Escherichia coli hemolysin causes thromboxane-mediated hypertension and vascular leakage in rabbit lungs.
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Animals↗
Biomedical subjects
S Bhakdi
Publications and source records attributed to S Bhakdi.
In vivo clearance studies of the terminal fluid-phase complement complex in rabbits.
The present study was directed at obtaining information on the in vivo elimination rate of SC5b-9, the terminal fluid-phase product of complement activation. A sandwich ELISA based on the use of mono- and polyclonal antibodies was constructed that permitted quantitation of rabbit SC5b-9 in plasma. Rabbit serum was activated with inulin in vitro to generate SC5b-9, and the activated serum was applied intravenously in normal and C6-deficient rabbits. Elimination of SC5b-9 in normal rabbits was rapid, half-life in the range of 30-50 min. No differences were noted between the clearance of homologous rabbit and heterologous human SC5b-9, SC5b-9 concentrations returned to basal levels 2-3 h after application. Plasma of C6-deficient rabbits contained no SC5b-9 and these animals displayed an even more effective clearance capacity for the complex. Quantitative considerations indicated that basal plasma SC5b-9 levels in healthy animals result from a spontaneous turnover rate of approximately 0.2% of C5-C9 components per h. When multiple doses of SC5b-9 were injected in sequence, the same half-life and total elimination time were found as with single-dose experiments. The results demonstrate the existence of an effective clearance mechanism for SC5b-9, consistent with recent findings that SC5b-9 plasma levels are very low not only in healthy adults, but also in the majority of patients with complement-consuming diseases.
Animals↗
Relative inefficiency of terminal complement activation.
The efficiency of generation of fluid-phase SC5b-9 and membrane C5b-9(m) complexes relative to cleavage of C3 and C5 was studied. Fluid-phase C activation was induced through addition of purified bacterial Ag to human serum. Sephadex beads were used as particulate activators of the alternative pathway. Rabbit or antibody-coated sheep or human E were used to study formation of cytolytic C5b-9(m) complexes. The molar ratios of C3a:C5a generated in the model systems were found to be in the range of 60 to 200:1 in the case of soluble immune complex activators, and 70 to 150:1 with particulate activators and cells. The efficiency of C5 cleavage relative to C3 cleavage increased on surfaces with the density of antibody and/or C3b-binding sites. With soluble immune complexes, the efficiency of subsequent SC5b-9 generation displayed wide variations dependent on Ag and donor with molar ratios of C5a:SC5b-9 ranging from 30:1 for teichoic acid and sometimes approaching 1:1 for streptolysin-O. In contrast, activation on particles or cells always led to C5a:C5b-9 (calculated as the sum of generated moles SC5b-9 and C5b-9(m] ratios approaching 1:1. Hence, there is an overall inefficiency of terminal sequence activation in the C cascade due first to a dissociation at the level of C5 convertase formation/C5-cleavage and second, to a frequent inefficiency of C5b-utilization in the fluid-phase. The results provide an explanation for the very low levels of SC5b-9 found in plasma of healthy individuals and in patients with C-consuming immune complex disease.
Animals↗
Staphylococcal alpha toxin promotes blood coagulation via attack on human platelets.
