Human monocytes and polymorphonuclear granulocytes (PMN) differ in their lipid species pattern and their pathologic lipidomic response upon E-LDL loading

Altered pore-forming properties of proteolytically nicked staphylococcal alpha-toxin

Staphylococcal alpha-toxin is a single-chain polypeptide with a molecular weight of 34,000 that hexamerizes in lipid bilayers to form pores of 1-1.5 nm effective diameter in membranes. We demonstrate that limited proteolysis of purified alpha-toxin with proteinase K generates a hemolytically active product that yields one major protein band of 17-18 kDa in SDS-polyacrylamide gel electrophoresis. The 17-18-kDa protein band harbors two major fragments of similar size representing the N- and C-terminal halves, which remain associated with each other in non-denaturing buffers but dissociate in 6 M urea. Dissociation in urea leads to loss of hemolytic activity. In contrast, unnicked alpha-toxin …

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 10 5 –10 6 molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca 2+ -calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clo…

PLASMA PROTEIN LOSS DURING SURGERY: BENEFICIAL EFFECTS OF ALBUMIN SUBSTITUTION

Plasma protein loss during abdominal surgery is a known phenomenon, but its possible pathophysiological relevance has remained unknown. The present study evaluates the effects of albumin substitution on systemic and local hemodynamics and cellular interactions in the mesenteric microcirculation. Rats underwent median laparotomy and exteriorization of an ileal loop for intravital microscopy of the mesenteric microcirculation. Plasma protein concentrations, systemic and local hemodynamics were recorded during the follow up period, with or without albumin substitution. Depending on the time course of plasma protein loss in control experiments, 80% of the calculated protein loss was infused dur…

Proteinaceous bacterial toxins and pathogenesis of sepsis syndrome and septic shock: the unknown connection

Influence of the terminal complement-complex on reperfusion injury, no-reflow and arrhythmias: a comparison between C6-competent and C6-deficient rabbits

Objective: The complement system has been suggested to play a role in reperfusion injury which may result from an enhanced destruction of myocardial tissue or from an impairment of reflow. We investigated the influence of the C5b-9 complement complex on infarct size, reflow and arrhythmogenesis. Methods: Twenty-eight C6-competent rabbits and 18 rabbits with congenital C6 deficiency were subjected to either 30 min or 2 h of coronary artery occlusion followed by reperfusion. C6 deficiency was confirmed by the complement titration test and immunohistology. The triphenyl tetrazolium chloride method was used to delineate infarct size. Reflow into infarcted areas was evaluated histologically afte…

Recovery of human fibroblasts from attack by the pore-forming alpha-toxin of Staphylococcus aureus.

When applied at low concentrations (10 micrograms/ml), staphylococcal alpha-toxin generates a small channel in keratinocyte and lymphocyte membranes that permits selective transmembrane flux of monovalent ions. Here we show that a moderate concentration (1-50 micrograms/ml) of alpha-toxin similarly produces a small pore in membranes of human fibroblasts. This process leads to rapid leakage of K+ and to a drop in cellular ATP to 10-20% of normal levels in 2 h. In the presence of medium supplemented with serum and at pH 7.4, the cells are able to recover from toxin attack, so that normal levels of K+ and ATP are reached after 6-8 h at 37 degrees C. The repair process is dependent on the prese…

In vivo effects of intravascularly applied Escherichia coli hemolysin: dissociation between induction of granulocytopenia and lethality in monkeys.

The effects of intravascular application of endotoxin-depleted Escherichia coli hemolysin (HlyA) was studied in rabbits and monkeys. In rabbits, bolus application of HlyA calculated to effect final blood levels of approximately 2-3 HU/ml (200-300 ng/ml) caused an acute fall of polymorphonuclear blood leukocytes to less than 20% of starting levels within 5 min. Additionally, platelet counts dropped to approximately 30% of starting levels, whereas lymphocyte counts varied considerably and seldom fell to less than 50%. Nine out ten animals that received 2-4 HU/ml toxin died within 90 min post application. These animals presented with signs of acute respiratory failure and post mortem inspectio…

Surgical procedure affects physiological parameters in rat myocardial ischemia: need for mechanical ventilation.

Several surgical approaches are being used to induce myocardial ischemia in rats. The present study investigated two different operative procedures in spontaneously breathing and mechanically ventilated rats under sham conditions. A snare around the left coronary artery (LCA) was achieved without occlusion. Left lateral thoracotomy was performed in spontaneously breathing and mechanically ventilated rats (tidal volume 8 ml/kg) with a respiratory rate of 90 strokes/min at different levels of O2 supplementation (room air and 30, 40, and 90% O2). All animals were observed for 60 min after thoracotomy. Rats operated with exteriorization of the heart through left lateral thoracotomy while breat…

Hypersusceptibility of neutrophil granulocytes towards lethal action of free fatty acids contained in enzyme-modified atherogenic low density lipoprotein.

