Membrane-mediated assembly of the prothrombinase complex

Prothrombinase assembly was studied on macroscopic planar bilayers consisting of 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC). The dissociation constant for the binding of factor Xa to the bilayer, measured by ellipsometry, was Kd = 47 +/- 8 nM (mean +/- S.D.) and this value was lowered to Kd = 2.2 +/- 0.3 pM by preadsorption of factor Va. This latter value was determined from direct measurement of steady-state thrombin production. A comparable value of Kd = 1.0 +/- 0.1 pM was found by repeating these experiments in suspensions of phospholipid vesicles, and it was verified that prothrombinase assembly was not influenced by the addition of prothrombin. Using a minute amount (0.094 fmol cm-2) of preadsorbed factor Va, it was found that the rate of prothrombinase assembly exceeds the rate of collisions between Xa molecules from the buffer and the sparse Va molecules on the bilayer. Apparently, factor Xa adsorbs first to the membrane and then associates rapidly with factor Va by lateral diffusion. The data indicate almost instantaneous equilibrium of this complex formation on the surface with a lower limit for the bimolecular rate constant of kon = 2.8 x 10(13) (mol/cm2)-1 s-1. In suspensions of small phospholipid vesicles, prothrombinase assembly is collisionally limited and the value of kon should be proportional to vesicle diameter. This was verified with a method for estimation of kon values from thrombin generation curves. Values of 0.36 x 10(9) and 1.6 x 10(9) M-1 s-1 were found for vesicles of 20-30- and 60-80-nm diameter, respectively.     

Genetic recombination between alpha-toxin mutants of Staphylococcus aureus

McClatchy, J. K. (The University of Texas Southwestern Medical School, Dallas), and E. D. Rosenblum. Genetic recombination between alpha-toxin mutants of Staphylococcus aureus. J. Bacteriol. 92:580-583. 1966.-A demonstration of genetic recombination between Staphylococcus aureus nonhemolytic mutants was attempted by means of transduction. The results of two-point reciprocal transductions placed the mutants into two genetic groups. Recombination within each group was not detectable within the limits of the method, but hemolytic recombinants were obtained in transductional crosses when donor and recipient were from different groups. At least two genetic loci are therefore involved in alpha-toxin production. The 11 mutants of group II were fibrinolysin-negative. The recombinants were always found to be restored to fibrinolysin production as well as to alpha-toxin production. These data suggest the existence of a pleiotropic gene simultaneously affecting the synthesis of both alpha toxin and fibrinolysin. The nine mutants of group I were fibrinolysin-positive. Group I members are postulated to be alpha-toxin structural mutants. Three mutants were also negative for bound coagulase, but no linkage was observed between the locus controlling bound coagulase and the loci for either fibrinolysin or alpha-toxin production.     

Staphylococcus aureus alpha-toxin induces apoptosis in endothelial cells

The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of alpha-toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified alpha-toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two alpha-toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce alpha-toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of alpha-toxin during endothelial cell invasion by S. aureus enhances apoptosis.     

Crystalline layers and three-dimensional structure of Staphylococcus aureus alpha-toxin

Interaction of the pore-forming protein alpha-toxin from Staphylococcus aureus with lipid components from platelet membranes induces crystal formation of the toxin oligomers. Structure analysis of crystalline areas in either sodium phosphotungstic acid or a sodium phosphotungstic acid/glucose mixture has been performed with electron microscopy and image processing. Ordered domains extending up to a few micrometers were observed, particularly after application of alpha-toxin to pre-formed lipid layers. The crystals, showing tetragonal symmetry, formed either separate two-dimensional sheets or three-dimensional piles of layers. The corresponding unit cell parameter of the single layer was a = b = 109.4 A (standard deviation 2.1 A, n = 21). Incubation of the toxin with intact membranes or extracted lipids as well as application of the lipid layer technique resulted in congruous crystalline properties. The projected averaged alpha-toxin oligomer shows cyclic symmetry with a stain-filled space in the centre. The bulk of the three-dimensional model consists of four asymmetric protein units forming a ring. In addition, a small domain covers the central cavity at the face of the protein opposite to the underlying lipid. The conditions under which the tetragonal arrays are formed on the lipid layers suggest that the alpha-toxin molecule is in a conformation binding to a hydrophobic surface rather than fully inserted into a lipid bilayer.     