Staphylococcus aureus plays a major role as a bacterial pathogen in human medicine, causing diseases that range from superficial skin and wound to systemic nosocomial infections . The majority of S. aureus strains produces a toxin, a proteinaceous exotoxin whose hemolytic, dermonecrotic, and lethal properties have long been known (1-6). The toxin is secreted as a single- chained, nonglycosylated polypeptide with a M(r) of 3.4 x 10(4) (7, 8). The protein spontaneously binds to lipid monolayers and bilayers (9-14), producing functional transmembrane pores that have been sized to 1.5-2.0-nm diameters (15-18). The majority of pores formed at high toxin concentrations (20 mug/ml) is visible in the electron microscope as circularized rings with central pores of approximately 2 nm in diameter. The rings have been isolated, and molecular weight determinations indicate that they represent hexamers of the native toxin (7). We have proposed that transmembrane leakiness is due to embedment of these ring structures in the bilayer, with molecular flux occurring through the central channels (15, 19). Pore formation is dissectable into two steps (20, 21). Toxin monomers first bind to the bilayer without invoking bilayer leakiness . Membrane-bound monomers then laterally diffuse and associate to form non-covalently bonded oligomers that generate the pores. When toxin pores form in membranes of nucleated cells, they may elicit detrimental secondary effects by serving as nonphysiologic calcium channels, influx of this cation triggering diverse reactions, including release of potent lipid mediators originating from the arachidonate cascade (22-24). That alpha toxin represents an important factor of staphylococcal pathogenicity has been clearly established in several models of animal infections through the use of genetically engineered bacterial strains deleted of an active alpha toxin gene (25-27). Whether the toxin is pathogenetically relevant in human disease, however, is a matter of continuing debate. Doubts surrounding this issue originate from two main findings. First, whereas 60 percent hemolysis of washed rabbit erythrocytes is effected by approximately 75 ng/ml alpha toxin, approximately 100-fold concentrations are required to effect similar lysis of human cells (4-6, 13). The general consensus is that human cells display a natural resistance towards toxin attack. The reason for the wide inter-species variations in susceptibility towards alpha toxin is unknown but does not seem to be due to the presence or absence of high-affinity binding sites on the respective target cells (20, 21). Second, low-density lipoprotein (28) and neutralizing antibodies present in plasma of all healthy human individuals inactivate a substantial fraction of alpha toxin in vitro. These inactivating mechanisms presumably further raise the concentration threshold required for effective toxin attack, and it is most unlikely that such high toxin levels will ever be encountered during infections in the human organism. The aforegoing arguments rest on the validity of two general assumptions. First, the noted natural resistance of human erythrocytes to alpha toxin must be exhibited by other human cells. Second, toxin neutralization by plasma components, usually tested and quantified after their preincubation with toxin in vitro, must be similarly effective under natural conditions, and protection afforded by these components must not be restricted to specific cell species.
Adenosine Triphosphate↗
Deposition of terminal C5b-9 complement complexes on erythrocytes and leukocytes during cardiopulmonary bypass.
Hemolysis, leukopenia, a hemostatic deficit, and nonspecific systemic reactions collectively known as the postperfusion syndrome develop in patients who undergo cardiopulmonary bypass. We now report that terminal C5b-9 complement complexes are deposited on erythrocytes and polymorphonuclear neutrophilic leukocytes during cardiopulmonary bypass. Plasma samples taken from 48 unselected patients during and at the end of cardiopulmonary bypass contained raised levels of fluid-phase SC5b-9 complement complexes, indicating that the complement sequence had been activated to completion. Various degrees of overt intravascular hemolysis were observed in all the patients, and lysed erythrocyte membranes were recovered from the blood samples. Immunoassays performed with use of antibodies to C5b-9 neoantigens demonstrated the presence of C5b-9 on red-cell ghosts but not on intact erythrocytes. The appearance of ghosts carrying C5b-9 always coincided with hemolysis. Furthermore, granulocytes isolated from 20 patients during bypass were all found to carry C5b-9 complexes, whereas cells isolated before or 24 hours after surgery carried no C5b-9. The neoantigen-positive material present in detergent extracts of granulocytes sedimented in a broad peak (25 to 40 sedimentation coefficient [S]) in sucrose-density gradients, exactly as did pore-forming C5b-9 complexes. Deposition of C5b-9 on blood cells during cardiopulmonary bypass may be partly responsible for the hemolysis and may augment granulocyte activation by the stimulation of arachidonate metabolism in those cells.
Autoantibodies↗
The hemolysin of Escherichia coli.
Many strains of E. coli elaborate a hemolysin which is responsible for the zone of beta-hemolysis surrounding bacterial colonies on blood agar. The significance of this cytolysin as a determinant of bacterial pathogenicity has been established in animal models with the use of genetically engineered, isogenic bacterial strains. An analogous role in human infections has been inferred from the high association of hemolysin production with disease. Studies at a molecular genetical level have defined 4 genes that are required for the synthesis, post-translational modification and secretion of the hemolysin. The structural gene hlyA encodes for a 107-110,000 polypeptide, which must be modified in an unknown manner to its active form by the product of the neighboring hlyC gene. Genes hlyB and hlyD encode for proteins that export the molecule to the extracellular medium. The signal for secretion is contained in the C-terminal portion of the toxin molecule. The secreted hemolysin attacks plasma membranes of target mammalian cells by inserting as a monomer into the bilayer and generating hydrophilic transmembrane pores of approximately 2 nm effective diameter. The pores display a marked selectivity for cations over anions and pore-opening is dependent on the presence of a correct transmembrane potential. Binding to a membrane target does not require the presence of a specific receptor, and pores may be generated in planar lipid membranes consisting solely of phosphatidylcholine. Pore formation in nucleated cells can trigger secondary reactions such as stimulation of arachidonate metabolism with release of lipid mediators, probably initiated by passive influx of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Incorporation of toxin pores into liposomes.