Abstract Objective The bulk of LDL entrapped in the arterial intima is modified by hydrolytic enzymes, leading to extensive cleavage of cholesterylesters and liberation of fatty acids. The latter induce apoptosis in endothelial cells but are far less cytotoxic towards macrophages. We have compared the cytotoxic effects of enzymatically modified LDL (E-LDL) on macrophages and polymorphonuclear granulocytes (PMN). Methods and results E-LDL displayed toxicity towards PMN at far lower concentrations than towards monocyte-derived macrophages. Native or oxidized LDL had no effect. Free fatty acids contained in E-LDL were the cause of the observed toxicity, which could be mimicked by linoleic acid…

CD59 (homologous restriction factor 20), a plasma membrane protein that protects against complement C5b-9 attack, in human atherosclerotic lesions

Blood cells express a cell membrane protein, termed homologous restriction factor 20 (HRF20) and identical to CD59, that can inhibit complement C5b-9 insertion into their membranes. In this report, we investigated by immunohistochemistry whether CD59 was present on cells in human atherosclerotic lesions since membranous C5b-9(m) has been found in lesions. Using a monoclonal anti-CD59 antibody, a cellular CD59 staining pattern was apparent in nearly all lesion specimens. CD59 stain co-localised with macrophage (CD14), T lymphocyte (CD7), endothelial cell (anti-factor VIII related antigen) and smooth muscle cell cytoskeletal-specific antigens (anti-alpha actin and muscle myosin). Endothelial …

Immunopathogenesis of atherosclerosis: endotoxin accelerates atherosclerosis in rabbits on hypercholesterolemic diet.

Background—On the basis of our concept that atherosclerosis has an immunopathological background, we tested whether activation of the innate immune system influences its progression.Methods and Results—Hypercholesterolemic (0.5% wt/wt diet) rabbits received either repeated intravenous injections of endotoxin (Escherichia colilipopolysaccharide 1.25 to 2.5 μg, once per week) or a self-limiting cutaneousStaphylococcus aureusinfection with or without a quinolone antibiotic. Measured laboratory parameters, including LDL and HDL cholesterols, were similar in the different groups of hypercholesterolemic animals. All endotoxin-treated animals developed transient episodes of fever after endotoxin a…

One-step polymerase chain reaction-based typing of Helicobacter pylori vacA gene: association with gastric histopathology.

Heterogeneity of the Helicobacter pylori vacA gene may be associated with bacterial virulence and presentation. In this study, the possible correlation between vacA genotypes and gastric histopathology was investigated. Using a modified one-step polymerase chain reaction (PCR)-based method, 122 of 131 H. pylori isolates obtained from 63 of 67 patients from Germany were classified into distinct vacA genotypes according to their signal sequence (s1 or s2) and their midregion alleles (m1 or m2). A possible subtype of m1, now alluded to as m3, was identified in one-third of the isolates. Signal sequence s1 was significantly associated with higher H. pylori density but not with gastric inflammat…

Digestive vacuole of Plasmodium falciparum released during erythrocyte rupture dually activates complement and coagulation.

Abstract Severe Plasmodium falciparum malaria evolves through the interplay among capillary sequestration of parasitized erythrocytes, deregulated inflammatory responses, and hemostasis dysfunction. After rupture, each parasitized erythrocyte releases not only infective merozoites, but also the digestive vacuole (DV), a membrane-bounded organelle containing the malaria pigment hemozoin. In the present study, we report that the intact organelle, but not isolated hemozoin, dually activates the alternative complement and the intrinsic clotting pathway. Procoagulant activity is destroyed by phospholipase C treatment, indicating a critical role of phospholipid head groups exposed at the DV surfa…

Potent membrane-permeabilizing and cytocidal action of Vibrio cholerae cytolysin on human intestinal cells

Many strains of Vibrio cholerae non-O1 and O1 El Tor that cause diarrhea do not harbor genes for a known secretogenic toxin. However, these strains usually elaborate a pore-forming toxin, hitherto characterized as a hemolysin and here designated V. cholerae cytolysin, whose action on intestinal cells has not yet been described. We report that V. cholerae cytolysin binds as a monomer to Intestine 407 cells and then assembles into detergent-stable oligomers that probably represent tetra- or pentamers. Oligomer formation is accompanied by generation of small transmembrane pores that allow rapid flux of K+ but not influx of Ca2+ or propidium iodide. Pore formation is followed by irreversible AT…

Superoxide generation by human neutrophils induced by low doses of Escherichia coli hemolysin.

Escherichia coli hemolysin (Hly) was isolated from bacterial culture supernatants by polyethylene glycol precipitation and centrifugation in glycerol density gradients. The toxin preparations contained less than 1 mol of lipopolysaccharide per 10 mol of protein, and they had no fatty acids. The capacity of purified hemolysin to stimulate superoxide anion production in polymorphonuclear leukocytes was monitored kinetically in a lumimeter by using the lucigenin assay and was correlated with the kinetics of transmembrane pore formation. When applied to leukocytes suspended in protein-free buffer, very low concentrations (0.02 to 0.1 HU/ml) of the toxin strongly stimulated the production of sup…

Binding of Escherichia coli hemolysin and activation of the target cells is not receptor-dependent.

Abstract Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (HlyA) to be radioactively labeled with tritiated N-ethylmaleimide without affecting biological activity. It thus became possible to study the binding characteristics of HlyA as well as of toxin mutants in which one or both acylation sites were deleted. All toxins bound to erythrocytes and granulocytes in a nonsaturable manner. Only wild-type toxin and the lytic monoacylated mutant stimulated production of superoxide anions in granulocytes. An oxidative burst coincided with elevation of intracellular Ca2+, which was likely because of passive influx of Ca2+ through the toxin pores. Competi…

Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin.

