O (PFO), a pore-forming toxin, with a highly membrane selective insertion into cholesterol-rich membranes is used. PFO inserts only into liposomes with a cholesterol concentration >30%. Twenty mole-percent cholesterol results in neither SHS-signal deviation nor pore formation as seen by cryo-electron microscopy of PFO and liposomes. PFO inserts into cholesterol-rich membranes of large unilamellar vesicles in an aqueous solution with Kd = (1.5 ± 0.2) × 10–12 M. Our results demonstrate a promising approach to probe protein–membrane interactions below sub-picomolar concentrations in a label-free and noninvasive manner on 3D systems. More importantly, the volume of protein sample is ultrasmall (<10 μL). These findings enable the detection of low-abundance proteins and their interaction with membranes.UPDALPELB