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Animals↗
The cytolytic C5b-9 complement complex: feedback inhibition of complement activation.
We describe a regulatory function of the terminal cytolytic C5b-9 complex [C5b-9(m)] of human complement. Purified C5b-9(m) complexes isolated from target membranes, whether in solution or bound to liposomes, inhibited lysis of sensitized sheep erythrocytes by whole human serum in a dose-dependent manner. C9 was not required for the inhibitory function since C5b-7 and C5b-8 complexes isolated from membranes were also effective. No effect was found with the cytolytically inactive, fluid-phase SC5b-9 complex. However, tryptic modification of SC5b-9 conferred an inhibitory capacity to the complex, due probably to partial removal of the S protein. Experiments using purified components demonstrated that C5b-9(m) exerts a regulatory effect on the formation of the classical- and alternative-pathway C3 convertases and on the utilization of C5 by cell-bound C5 convertases. C5b-9(m) complexes were unable to inhibit the lysis of cells bearing C5b-7(m) by C8 and C9. Addition of C5b-9(m) to whole human serum abolished its bactericidal effect on the serum-sensitive Escherichia coli K-12 strain W 3110 and suppressed its hemolytic function on antibody-sensitized, autologous erythrocytes. Feedback inhibition by C5b-9(m) represents a biologically relevant mechanism through which complement may autoregulate its effector functions.
Complement Activation↗
Damage to cell membranes by pore-forming bacterial cytolysins.
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Animals↗
Complement S-protein (vitronectin) is associated with cytolytic membrane-bound C5b-9 complexes.
It has been assumed that S-protein (vitronectin) associates with terminal C5b-9 complement complexes only when the latter fail to attach to target lipid bilayers, thereby forming inactive fluid-phase SC5b-9 complexes. Using monoclonal anti-S-protein antibodies, we show here that a minor portion of C5b-9 complexes associated with both homologous and heterologous cells contain S-protein. This conclusion derives from Western blot analyses, from the sedimentation behaviour of solubilized S-protein, and from the fact that the protein co-immunoprecipitates with C5b-9(m). Association of S-protein with C5b-9(m) takes place primarily at the stage of C9-binding. An average of less than or equal to 0.4 moles of S-protein are estimated to be present per mole C5b-9(m). Hence, only a fraction of C5b-9 complexes contain S-protein. The function of cell-bound S-protein is unknown. Haemolytic titrations with purified components failed to demonstrate any protective effect of S-protein on the lysis of sheep or human erythrocytes by C5b-9. S-protein bound to complement-lysed homologous or heterologous cells is readily detectable by conventional immunocytochemical staining. We conclude that differentiation between tissue-deposited fluid-phase C5b-9 and membrane C5b-9 complexes cannot be made on the basis of immunohistological stainings for S-protein alone.
Animals↗
[Mechanism of cell damage by E. coli hemolysin].
Many strains of E. coli elaborate a hemolysin which is responsible for the zone of beta-hemolysis surrounding bacterial colonies on blood agar. The significance of this cytolysin as a determinant of bacterial pathogenicity has been established in animal models with the use of genetically engineered, isogenic bacterial strains. An analogous role in human infections has been inferred from the high association of hemolysin production with disease. Studies at a molecular genetical level have defined 4 genes that are required for the synthesis, post-translational modification and secretion of the hemolysin. The structural gene hlyA encodes for a 107-110,000 polypeptide which must be modified in an unknown manner to its active form by the product of the neighboring hlyC gene. Genes hlyB and hlyD encode for proteins that export the molecule to the extracellular medium. The signal for secretion is contained in the C-terminal portion of the toxin molecule. The secreted hemolysin attacks plasma membranes of target mammalian cells by inserting as a monomer into the bilayer and generating a hydrophilic transmembrane pore of approximately 2 nm effective diameter. The pore displays a marked selectivity for cations over anions and pore-opening is dependent on the presence of a correct transmembrane potential. Binding to a membrane target does not require the presence of a specific receptor, and pores may be generated in planar lipid membranes consisting solely of phosphatidylcholine. Pore formation in nucleated cells can trigger secondary reactions such as stimulation of arachidonate metabolism with release of lipid mediators, probably initiated by passive influx of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes.