Staphylococcus aureus alpha-toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores. During assembly, the formation of a pre-pore precedes membrane permeabilization; the latter is linked to a conformational change in the oligomer. Here, 41 single-cysteine replacement toxin mutants were thiol-specifically labelled with the polarity-sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attached to residues in the sequence 118-140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluorophore to a hydrophobic environment. Within this region, two functio…

Histidine residues near the N terminus of staphylococcal alpha-toxin as reporters of regions that are critical for oligomerization and pore formation.

Chemical modification of histidine residues in staphylococcal alpha-toxin leads to loss of functional activity. Site-directed mutants of the toxin in which each of the four histidine residues was replaced by several amino acids were therefore produced. The mutant proteins were purified and characterized. Exchange of H-259 or H-144 was sometimes tolerated without reduction in hemolytic activity. These histidine residues are thus not essential for toxin function. Exchange of H-35 and H-48, however, had marked effects. H-35 mutant toxins bound with high affinity to rabbit erythrocytes but displayed faulty oligomerization and were unable to form pores. H-48 mutant toxins also had severely impai…

Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a β-barrel structure

Summary Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at…

Isolation and characterization of a complement-activating lipid extracted from human atherosclerotic lesions.

The major characteristics of human atherosclerotic lesions are similar to those of a chronic inflammatory reaction, namely fibrosis, mesenchymal cell proliferation, the presence of resident macrophages, and cell necrosis. Atherosclerosis exhibits in addition the feature of lipid (mainly cholesterol) accumulation. The results of the present report demonstrate that a specific cholesterol-containing lipid particle present in human atherosclerotic lesions activates the complement system to completion. Thus, lipid could represent a stimulatory factor for the inflammatory reaction, whose underlying mechanistic basis may be, at least in part, complement activation. The complement-activating lipid …

Complement C6 deficiency protects against diet-induced atherosclerosis in rabbits.

Abstract —Low-density lipoprotein (LDL) can be transformed to an atherogenic moiety by nonoxidative, enzymatic degradation. Enzymatically degraded LDL induces macrophage foam cell formation, provokes release of cytokines, and also activates complement. To determine whether complement activation may contribute to atherogenesis, 6 pairs of homozygous C6-deficient rabbits and their non–C6-deficient heterozygous siblings were fed a cholesterol-rich diet for 14 weeks. Cholesterol levels and plasma lipoprotein profiles of the animals in the C6-competent and C6-deficient groups did not significantly differ, and the high density lipoprotein and LDL cholesterol ratios at the end of the experiment w…

GyrA sequence-based typing of Legionella.

Comparative sequence analysis of a 423-bp segment of the gyrA gene including a region homologous to the quinolone resistance-determining region (QRDR) of other species was evaluated as a novel typing method for Legionella strains. The study was performed with 29 reference strains representing 11 different Legionella species, with various serogroups, and with 13 clinical isolates of L. pneumophila. Pulsed-field gel electrophoresis and serotyping were employed for comparison of the clinical isolates. QRDR sequencing proved to be a highly discriminative tool for typing Legionellae, and permitted identification of species, serogroups and even different strains within serogroup 1. None of the is…

Infarct Size Measurement by Triphenyltetrazolium Chloride StainingVersus In VivoInjection of Propidium Iodide

Infarct size delineation by triphenyltetrazolium chloride (TTC) staining is dependent on sufficient reperfusion. We therefore evaluated the possibility of using propidium iodide (PI), a reagent conventionally used in flow cytometry to fluorescently stain dead cells, for infarct size analysis after short periods of reperfusion. Forty-five rabbits were subjected to either 15 min, 2 h or 4.5 h of coronary artery occlusion without reperfusion, or to 15 min, 30 min and 2 h of coronary artery occlusion followed by 30 min, 1 h and 3 h of reperfusion. Fifteen min before terminating the experiment, PI was injected into the left atrium. Patent blue violet was used to delineate the area at risk. Follo…

Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins.

Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin are well-studied prototypes of pore-forming bacterial cytotoxins. Each is produced as a water-soluble single-chain polypeptide that inserts into target membranes to form aqueous transmembrane pores. This review will compare properties of the three toxin prototypes, highlighting the similarities and also the differences in their structure, mode of binding, mechanism of pore formation, and the responses they elicit in target cells. Pore-forming toxins represent the most potent and versatile weapons with which invading microbes damage the host macroorganism.

Atherogenic properties of enzymatically degraded LDL: selective induction of MCP-1 and cytotoxic effects on human macrophages.

Abstract —The mechanisms underlying the selective accumulation of macrophages in early atherosclerotic lesions are poorly understood but are likely to be related to specific properties of altered low density lipoprotein (LDL) deposited in the subendothelium. Enzymatic, nonoxidative degradation of LDL converts the lipoprotein to a potentially atherogenic moiety, enzymatically altered LDL (E-LDL), which activates complement and is rapidly taken up by human macrophages via a scavenger receptor–dependent pathway. Immunohistological evidence indicates that E-LDL is present in an extracellular location in the early lesion. We report that E-LDL causes massive release of monocyte chemotactic prote…

Expression of Active Streptolysin O in Escherichia coli as a Maltose-Binding-Protein-Streptolysin-O Fusion Protein. The N-Terminal 70 Amino Acids are not Required for Hemolytic Activity

Streptolysin 0 (SLO) is the prototype of a family of cytolysins that consists of proteins which bind to cholesterol and form very large transmembrane pores. Structure/function studies on the pore-forming cytolysin SLO have been complicated by the proteolytic inactivation of a substantial portion of recombinant SLO (rSLO) expressed in Escherichia coli. To overcome this problem, translational fusions between the E. coli maltose-binding protein (MBP) gene and SLO were constructed, using the vectors pMAL-p2 and pMAL-c2. MBP-SLO fusion proteins were degraded if secreted into the E. coli periplasm, but intact, soluble MBP-SLO fusion proteins were produced at high levels in the cytoplasm. Active S…

