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To promote infections, pathogens exploit host cell machineries including structural elements of the plasma membrane. Studying these interactions and identifying involved molecular players is an ideal way to gain insights into the fundamental biology of the host cell. Here, using the anthrax toxin, we screened a 1500-gene library of regulatory, cell surface, and membrane trafficking genes for their involvement in the intoxication process. We found that the ER–Golgi-localized proteins TMED2 and TMED10 are required for toxin oligomerization at the cell surface, an essential step for anthrax intoxication that depends on localization to cholesterol-rich lipid nanodomains. Further biochemical, morphological and mechanistic analyses showed that TMED2 and TMED10 are essential components of a multiprotein supercomplex that operates exchange of both cholesterol and ceramides at ER-Golgi membrane contact sites. Overall, this study of anthrax intoxication led to the discovery that lipid compositional remodelling at ER-Golgi interfaces fully controls the formation of functional membrane nanodomains at the cell surface.

SARS-CoV-2 virions are surrounded by a lipid bilayer which contains membrane proteins such as Spike, responsible for target-cell binding and virus fusion, the envelope protein E and the accessory protein Orf3a. Here, we show that during SARS-CoV-2 infection, all three proteins become lipid modified, through action of the S- acyltransferase ZDHHC20. Particularly striking is the rapid acylation of Spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics and biochemical approaches, we show that this massive lipidation controls Spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid rich lipid nanodomains, in the early Golgi where viral budding occurs. ZDHHC20-mediated acylation allows the formation of viruses with enhanced fusion capacity and overall infectivity. Our study points towards S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets. Funding: This work was supported by the Swiss National Science Foundation Corona Call, the CARIGEST foundation and the EPFL Corona Research task force to F.G.v.d.G. MD simulations were carried out on the Piz Daint computer at the Swiss Supercomputing Center (CSCS) thanks to access granted by PRACE Covid19 fast track project #17 (pr97) to M.D.P. Conflict of Interest: The authors declare no competing interests.

Pore-forming toxins (PFTs) form holes in membranes causing one of the most catastrophic damages to a target cell. Target organisms have evolved a regulated response against PFTs damage including cell membrane repair. This ability of cells strongly depends on the toxin concentration and the properties of the pores. It has been hypothesized that there is an inverse correlation between the size of the pores and the time required to repair the membrane, which has been for long a non-intuitive concept and far to be completely understood. Moreover, there is a lack of information about how cells react to the injury triggered by eukaryotic PFTs. Here, we investigated some molecular events related with eukaryotic cells response against the membrane damage caused by sticholysin II (all), a eukaryotic PFT produced by a sea anemone. We evaluated the change in the cytoplasmic potassium, identified the main MAPK pathways activated after pore-formation by Stll, and compared its effect with those from two well-studied bacterial PFTs: aerolysin and listeriolysin O (LLO). Strikingly, we found that membrane recovery upon all damage takes place in a time scale similar to LLO in spite of the fact that they form pores by far different in size. Furthermore, our data support a common role of the potassium ion, as well as MAPKs in the mechanism that cells use to cope with these toxins injury. (C) 2017 Elsevier B.V. All rights reserved.

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.

The plasma membrane defines the boundary of every living cell, and its integrity is essential for life. The plasma membrane may, however, be challenged by mechanical stress or pore-forming proteins produced by the organism itself or invading pathogens. We will here review recent findings about pore-forming proteins from different organisms, highlighting their structural and functional similarities, and describe the mechanisms that lead to membrane repair, since remarkably, cells can repair breaches in their plasma membrane of up to 10,000 microm(2).

Following endocytosis, ubiquitinated signaling receptors are incorporated within intraluminal vesicles of forming multivesicular endosomes. These vesicles then follow the pathway from early to late endosomes, remaining within the endosomal lumen, and are eventually delivered to lysosomes, where they are degraded together with their protein cargo. However, intraluminal vesicles do not always end up in lysosomes for degradation; they can also fuse back with the limiting membrane of late endosomes. This route, which might be regulated by lyso-bisphosphatidic acid and its putative effector Alix, can be hijacked by the anthrax toxin and vesicular stomatitis virus and is presumably exploited by proteins and lipids that transit through intraluminal vesicles. Alternatively, these vesicles can be released extracellularly, like HIV in macrophages, upon fusion of endosomes or lysosomes with the plasma membrane.

SummaryMany pathogenic organisms produce pore-forming toxins as virulence factors. Target cells however mount a response to such membrane damage. Here we show that toxin-induced membrane permeabilization leads to a decrease in cytoplasmic potassium, which promotes the formation of a multiprotein oligomeric innate immune complex, called the inflammasome, and the activation of caspase-1. Further, we find that when rendered proteolytic in this context caspase-1 induces the activation of the central regulators of membrane biogenesis, the Sterol Regulatory Element Binding Proteins (SREBPs), which in turn promote cell survival upon toxin challenge possibly by facilitating membrane repair. This study highlights that, in addition to its well-established role in triggering inflammation via the processing of the precursor forms of interleukins, caspase-1 has a broader role, in particular linking the intracellular ion composition to lipid metabolic pathways, membrane biogenesis, and survival.