The action of the 107 kDa hemolysin from Escherichia coli on planar lipid membranes was investigated. We report that a single toxin molecule can form a cation-selective, ion-permeable channel of large conductance in a planar phospholipid bilayer membrane. The conductance of the pore is proportional to that of the bulk solution, indicating that the channel is filled with water. A pore diameter of about 2 nm can be evaluated. The pore formation mechanism is voltage-dependent and essentially resembles that of pore-forming colicins; this implies that opening of the channel is dependent on transfer of an electrical charge through the membrane. We propose that the physiological effects of E. coli hemolysin result from its ability to form ion channels in the membrane of attacked cells, and show that there is quantitative agreement between the effects of this toxin on model membranes and its hemolytic properties.
Calcium↗
Sensitive ELISA for quantitating the terminal membrane C5b-9 and fluid-phase SC5b-9 complex of human complement.
Activation of the complement system to completion results either in the generation of a pore-forming, cytolytic C5b-9(m) complex, or of a cytolytically inactive, fluid-phase SC5b-9 complex. In this paper, we describe a sensitive and reliable, sandwich ELISA for C5b-9(m) and SC5b-9, which is based on the use of a monoclonal antibody to a neoantigen of C5b-9 in combination with affinity-purified, polyclonal rabbit antibodies. The ELISA has been calibrated with purified C5b-9(m) and SC5b-9, and can detect 3 ng/ml C5b-9(m) and 20 ng/ml SC5b-9. We show that maximal conversion of C5-C9 in pooled human serum by insulin or zymosan activation generates 220 +/- 40 micrograms/ml SC5b-9. 65 of 100 normal human EDTA plasma samples analyzed in this study contained 100-600 ng/ml SC5b-9, corresponding to 0.04-0.24% of maximal conversion. Levels of circulating SC5b-9 in other donors were below the limit of detection. Incubation of serum at 37 degrees C always led to spontaneous generation of SC5b-9; concentrations ranged from 490-4725 ng/ml after 60 min, 37 degrees C, with a mean of 1848 +/- 1031 (SD) ng/ml amongst 25 donors studied. The terminal complement complex present in EDTA plasma was partially purified by PEG precipitation, DEAE-ion exchange chromatography and sucrose density gradient centrifugation, and was found to contain C8, C9 and S-protein as demonstrable by SDS-PAGE immunoblotting. Thus, the material most probably represented genuine SC5b-9. No significant age- or sex-dependent variations in SC5b-9 levels were noted. The present data call for a critical re-appraisal of several previously published methods for the determination of SC5b-9 levels in human plasma and serum.
Antibodies, Monoclonal↗
Damage to mammalian cells by proteins that form transmembrane pores.
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Animals↗
A natural auto-inhibitory factor of the terminal complement pathway in serum of Ctenodactylus gondi.
The serum of Ctenodactylus gondi, a Tunisian rodent, contains a unique inhibitor of the terminal complement pathway. The auto-inhibitor has been partially characterized as a heat-stable euglobulin that is slightly retarded on a DEAE-ion exchange column at pH 7 and elutes as a symmetrical peak on Sephacryl S-300 in the mol. wt region of approximately 200,000. The inhibitor acts by preventing attachment of cytolytic C5b-9 complexes to natural target cells. It does not appear to affect formation and function of C3-convertase, does not exert inhibitory effects at stages later than C5b-7 formation, and also does not prevent formation of SC5b-9 in serum. That the factor prevents attachment of C5b-7/C5b-9 to cells has been demonstrated in hemolysis model systems using sheep EA + human serum, and in the C3-independent reactive lysis system with the use of ELISA methods and quantitative assays with radioiodinated C8. Addition of partially purified inhibitory factor to human sera or to sera of other animal species abolishes the hemolytic activities of these sera. The inhibitory factor of Gondi serum is the first inhibitor of the terminal pathway which has been shown to be capable of preventing cytolysis of cells undergoing complement attack under physiological conditions. The presence of this factor is probably partially responsible for the remarkable susceptibility of C. gondi towards bacterial and parasitic infections.
Animals↗
Haemolytic 'efficiency' of C5b-9 complexes in drug-induced immune haemolysis: role of cellular C5b-9 distribution.