Immunpathogenese der Atherosklerose

Atherosclerosis is widely regarded as a chronic inflammatory disease that develops as a consequence of entrapment of low density lipoprotein (LDL) in the arterial intima. Native LDL lacks inflammatory properties, so the lipoprotein must undergo biochemical alterations in order to become atherogenic. Modification is commonly regarded as being dangerous because it bestows inflammatory properties onto the lipoprotein. Most current models regard oxidation to be the decisive modifying event. However, we have obtained experimental evidence in support of a different concept. We propose that modification of tissue-entrapped LDL is required because it enables the lipoprotein to signal to the immune …

Mode of primary binding to target membranes and pore formation induced by Vibrio cholerae cytolysin (hemolysin).

Vibrio cholerae cytolysin (VCC) is produced by many non-choleratoxigenic strains of V. cholerae, and possibly represents a relevant pathogenicity determinant of these bacteria. The protein is secreted as a pro-toxin that is proteolytically cleaved to yield the active toxin with a molecular mass of approximately 63 kDa. We here describe a simple procedure for preparative isolation of mature VCC from bacterial culture supernatants, and present information on its mode of binding and pore formation in biological membranes. At low concentrations, toxin monomers interact with a high-affinity binding site on highly susceptible rabbit erythrocytes. This as yet unidentified binding site is absent on…

Isolation of Abiotrophia adiacens from a brain abscess which developed in a patient after neurosurgery.

ABSTRACT We report the case of a patient who developed a large brain abscess after neurosurgery. Cerebrospinal fluid from the abscess drainage yielded Abiotrophia adiacens -specific PCR products and microorganisms that were identified by conventional microbiological methods and by 16S ribosomal DNA analysis as Abiotrophia adiacens , which was formerly classified as a member of nutritionally variant streptococci.

No effect of C-reactive protein on early atherosclerosis in LDLR-/- / human C-reactive protein transgenic mice

summaryThe association between increased concentrations of C-reactive protein (CRP) and future cardiovascular events is well established. However, it is currently unclear whether this clinical observation represents an epiphenomenon or whether the pentraxin may actively promote the development of atherosclerosis. Experimental studies with knockout mice with a defect in apolipoprotein E (ApoE-/-) have been used to investigate the role of CRP in atherogenesis, but the results obtained have been contradictory so far. Since knockout mice with a defect in low density lipoprotein receptor (LDLR-/-) may represent a better model of atherogenesis compared to ApoE-/- animals, we undertook experiments…

Complement and Atherogenesis

Abstract —Complement activation occurs in temporal correlation with the subendothelial deposition of LDL during early atherogenesis, and complement also plays a pathogenetic role in promoting lesion progression. Two lesion components have been identified that may be responsible for complement activation. First, enzymatic degradation of LDL generates a derivative that can spontaneously activate complement, and enzymatically degraded LDL (E-LDL) has been detected in the lesions. Second, C-reactive protein (CRP) colocalizes with complement C5b-9, as evidenced by immunohistological studies of early atherosclerotic lesions, so the possibility exists that this acute phase protein also fulfills a…

Complement and atherogenesis: The unknown connection

The question why low-density lipoprotein (LDL) stranded in the subendothelium of arteries should acquire the proinflammatory properties that initiate and sustain atherogenesis has puzzled researchers for decades. The most popular concept contends that oxidative processes are crucial because oxidized LDL (ox-LDL) produced in vitro has atherogenic properties and small amounts of it are found in atherosclerotic lesions. Recently, a possible role for vascular infections has also been considered because infectious agents, in particular Chlamydia pneumoniae, are sometimes present in the lesions. Here, evidence is summarized for a different concept of atherogenesis, which evolves from the fact tha…

Bakterielle Exotoxine und ihre mögliche Relevanz für die Pathogenese des septischen Schocks

Die Mehrzahl menschenpathogener Bakterien produziert Exotoxine, deren primarer Angriff auf die Plasmamembran der Zielzellen gerichtet ist. Ein Hauptmechanismus fur diese Schadigung besteht in der Bildung von Poren, welche die Membran durchlochern und zum raschen Zusammenbruch des lebensnotwendigen „milieu interieur“ der Zelle fuhren. Prototypen solcher Porenbildner sind das α-Toxin von S. aureus, die Familie der sulfhydryl-aktivierten Toxine (mit Streptolysin-O als ein Hauptvertreter) sowie die in jungster Zeit entdeckte Familie der mit dem E. coli-Hamolysin verwandten Toxine [1, 2]. Bislang wurde membranschadigenden Toxinen wenig Beachtung als moglichen Pathogenitatsfaktoren geschenkt. In …

15. Mainzer Allergie-Workshop 2003

Pore-forming Staphylococcus aureus alpha-toxin triggers epidermal growth factor receptor-dependent proliferation.

