Marc Fivaz

MEMBRANE INSERTION : THE STRATEGIES OF TOXINS (REVIEW)

Protein toxins are soluble molecules secreted by pathogenic bacteria which act at the plasma membrane or in the cytoplasm of target cells. They must therefore interact with a membrane at some point, either to modify its permeability properties or to reach the cytoplasm. As a consequence, toxins have the built-in capacity to adopt two generally incompatible states: water-soluble and transmembrane. Irrespective of their origin or function, the membrane interacting domain of most protein toxins seems to have adopted one out of two structural strategies to be able to undergo this metamorphosis. In the first group of toxins the membrane interacting domain has the structural characteristics of most known membrane proteins, i.e. it contains hydrophobic and amphipathic alpha-helices long enough to span a membrane. To render this membrane protein water-soluble during the initial part of its life the hydrophobic helices are sheltered from the solvent by a barrel of amphipathic helices. In the second group of toxins the opposite strategy is adopted. The toxin is an intrinsically soluble protein and is composed mainly of beta-structure. These toxins manage to become membrane proteins by oligomerizing in order to combine amphipathic beta-sheet to generate sufficient hydrophobicity for membrane insertion to occur. Toxins from this latter group are thought to perforate the lipid bilayer as a beta-barrel such as has been described for bacterial porins, and has recently been shown for staphylococcal alpha-toxin. The two groups of toxins will be described in detail through the presentation of examples. Particular attention will be given to the beta-structure toxins, since four new structures have been solved over the past year: the staphyloccocal alpha-toxin channel, the anthrax protective antigen protoxin, the anthrax protective antigen-soluble heptamer and the CytB protoxin. Structural similarities with mammalian proteins implicated in the immune response and apoptosis will be discussed. Peptide toxins will not be covered in this review.

Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking.

Background Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. Methodology/Principal Findings Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. Conclusions/Significance These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.

Adventures of a pore-forming toxin at the target cell surface

The past three years have shed light on how the pore-forming toxin aerolysin binds to its target cell and then hijacks cellular devices to promote its own polymerization and pore formation. This selective permeabilization of the plasma membrane has unexpected intracellular consequences that might explain the importance of aerolysin in Aeromonas pathogenicity.

Aerolysin Induces G-protein Activation and Ca2+Release from Intracellular Stores in Human Granulocytes

Aerolysin is a pore-forming toxin that plays a key role in the pathogenesis of Aeromonas hydrophilainfections. In this study, we have analyzed the effect of aerolysin on human granulocytes (HL-60 cells). Proaerolysin could bind to these cells, was processed into active aerolysin, and led to membrane depolarization, indicating that granulocytes are potential targets for this toxin. Fura-2 measurements were used to analyze the effect of aerolysin on cytosolic [Ca2+] homeostasis. As expected for a pore-forming toxin, aerolysin addition led to Ca2+influx across the plasma membrane. In addition, the toxin triggered Ca2+ release from agonist and thapsigargin-sensitive intracellular Ca2+ stores. This Ca2+ release was independent of the aerolysin-induced Ca2+ influx and occurred in two kinetically distinct phases: an initial rapid and transient phase and a second, more sustained, phase. The first, but not the second phase was sensitive to pertussis toxin. Activation of pertussis toxin-sensitive G-proteins appeared to be a consequence of pore formation, rather than receptor activation through aerolysin-binding, as it: (i) was not observed with a binding competent, insertion-incompetent aerolysin mutant, (ii) had a marked lag time, and (iii) was also observed in response to other bacterial pore-forming toxins (staphylococcal α-toxin, streptolysin O) which are thought to bind to different receptors. G-protein activation through pore-forming toxins stimulated cellular functions, as evidenced by pertussis toxin-sensitive chemotaxis. Our results demonstrate that granulocytes are potential target cells for aerolysin and that in these cells, Ca2+ signaling in response to a pore-forming toxin involves G-protein-dependent cell activation and Ca2+ release from intracellular stores.

Not as simple as just punching a hole.

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.

Aerolysin from Aeromonas hydrophila and related toxins.

Aeromonads are ubiquitous gram-negative bacteria found in aqueous environments. Some members of the genus are pathogenic for fish, reptiles and cows. In humans, Aeromonas infection is mainly associated with grastrointestinal diseases, but in immuno-compromised individuals infection can lead to septicemia and meningitis (Austin et al. 1996). Aeromonas secretes a variety of virulence factors amongst which aerolysin is the best characterized. Using marker exchange mutagenesis, aerolysin was demonstrated to be required not only for the establishment but also for the subsequent maintenance of systemic infections associated with the bacterium (Chakraborty et al. 1987). Furthermore, specific neutralizing antibodies to aerolysin have been detected in animals surviving Aeromonas infection.

Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis

The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)‐anchored proteins y two‐dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent‐resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI‐anchored proteins. Although the GPI‐anchored proteins were readily separated by one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), these proteins were undetectable on two‐dimensional (2‐D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent‐resistant membranes such as caveolin‐1 and flotillin‐1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI‐anchored proteins required cleavage of the lipid tail with phosphatidylinositol‐specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI‐anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI‐anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N‐glycosylation. Pretreatment with N‐glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N‐linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray‐tandem mass spectrometry as illustrated for the GPI‐anchored protein, Thy‐1.

Dimer dissociation of the pore-forming toxin aerolysin precedes receptor binding

The pore-forming toxin aerolysin is secreted byAeromonas 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.

The tip of a molecular syringe

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

Pathogens, toxins and lipid rafts.

SummaryThe plasma membrane is not a uniform two-dimensional space but includes various types of specialized regions containing specific lipids and proteins. These include clathrin-coated pits and caveolae. The existence of other cholesterol- and glycosphingolipid-rich microdomains has also been proposed. The aim of this review is to illustrate that these latter domains, also called lipid rafts, may be the preferential interaction sites between a variety of toxins, bacteria, and viruses and the target cell. These pathogens and toxins have hijacked components that are preferentially found in rafts, such as glycosylphosphatidylinositol-anchored proteins, sphingomyelin, and cholesterol. These molecules not only allow binding of the pathogen or toxin to the proper target cell but also appear to potentiate the toxic action. We briefly review the structure and proposed functions of cholesterol- and glycosphingolipid-rich microdomains and then describe the toxins and pathogens that interact with them. When possible the advantage conferred by the interaction with microdomains will be discussed.