We report on quantitative analyses of C5b-9 binding to target red blood cells (RBC) lysed through the action of drug (nomifensine)-dependent antibodies (ddab) and anti-RBC antibodies (warm plus cold agglutinins). Immunoradiometric assays showed that, at any given degree of haemolysis, more C5b-9 was bound to cells sensitized with anti-RBC antibodies compared to cells lysed via ddab. Thus, C5b-9 complexes deposited through the action of ddab appeared to be haemolytically more 'efficient' than those deposited via anti-RBC antibodies. However, data obtained from flow-cytometric assays and immunoelectron microscopy demonstrated a major role for the distribution (total number) of C5b-9 lesions among the cells within a given cell population. Enhanced haemolysis with a given total amount of C5b-9 complexes appeared to be derived from a more even distribution of these complexes among the cells rather than from higher lytic efficiency of individual complexes. Since ddab bind with low affinity to cells, we suggest that these antibodies diffuse from cell to cell and cause extensive haemolysis through the deposition of relatively few C5b-9 complexes at each site. In contrast, high affinity antibodies remain largely bound to relatively restricted sites causing deposition of larger amounts of C5b-9 on, and hence lysis of, numerically fewer cells. With respect to the net intravascular haemolytic effect, our findings may explain why complement-activating antibodies of low affinity are often more detrimental than high affinity antibodies.
Antibody Affinity↗
Mechanism of leukotriene generation in polymorphonuclear leukocytes by staphylococcal alpha-toxin.
The effects of staphylococcal alpha-toxin on arachidonic acid metabolism in rabbit polymorphonuclear leukocytes (PMNs) were investigated and compared with those of the ionophore A23187 and the chemotactic tripeptide formylmethionyl-leucyl-phenylalanine (fMLP). Sublytic amounts of alpha-toxin stimulated the release of leukotriene B4 (LTB4) in PMNs in a dose-dependent manner. The toxin was several times more potent than fMLP but was not as effective as the ionophore. Preincubation of the toxin with neutralizing antibodies abolished the effect. Extracellular calcium was strictly required for eliciting LTB4 generation. Verapamil, a calcium channel blocker, inhibited fMLP-mediated LTB4 generation but had no effect on alpha-toxin- or A23187-exposed PMNs. Agents such as trifluoperazine and N-6(aminohexyl)-5-chloro-1-naphthalene sulfonamid that interfered with calmodulin activity, however, inhibited LTB4 generation in all cases. One minute after the addition of alpha-toxin, PMNs exhibited a severalfold enhancement in passive permeability to 45Ca2+. In addition, these cells became permeable to sucrose but not to inulin or dextran. The influx pattern was consistent with the previous observation that alpha-toxin creates discrete transmembrane channels in erythrocytes with an effective internal diameter of 2 to 3 nm. The results suggest that alpha-toxin triggers the arachidonic acid pathway in PMNs by facilitating calcium influx into the cells, possibly via transmembrane toxin pores that serve as calcium gates. Generation of arachidonic acid metabolites in PMNs by sublytic amounts of alpha-toxin may represent an important cellular reaction that generally occurs during infections with Staphylococcus aureus.
Animals↗
Formation of transmural complement pores in serum-sensitive Escherichia coli.
The binding of C9 at 0 and 37 degrees C to viable Escherichia coli K-12 cells carrying C5b-8 complexes was quantified. At low temperature, limited average binding of only 1 to 1.4 molecules of C9 per C8 molecule occurred, whereas 6 to 8 C9 molecules were bound per C8 molecule at 37 degrees C. Despite incorporation of C9 into C5b-9 complexes at 0 degrees C, these terminal complexes caused no loss of bacterial viability even when present in very large numbers (1,000 to 1,500 per CFU) on the bacterial cells. In contrast, generation of 50 to 100 C5b-9 complexes carrying multiple C9 molecules per CFU caused loss of viability. The failure of C5b-81C91 complexes to generate transmural pores was confirmed by measurements of o-nitrophenyl-beta-D-galactoside influx into the cells. Whereas treatment of C5b-8-laden cells with C9 at 32 degrees C caused virtually instantaneous influx of the marker, almost no influx was registered in cells receiving C9 at 0 degrees C. When cells carrying C5b-7 were brought into the stationary phase and given C8 and C9 at 32 degrees C, a C9-dependent disruption of the outer membrane permeability barrier immediately occurred as demonstrated by cleavage of a chromogenic substrate by periplasmic beta-lactamase. In sharp contrast, o-nitrophenyl-beta-D-galactoside influx was markedly retarded over a prolonged period, with abrupt permeability increases of the inner membrane toward this molecule being noted just before bacterial cell division occurred. We conclude that killing of E. coli requires binding of C5b-9 complexes containing C9 oligomers to the outer membrane and suggest that formation of pores in the inner membrane occurs when these complexes are "hit" by transiently forming zones of bioadhesion. Formation of the latter may be a dynamic process that is accentuated during cell division and quiescent during the stationary phase.
Blood Bactericidal Activity↗