Staphylococcal alpha-toxin is an archetypal killer protein that homo-oligomerizes in target cells to create small transmembrane pores. The membrane-perforating beta-barrel motif is a conserved attack element of cytolysins of Gram-positive and Gram-negative bacteria. Following the recognition that nucleated cells can survive membrane permeabilization, a profile of abundant transcripts was obtained in transiently perforated keratinocytes. Several immediate early genes were found to be upregulated, reminiscent of the cellular response to growth factors. Cell cycle analyses revealed doubling of S + G2/M phase cells 26 h post toxin treatment. Determination of cell counts uncovered that after an …

Selective permeabilization of infected host cells with pore-forming proteins provides a novel tool to study protein synthesis and viability of the intracellular apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii

Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gradually during oligomerization

Streptolysin O (SLO) is a bacterial exotoxin that binds to cell membranes containing cholesterol and then oligomerizes to form large pores. Along with rings, arc-shaped oligomers form on membranes. It has been suggested that each arc represents an incompletely assembled oligomer and constitutes a functional pore, faced on the opposite side by a free edge of the lipid membrane. We sought functional evidence in support of this idea by using an oligomerization-deficient, non-lytic mutant of SLO. This protein, which was created by chemical modification of a single mutant cysteine (T250C) with N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid, formed hybrid oligomers with active SLO on memb…

Differential role of p38 mitogen activated protein kinase for cellular recovery from attack by pore-forming S. aureus alpha-toxin or streptolysin O.

Following the observation that cells are able to recover from membrane lesions incurred by Staphylococcus aureus alpha-toxin and streptolysin O (SLO), we investigated the role of p38 in this process. p38 phosphorylation occurred in response to attack by both toxins, commencing within minutes after toxin treatment and waning after several hours. While SLO reportedly activates p38 via ASK1 and ROS, we show that this pathway does not play a major role for p38 induction in alpha-toxin-treated cells. Strikingly divergent effects of p38 blockade were noted depending on the toxin employed. In the case of alpha-toxin, inhibition of p38 within the time frame of its activation led to disruption of th…

Fatty acids liberated from low-density lipoprotein trigger endothelial apoptosis via mitogen-activated protein kinases.

Enzymatic modification of low-density lipoprotein (LDL) as it probably occurs in the arterial intima drastically increases its cytotoxicity, which could be relevant for the progression of atherosclerotic lesions. LDL was treated with a protease and cholesterylesterase to generate a derivative similar to lesional LDL, with a high content of free cholesterol and fatty acids. Exposure of endothelial cells to the enzymatically modified lipoprotein (E-LDL), but not to native or oxidized LDL, resulted in programmed cell death. Apoptosis was triggered by apoptosis signal-regulating kinase 1 dependent phosphorylation of p38. Depletion and reconstitution experiments identified free fatty acids (FFA)…

Permeabilization of the erythrocyte membrane with streptolysin O allows access to the vacuolar membrane of Plasmodium falciparum and a molecular analysis of membrane topology

Membrane Insertion of the Heptameric Staphylococcal α-Toxin Pore

Abstract Staphylococcal α-toxin forms heptameric pores on eukaryotic cells. After binding to the cell membrane in its monomeric form, the toxin first assembles into a heptameric pre-pore. Subsequently, the pre-pore transforms into the final pore by membrane insertion of an amphipathic β-barrel, which comprises the “central loop” domains of all heptamer subunits. The process of membrane insertion was analyzed here using a set of functionally altered toxin mutants. The results show that insertion may be initiated within an individual protomer when its NH2 terminus activates its central loop. The activated state is then shared with the central loops of the residual heptamer subunits, which res…

Potassium-inhibited processing of IL-1 beta in human monocytes.

Agents that deplete cells of K+ without grossly disrupting the plasma membrane were found to stimulate the cleavage of pro-interleukin (IL)-1 beta to mature IL-1 beta. Agents examined in this study included staphylococcal alpha-toxin and gramicidin, both of which selectively permeabilize plasma membranes for monovalent ions, the ionophores nigericin and valinomycin, and the Na+/K+ ATPase inhibitor ouabain. K+ depletion by brief hypotonic shock also triggered processing of pro-IL-1 beta. The central role of K+ depletion for inducing IL-1 beta maturation was demonstrated in cells permeabilized with alpha-toxin: processing of pro-IL-1 beta was totally blocked when cells were suspended in mediu…

C1-Esterase-Inhibitor Treatment at Early Reperfusion of Hemorrhagic Shock Reduces Mesentery Leukocyte Adhesion and Rolling

Objective: Complement activation probably plays a pathogenic role in multiple organ failure in shock. This study evaluates the effects of C1-esterase-inhibitor treatment on leukocyte-endothelial interaction in the mesenteric microcirculation in hemorrhagic shock. Methods: Rats underwent median laparotomy and exteriorization of an ileal loop for intravital microscopy of the mesenteric microcirculation. Volume controlled hemorrhagic shock was provoked by arterial blood withdrawal (2.5 mL/ 100 g body wt. for 60 minutes) followed by a 4-hour reperfusion period. C1-INH (100 IU/kg body wt. i.v.) or 0.9% NaCl i.v. were administered as a bolus at the beginning of reperfusion. Reperfusion time mimic…

Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186

Staphylococcal alpha-toxin, the prototype of an oligomerizing, pore-forming cytotoxin, is sensitive to biochemical modifications and cannot be labeled with biotin or fluorescein under preservation of its biological activity. In this study, we have used site-directed mutagenesis to introduce cysteine residues at positions 69, 130, and 186. Each mutant was fully and rapidly reactive with several sulfhydryl-specific reagents, indicating superficial location. Coupling of SH-groups with fluorescein-maleimide or biotin-maleimide was tolerated without loss of hemolytic activity at position 130, and the formed hexamers were visible on target cells by fluorescence microscopy and could be detected on…

Enzyme-linked immunoassay for detection of PCR-amplified DNA of legionellae in bronchoalveolar fluid.