The three-dimensional structure of trypsin-treated Staphylococcus aureus alpha-toxin.

Trypsin treatment of staphylococcal alpha-toxin cleaves the molecule into two roughly equally sized parts, which results in inactivation of the toxin. Tetragonal arrays of oligomers, closely resembling the native ones, can however be formed on lipid layers. From tilted views of negatively stained crystals a 3D structure to 23 A resolution has been determined by electron microscopy and image processing. On comparison with the 3D structure of the native alpha-toxin (Olofsson et al., J. Mol. Biol. 214, 299-306, 1990) the subdomains are more separated, confirming the differences found when comparing the projection maps (Olofsson et al., J. Struct. Biol. 106, 199-204, 1991). The tryptic cleavage takes place in a postulated hinge region. The results are consistent with the hypothesis that the conformational change required for inducing the membrane permeabilizing property takes place in this region. Furthermore, we present a refined projection map at approximately 10 A resolution based on the analysis of a large number of crystals using unbending methods.     

Interaction of the alpha-toxin of Staphylococcus aureus with the liposome membrane

When the liposome membrane is exposed to the alpha-toxin of Staphylococcus aureus, fluorescence of the tryptophan residue(s) of the toxin molecule increases concomitantly with the degree of toxin-hexamer formation (Ikigai, H., and Nakae, T. (1985) Biochem. Biophys. Res. Commun. 130, 175-181). In the present study, the toxin-membrane interaction was distinguished from the hexamer formation by the fluorescence energy transfer from the tryptophan residue(s) of the toxin molecule to the dansylated phosphatidylethanolamine in phosphatidylcholine liposome. Measurement of these two parameters yielded the following results. The effect of the toxin concentration and phospholipid concentration on these two parameters showed first order kinetics. The effect of liposome size on the energy transfer and the fluorescence increment of the tryptophan residue(s) was only detectable in small liposomes. Under moderately acidic or basic conditions, the fluorescence energy transfer always preceded the fluorescence increment of the tryptophan residue(s). The fluorescence increment at 336 nm at temperatures below 20 degrees C showed a latent period, whereas the fluorescence energy transfer did not. These results were thought to indicate that when alpha-toxin damages the target membrane, the molecule interacts with the membrane first, and then undergoes oligomerization within the membrane.     

Functional prothrombinase complex assembly on isolated monocytes and lymphocytes

Isolated peripheral blood monocytes and lymphocytes interact with Factor Va and Factor Xa to form a functional catalytic complex which proteolytically activates prothrombin to thrombin. The kinetics of prothrombin activation were monitored continuously using the fluorescent, reversible thrombin inhibitor, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide, which displays enhanced fluorescence upon binding to thrombin. Incubation of monocytes or lymphocytes with prothrombin, the cofactor (Factor Va), and the enzyme (Factor Xa) in the presence of Ca2+ generated thrombin at rates/cell exceeding those previously obtained with either bovine or human platelets. The rate of thrombin generation by monocytes exceeded that of lymphocytes and increased as monocytes adhered to a surface. Monocyte prothrombinase activity appears to be mediated through interactions, whereby Factor Va forms a receptor for Factor Xa at the monocyte surface. Monocytes possess approximately 16,100 Factor Va binding sites with a dissociation constant (Kd) of 4 X 10(-11) M. In addition, isolated, well washed monocytes and lymphocytes, respectively, contain approximately 61,400 +/- 9,900 and 24,500 +/- 4,800 molecules of Factor V/cell as determined by radioimmunoassay. Bioassay data of mononuclear cell preparations paralleled the radioimmunoassay data. The Factor V associated with washed mononuclear cells appears to be intracellular and not membrane-associated. The release of Factor V, and perhaps other sequestered coagulation factors, by these immunoreactive cells at an inflammatory site, coupled with the ability of these cells to effect thrombin generation may explain the relationship between extravascular fibrin deposition and mononuclear cell accumulation in the pathogenesis of inflammatory lesions.     

Influence of membrane fluidity on the assembly of Staphylococcus aureus alpha-toxin, a channel-forming protein, in liposome membrane.