Rather than being distributed homogeneously on the cell surface, proteins are probably aggregated in clusters or in specific domains. Some of these domains (lipid rafts) have lipid compositions, which differ from their surrounding membrane. They have been implicated in cell signaling, cell adhesion, and cholesterol homeostasis. Estimates of their size vary from 40 to 350 nm in diameter depending on the study and cell type used. Rafts are enriched in glycosphingolipids and cholesterol and appear to be in a more ordered lipid phase. Although there is some knowledge of their function in cell signaling, less is known about their assembly and dynamics in cells at various temperatures. We use image correlation spectroscopy and dynamic image correlation spectroscopy to study the clustering and diffusion of glycosylphosphatidylinositol (GPI)-anchored proteins within the plasma membrane of living cells at various temperatures. We find that GPI-anchored proteins occur both as monomers and in clusters at the cell surface. The propensities to cluster as well as the diffusion coefficient of these clusters are strongly temperature dependent. At 37 degrees C the GPI-anchored proteins are highly dynamic with a lower state of clustering than at lower temperatures.

The past five years have led to a tremendous increase in our molecular understanding of the mode of action of the anthrax toxin, one of the two main virulence factors produced by Bacillus anthracis . The structures of each of the three components of the toxin – lethal factor (LF), edema factor (EF) and protective antigen (PA) – have been solved not only in their monomeric forms but, depending on the subunit, in a heptameric form, bound to their substrate, co-factor or receptor. The endocytic route followed by the toxin has also been unraveled and the enzymatic mechanisms of EF and LF elucidated.

An ever more complete picture of the organization and function of the endocytic pathway is emerging. New mechanisms, and in particular lipid-based mechanisms that couple membrane dynamics and sorting, are being unraveled. But the final picture is still coming into focus as new membrane domains, cell entry pathways and compartments come into view. Of special interest are the recent findings that pathogenic agents, in contrast to scientists, seem to have long discovered how to subvert membrane specialization to their own advantage.

Like a variety of other pathogenic bacteria, Aeromonas hydrophila secretes a pore-forming toxin that contribute to its virulence. The last decade has not only increased our knowledge about the structure of this toxin, called aerolysin, but has also shed light on how it interacts with its target cell and how the cell reacts to this stress. Whereas pore-forming toxins are generally thought to lead to brutal death by osmotic lysis of the cell, based on what is observed for erythrocytes, recent studies have started to reveal far more complicated pathways leading to death of nucleated mammalian cells.

The pore-forming toxin aerolysin is secreted by Aeromonas hydrophila as an inactive precursor. Based on chemical cross-linking and gel filtration, we show here that proaerolysin exists as a monomer at low concentrations but is dimeric above 0.1 mg/ml. At intermediate concentrations, monomers and dimers appeared to be in rapid equilibrium. All together our data indicate that, at low concentrations, the toxin is a monomer and that this species is competent for receptor binding. In contrast, a mutant toxin that forms a covalent dimer was unable to bind to target cells.

Aerolysin is a bacterial pore-forming toxin that is secreted as an inactive precursor, which is then processed at its COOH terminus and finally forms a circular heptameric ring which inserts into membranes to form a pore. We have analyzed the stability of the precursor proaerolysin and the heptameric complex. Equilibrium unfolding induced by urea and guanidinium hydrochloride was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to the unfolding of the large COOH-terminal lobe followed by the unfolding of the small NH(2)-terminal domain. We show that proaerolysin contains two disulfide bridges which strongly contribute to the stability of the toxin and protect it from proteolytic attack. The stability of aerolysin was greatly enhanced by polymerization into a heptamer. Two regions of the protein, corresponding to amino acids 180-307 and 401-427, were identified, by limited proteolysis, NH(2)-terminal sequencing and matrix-assisted laser desorption ionization-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer interactions in the heptameric protein and are exclusively composed of beta structure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenerative diseases.

Aerolysin is a cytolytic toxin which forms channels in the plasma membranes of eucaryotic cells. The protein is secreted by Aeromonas hydrophila as an inactive protoxin. Its stability and water solubility are conferred by its ability to dimerize. Maturation of the protein occurs through proteolytic removal of a C-terminal peptide outside the secreting cell. Although the aerolysin which is so produced is still a dimer, it then has the ability to oligomerize. The oligomer is the active form of the toxin, capable of forming the transmembrane channels that disrupt cells. We review here the present knowledge about the structure of aerolysin in relation to the various steps in channel formation.

The endoplasmic reticulum (ER) forms functional contacts with several cellular organelles and regulates processes such as mitochondrial fission. In a recent issue of Cell, Rowland et al. (2014) extend these findings to endosomes, showing that the ER contacts endosomes at sites containing the WASH subunit FAM21, where it forecasts fission events.

Keywords: Gram-Negative Bacteria/genetics/*metabolism/pathogenicity ; Gram-Negative Bacterial Infections/*microbiology ; Membrane Proteins/*genetics/*metabolism ; Plant Diseases/*microbiology ; Virulence Note: Dept of Biochemistry, University of Geneva, 30 quai E. Ansermet, 1211 Geneva 4, Switzerland. Reference VDG-ARTICLE-1999-008doi:10.1016/S0966-842X(99)01574-7 Record created on 2009-01-30, modified on 2017-05-12

Keywords: Bacterial Proteins ; Bacterial Toxins/*chemistry/metabolism ; Cell Membrane/chemistry/metabolism ; Hemolysin Proteins ; Prions/chemistry/metabolism ; Protein Structure ; Secondary ; Streptolysins/*chemistry/metabolism Note: Dept of Biochemistry, University of Geneva, 30 quai E. Ansermet, 1211 Geneva 4, Switzerland. gisou.vandergoot@biochem.unige.ch Reference VDG-ARTICLE-2000-002doi:10.1016/S0966-842X(99)01692-3 Record created on 2009-01-30, modified on 2017-05-12