A nonradioactive method is described that detects 10 to 100 legionellae in 1 ml of bronchoalveolar lavage fluid. DNA is purified by a proteinase K-phenol protocol or with a commercial DNA preparation kit and amplified by PCR with amplimers specific for the 16S rRNA gene of Legionella pneumophila. The upstream primer is 5' biotinylated. The amplification product is immobilized on streptavidin-coated microtiter plates. Because of the high binding capacity, no removal of nonincorporated biotin from the PCR product is required. After alkaline denaturation, the single-stranded PCR product is hybridized with a 5' digoxigenin-labeled probing oligomer. The amplification product is then detected by …

Cholesterol-Streptolysin O Interaction: An EM Study of Wild-Type and Mutant Streptolysin O

We present transmission electron microscopical data from negatively stained specimens of cholesterol following interaction with the thiol-activated bacterial toxin streptolysin O (SLO) (wild-type and a number of cysteine substitution mutants), with and without chemical modification of the cysteine residues. Two experimental systems were used, one with an aqueous suspension of cholesterol microcrystals and the other with immobilized thin planar cholesterol crystals attached to a carbon film. In both systems the wild-type SLO and two cytolytically active mutants, Cys 530 --Ala (C530A) and Ser 101 --Cys (S101C), readily generated the characteristic SLO arc- and ring-like oligomers on the surfa…

Identification of a Unique Helicobacter Species by 16S rRNA Gene Analysis in an Abdominal Abscess from a Patient with X-Linked Hypogammaglobulinemia

ABSTRACT A unique Helicobacter species, MZ640285, was isolated from a patient with X-linked hypogammaglobulinemia suffering from recurrent abdominal abscesses and was identified by 16S rRNA gene sequence analysis. In the phylogenetic tree, the isolate fell into a cluster which included Flexispira rappini , Helicobacter bilis , and Helicobacter sp. strain Mainz. Helicobacters are being increasingly recognized as pathogens in immunocompromised hosts. These fastidious bacteria are not easily cultured in the routine diagnostic laboratory, and this is the first report of their identification by 16S rRNA gene sequencing performed directly from a clinical specimen.

Evidence that clustered phosphocholine head groups serve as sites for binding and assembly of an oligomeric protein pore.

High susceptibility of rabbit erythrocytes toward the pore-forming action of staphylococcal alpha-toxin correlates with the presence of saturable, high affinity binding sites. All efforts to identify a protein or glycolipid receptor have failed, and the fact that liposomes composed solely of phosphatidylcholine are efficiently permeabilized adds to the enigma. A novel concept is advanced here to explain the puzzle. We propose that low affinity binding moieties can assume the role of high affinity binding sites due to their spatial arrangement in the membrane. Evidence is presented that phosphocholine head groups of sphingomyelin, clustered in sphingomyelin-cholesterol microdomains, serve th…

Enzymatically Degraded, Nonoxidized LDL Induces Human Vascular Smooth Muscle Cell Activation, Foam Cell Transformation, and Proliferation

Background —Enzymatic, nonoxidative modification transforms LDL to an atherogenic molecule (E-LDL) that activates complement and macrophages and is present in early atherosclerotic lesions. Methods and Results —We report on the atherogenic effects of E-LDL on human vascular smooth muscle cells (SMC). E-LDL accumulated in these cells, and this was accompanied by selective induction of monocyte chemotactic protein-1 in the absence of effects on the expression of interleukin (IL)-8, RANTES, or monocyte inflammatory proteins-1α and -β). Furthermore, E-LDL stimulated the expression of gp130, the signal-transducing chain of the IL-6 receptor (IL-6R) family, and the secretion of IL-6. E-LDL invok…

Staphylococcal alpha-toxin: formation of the heptameric pore is partially cooperative and proceeds through multiple intermediate stages.

Staphylococcal alpha-toxin is a 293 residue polypeptide that assembles into pore-forming heptamers, residues 118-140, thereby inserting to form an amphipathic beta-barrel in the lipid bilayer. Fluorometric analyses were here conducted using cysteine-substitution mutants site-specifically-labeled at positions 35 or 130 with the environmentally-sensitive fluorophore acrylodan. In conjunction with functional assays, three conformational states of the heptamer were defined, which may represent transitional configurations of the toxin molecule along its way to membrane insertion and pore formation. The first was the freshly assembled, SDS-sensitive heptamer alpha7*a, where a minor alteration in …

Simultaneous identification of campylobacters and prediction of quinolone resistance by comparative sequence analysis.

Comparative sequence analysis of a 30-bp segment in the quinolone resistance-determining region of campylobacters not only allows for the detection of base changes associated with resistance but also is a powerful tool for species identification based on silent mutations.

Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol esters, and cholesterol derivatives: a TEM study.