By use of multilamellar phosphatidylcholine (PC) liposomes of different acyl composition and cholesterol content as model membranes, we studied whether or not membrane fluidity affects the assembly process of Staphylococcus aureus alpha-toxin. Under conditions using fluid and solid membranes, we assayed accessibility (or hemolytic activity) of liposome-bound alpha-toxin to rabbit erythrocytes added, hexamerization of membrane-bound toxin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nondenaturating conditions, and susceptibility of liposome-bound toxin to trypsin digestion. Our data indicated 1) that alpha-toxin bound to PC membrane as a hemolytically active monomer (or reversibly bound state); 2) that when the membrane was fluidized either by phase transition of PC or by inclusion of cholesterol over 20 mol %, the hemolytically active monomer of the toxin was irreversibly converted to nonhemolytic monomer (and/or unstable oligomer) in a first-order kinetics with a t1/2 of about 1 min, and thereafter hexamerization of the toxin gradually proceeded in the following 60-90 min; 3) that alpha-toxin might have different topology and/or conformation in PC membrane, depending on the presence or absence of cholesterol in the PC membrane; and 4) that coexistence of unsaturated acyl chain-carrying PC and cholesterol was a prerequisite for efficient hexamerization of alpha-toxin in membrane. Thus, increase in membrane fluidity promoted the assembly process of S. aureus alpha-toxin.     

Pore formation by Staphylococcus aureus alpha-toxin in lipid bilayers

Staphylococcus aureus -toxin forms ionic channels of large size in lipid bilayer membranes. We have developed two methods for studying the mechanism of pore formation. One is based on measurement of the ionic current flowing through a planar lipid membrane after exposure to the toxin; the other is based on measuring the release of the fluorescent complex Tb-Dipicolinic acid from large unilamellar vesicles under similar conditions.Both methods indicate that the pore formation process is complex, showing an initial delay followed by non-linear kinetics. The power dependence of the pore formation rate on the toxin concentration in planar bilayers indicates that an aggregation mechanism underlies the channel assembly. Arrhenius plots, obtained with both techniques, show no deviation from linearity up to 50°C and the derived activation energies are found to be comparable to those for the binding and the lysis of rabbit erythrocytes by the same toxin.The temperature dependence of the conductance induced in planar bilayers by a large number of toxin channels indicates that the pores are filled with aqueous solution. The analysis of single conductance events shows that a heterogeneous population of pores exist and that smaller channels are preferred at low temperature. We attribute this heterogeneity to the existence of pores resulting from the aggregation of different numbers of monomers.     

Calcium ion-mediated regulation of the alpha-toxin pore of Staphylococcus aureus.

The water-soluble alpha-toxin monomers of Staphylococcus aureus become hexamers forming the transmembrane pore when exposed to the membranes. This pore is freely permeable to small hydrophilic molecules, e.g. carboxyfluorescein, and becomes less permeable in the presence of calcium ions. Calcium ion-mediated decrease of the carboxyfluorescein leakage could not be eliminated by EDTA added in the medium, but the carboxyfluorescein could be freed by EDTA added in the intraliposomal space. This result suggests that the alpha-toxin pore changes its conformation as the calcium ion is bound and that the binding site is exposed to the intraliposomal side of the membrane. The interaction between the alpha-toxin hexamer and 8-anilino-1-naphthalene-sulfonic acid (ANS) was monitored by determining the fluorescence in the presence and absence of calcium chloride. The mean distances between the tryptophan residues of the alpha-toxin hexamer and the bound ANS were calculated to be 1.90 and 1.80 nm in the absence and presence, respectively, of calcium ions. The results showed the calcium ion mediated conformational change of the membrane-embedded alpha-toxin hexamer.     

Molecular mechanisms of cytotoxic effect of alpha-toxin of Staphylococcus aureus

Mechanism of action of Staphylococcus aureus alpha-toxin has been reviewed. Data about molecular transformations during cytolytic action, dimensional structure of monomeric and oligomeric toxin, its characteristics in different molecular states are presented. Data about defects of oligomerization due to modification or site-induced mutagenesis are discussed.     

Interaction of Staphylococcus aureus alpha-toxin with eukaryotic cells and their target

In this review information on S. aureus alpha-toxin is expounded. The data on the targets of the action of the toxin on eukaryotic cells, its activity with respect to different membrane structures, effects established in the interaction of toxin and cell elements, as well as of its biological action on the macroorganism, are presented. Information on the factors inactivating the cytolytic action of the toxin is given.     