The Vibrio cholerae cytolysin (VCC) 63-kDa monomer has been shown to interact in aqueous suspension with cholesterol microcystals to produce a ring/pore-like heptameric oligomer approximately 8 nm in outer diameter. Transmission electron microscopy data were produced from cholesterol samples adsorbed to carbon support films, spread across the holes of holey carbon films, and negatively stained with ammonium molybdate. The VCC oligomers initially attach to the edge of the stacked cholesterol bilayers and with increasing time cover the two planar surfaces. VCC oligomers are also released into solution, with some tendency to cluster, possibly via the hydrophobic membrane-spanning domain. At th…

Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O

Plasmodium falciparum is an intracellular parasite of human red blood cells (RBCs). Like many other intracellular parasites, P. falciparum resides and develops within a parasitophorous vacuole which is bound by a membrane that separates the host cell cytoplasm from the parasite surface. Some parasite proteins are secreted into the vacuolar space and others are secreted, by an as yet poorly defined pathway, into the RBC cytosol. The transport of proteins from the parasite has been followed mainly using morphological methods. In search of an experimental system that would allow (i) dissection of the individual steps involved in transport from the parasite surface into the RBC cytosol, and (ii…

Intracoronary application of C1 esterase inhibitor improves cardiac function and reduces myocardial necrosis in an experimental model of ischemia and reperfusion.

Background Myocardial injury from ischemia can be aggravated by reperfusion of the jeopardized area. The precise underlying mechanisms have not been clearly defined, but proinflammatory events, including complement activation, leukocyte adhesion, and infiltration and release of diverse mediators, probably play important roles. The present study addresses the possibility of reducing reperfusion damage by the application of C1 esterase inhibitor (C1-INH). Methods and Results Cardioprotection by C1-INH 20 IU/kg IC was examined in a pig model with 60 minutes of coronary occlusion, followed by 120 minutes of reperfusion. C1-INH was administered during the first 5 minutes of coronary reperfusion…

Investigation of Sudan IV staining areas in aortas of infants and children: Possible prelesional stages of atherogenesis

Although atherosclerosis in infants and children is generally acknowledged, the temporal and spatial sequence of LDL insudation, modification and intimal monocyte accumulation has not been systematically studied. We have investigated herein very early stages of lesion formation in human aortas of individuals up to the age of 15 years. Aortic specimens from 61 cases (37 male, 24 female) were examined. 34 cases were1 year old, 16 cases were between 1 and 5 years old, and 11 cases were between 6 and 15 years old. Areas preselected under a dissection microscope after Sudan IV staining were investigated in depth by immunohistochemical staining for apolipoprotein B, monocytes/macrophages, smooth …

Membrane-penetrating Domain of Streptolysin O Identified by Cysteine Scanning Mutagenesis

Streptolysin O (SLO), a polypeptide of 571 amino acids, belongs to a family of highly homologous toxins that bind to cell membranes containing cholesterol and then polymerize to form large transmembrane pores. A conserved region close to the C terminus contains the single cysteine residue of SLO and has been implicated in membrane binding, which has been the only clear assignment of function to a part of the sequence. We have used a cysteine-less active mutant of SLO to introduce single cysteine residues at 19 positions distributed throughout the sequence. The cysteines were derivatized with the polarity-sensitive fluorophore acrylodan, and the fluorescence emission of the label was examine…

Plastic foil technique attenuates inflammation in mesenteric intravital microscopy.

Abstract Background. Interpretation of intravital microscopic observations is complicated by the “inflammatory”-type response to the trauma inflicted on the tissue by the surgical preparation. The present study evaluates different experimental conditions for prolonged observations of the mesenteric microcirculation in the rat. Methods. The mesentery was exteriorized through a median laparotomy and subjected to an organ bath or a modified plastic foil technique. Hemodynamic, metabolic, respiratory, and microcirculatory data were analyzed. Results. In contrast to the plastic foil technique, which yielded stable baseline values over a 5-h observation period, venular velocity and wall shear rat…

Staphylococcal α-toxin: the role of the N-terminus in formation of the heptameric pore — a fluorescence study1This work contains parts of the M.D. thesis of Judith Pongs.1

Staphylococcus aureus alpha-toxin forms heptameric pores on eukaryotic cell membranes. Assembly of the heptamer precedes formation of the transmembrane pore. The latter event depends on a conformational change that drives a centrally located stretch of 15 amino acid residues into the lipid bilayer. A second region of the molecule that has been implicated in the pre-pore to pore transition is the far N-terminus. Here, we used fluorescently labeled single cysteine replacement mutants to analyze the functional role of the far N-terminus of alpha-toxin. Pyrene attached to mutants S3C, I5C and 17C forms excimers within the toxin pore complex. This indicates that the distance of adjacent N-termin…

Early Diagnosis of Acanthamoeba Infection during Routine Cytological Examination of Cerebrospinal Fluid

ABSTRACT Early identification of Acanthamoeba in cerebrospinal fluid is mandatory to prevent fatal granulomatous amebic encephalitis. In the case presented here amebic trophozoites were detected in a routine cerebrospinal fluid sample. The antibiotic treatment and the apparently low virulence of this isolate were responsible for the benign progression of the infection.