Staphylococcus aureus alpha-toxin. Dual mechanism of binding to target cells

Staphylococcal alpha-toxin was radiolabeled to high specific radioactivity (1,500-3,000 Ci/mmol) under retention of its hemolytic activity. Binding studies with susceptible rabbit erythrocytes and highly resistant human erythrocytes revealed that binding of alpha-toxin to target cells can occur via two different mechanisms. Binding of alpha-toxin to rabbit erythrocytes initially involves specific binding sites and occurs at low concentrations, with half-maximal binding at 1-2 nM. In contrast, toxin binding to human erythrocytes is absorptive and nonspecific, in this case, significant binding as well as hemolysis occur only at alpha-toxin concentrations exceeding 1 microM. Autoradiographic analyses of membrane-associated alpha-toxin from either cell species proved that hemolysis was inevitably associated with the formation of toxin hexamers. Our data indicate that the high susceptibility of certain target cells toward alpha-toxin is caused by the presence of specific binding sites. However, membrane damage of both susceptible and nonsusceptible target cells occurs via a common mechanism involving toxin oligomerization and pore formation.     

Purification of alpha-toxin from Staphylococcus aureus and application to cell permeabilization

Crude alpha-toxin was produced by Staphylococcus aureus, strain Wood 46. The amount of exotoxin was monitored during growth and all subsequent purification steps by determination of its hemolytic activity against rabbit erythrocytes. The culture supernatant was treated with ammonium sulfate (75% saturation). The resulting precipitate was dialyzed and subjected to cation-exchange chromatography. The fractions containing the hemolytic activity were further purified by gel chromatography. The final product was enriched by a factor of 8.5 compared to the crude toxin. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified toxin exhibited one major band. It caused the release of 86Rb+ and ATP from rat insulinoma (RIN A2) as well as pheochromocytoma cells (PC12) in culture, indicating efficient permeabilization of their plasma membranes for small molecules.     

The Staphylococcus aureus alpha-toxin channel complex and the effect of Ca2+ ions on its interaction with lipid layers.

Using the techniques of two-dimensional crystallization on supported lipid bilayers together with computer image processing, two distinct two-dimensional crystal types of staphylococcal alpha-toxin complex are formed depending on the presence or absence of Ca2+ ions. Without Ca2+, these are hexagonally packed (in A, a = b = 89.5 +/- 2.5 A; theta = 119.7 degrees) With Ca2+ present, rectangular crystal packing is seen (in A, a = 114.8 +/- 1.6 A, b = 140.2 +/- 0.7 A; theta = 89.1 degrees). A third, banded crystal type is also seen which is interpreted as a side-to-side packing of regular tubules. We use these tubular crystals for cross-correlation searches with top and side-on views of the complex from single particle reconstructions, and with the repeating units from the two-dimensional crystal types. The results lead us to propose a model in which the different two-dimensional crystal types are formed as a result of alpha-toxin hexamers packing in different orientations. In the hexagonal crystals the hexamers lie end-on with a 6-fold axis in projection. On the addition of Ca2+, the hexamers reorient to lie tilted with respect to the support, thus giving rise to a rectangular projection.     

Hemoglobin promotes Staphylococcus aureus nasal colonization

Staphylococcus aureus nasal colonization is an important risk factor for community and nosocomial infection. Despite the importance of S. aureus to human health, molecular mechanisms and host factors influencing nasal colonization are not well understood. To identify host factors contributing to nasal colonization, we collected human nasal secretions and analyzed their ability to promote S. aureus surface colonization. Some individuals produced secretions possessing the ability to significantly promote S. aureus surface colonization. Nasal secretions pretreated with protease no longer promoted S. aureus surface colonization, suggesting the involvement of protein factors. The major protein components of secretions were identified and subsequent analysis revealed that hemoglobin possessed the ability to promote S. aureus surface colonization. Immunoprecipitation of hemoglobin from nasal secretions resulted in reduced S. aureus surface colonization. Furthermore, exogenously added hemoglobin significantly decreased the inoculum necessary for nasal colonization in a rodent model. Finally, we found that hemoglobin prevented expression of the agr quorum sensing system and that aberrant constitutive expression of the agr effector molecule, RNAIII, resulted in reduced nasal colonization of S. aureus. Collectively our results suggest that the presence of hemoglobin in nasal secretions contributes to S. aureus nasal colonization.