Novel pathogenic mechanism of microbial metalloproteinases: liberation of membrane-anchored molecules in biologically active form exemplified by studies with the human interleukin-6 receptor

Certain membrane-anchored proteins, including several cytokines and cytokine receptors, can be released into cell supernatants through the action of endogenous membrane-bound metalloproteinases. The shed molecules are then able to fulfill various biological functions; for example, soluble interleukin-6 receptor (sIL-6R) can bind to bystander cells, rendering these cells sensitive to the action of IL-6. Using IL-6R as a model substrate, we report that the metalloproteinase from Serratia marcescens mimics the action of the endogenous shedding proteinase. Treatment of human monocytes with the bacterial protease led to a rapid release of sIL-6R into the supernatant. This effect was inhibitable …

Streptolysin O-permeabilized granulocytes shed L-selectin concomitantly with ceramide generation via neutral sphingomyelinase

Abstract Cleavage of membrane-associated L-selectin regulates leukocyte rolling on vascular endothelium at sites of inflammation. We report that rapid and massive shedding of L-selectin occurs from granulocytes attacked by the pore-forming bacterial toxin streptolysin O (SLO). Shedding was not induced by an SLO mutant that retained binding capacity but lacked pore-forming activity. Cells permeabilized with SLO exhibited a 1.5-fold increase in the activity of neutral sphingomyelinase, which was accompanied by increased ceramide formation. L-selectin cleavage was inducible by treatment of cells with bacterial sphingomyelinase, and also through exogenous application of a cell-permeable ceramid…

Complement and atherosclerosis—united to the point of no return?

Atherosclerosis is widely regarded as a chronic inflammatory disease that develops as a consequence of entrapment of oxidized low-density lipoprotein (LDL) in the arterial intima and its interaction with components of both innate and adaptive immunity. This article reviews the role of the complement system in the context of a different concept on atherogenesis. Arguments are forwarded in support of the contention that enzymatic and not oxidative modification of LDL is the prerequisite for transforming the lipoprotein into a moiety that is recognized by the innate immune system. In a departure from general wisdom, it is proposed that these processes are initially not pathological. To the con…

Elimination of a bacterial pore-forming toxin by sequential endocytosis and exocytosis

Staphylococcus aureus alpha-toxin is the archetype of bacterial pore forming toxins and a key virulence factor secreted by the majority of clinical isolates of S. aureus. Toxin monomers bind to target cells and oligomerize to form small beta-barrel pores in the plasma membrane. Many nucleated cells are able to repair a limited number of lesions by unknown, calcium-independent mechanisms. Here we show that cells can internalize alpha-toxin, that uptake is essential for cellular survival, and that pore-complexes are not proteolytically degraded, but returned to the extracellular milieu in the context of exosome-like structures, which we term toxosomes.

A cellular metalloproteinase activates Vibrio cholerae pro-cytolysin.

Many strains of Vibrio cholerae produce a cytolysin (VCC) that forms oligomeric transmembrane pores in animal cells. The molecule is secreted as a procytolysin (pro-VCC) of 79 kDa that must be cleaved at the N terminus to generate the active 65-kDa toxin. Processing can occur in solution, and previous studies have described the action of mature VCC thus generated. However, little is known about the properties of pro-VCC itself. In this study, it is shown that pro-VCC exist as a monomer in solution and binds as a monomer to eukaryotic cells. Bound pro-VCC can then be activated either by exogenous, extracellular, or by endogenous, cell-bound proteases. In both cases, cleavage generates the 65…

Pathogenesis of Sepsis Syndrome: Possible Relevance of Pore-Forming Bacterial Toxins

This review focuses on a group of bacterial products whose very existence is known to only a minority of clinicians, and whose potential significance as inducers of the sepsis syndrome has eluded the attention of most microbiologists. This is unfortunate because pore-forming bacterial toxins possess all the properties for contributing to the pathogenesis of local and systemic inflammatory reactions. Because pore formers generally are highly immunogenic proteins, the prospects for immune intervention are described that may eventually be of benefit to patients. The subject is therefore of interest not only from a theoretical but also from a practical point of view.

Melittin Modulates Keratinocyte Function through P2 Receptor-dependent ADAM Activation

Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecu…

Enzymatically modified nonoxidized low-density lipoprotein induces interleukin-8 in human endothelial cells: role of free fatty acids.

Background— Treatment of low-density lipoprotein (LDL) with a protease and cholesterolesterase transforms the lipoprotein to an entity that resembles lipoprotein particles in atherosclerotic lesions, which have a high content of free cholesterol, reflecting extensive de-esterification in the intima. Because de-esterification would occur beneath the endothelium, we examined the effects of enzymatically modified LDL (E-LDL) on cultured endothelial cells. Methods and Results— Incubation of endothelial cells with E-LDL provoked selective accumulation of interleukin (IL)-8 mRNA and production of the cytokine. Chemical analyses and depletion experiments indicated that the effect was caused by th…

Pathogenesis of Atherosclerosis: The Alternative Hypothesis

The concept that oxidation is the major single event underlying the transformation of LDL to a proinflammatory molecule dominates the world literature. An alternative hypothesis on the pathogenesis of atherosclerosis will be presented here. We have found that nonoxidative, enzymatic modification of LDL with ubiquitous enzymes also transforms the molecule to an atherogenic moiety. Enzymatically altered LDL (E-LDL) shares major properties in common with lipoproteins that have been isolated from atherosclerotic lesions. It activates complement and is recognized by a scavenger receptor on human macrophages, thus inducing foam cell formation. Uptake of E-LDL is accompanied by induction of MCP−1 …

Helicobacter pylori: clonal population structure and restricted transmission within families revealed by molecular typing.

ABSTRACT Helicobacter pylori infects up to 50% of the human population worldwide. The infection occurs predominantly in childhood and persists for decades or a lifetime. H. pylori is believed to be transmitted from person to person. However, tremendous genetic diversity has been reported for these bacteria. In order to gain insight into the epidemiological basis of this phenomenon, we performed molecular typing of H. pylori isolates from different families. Fifty-nine H. pylori isolates from 27 members of nine families were characterized by using restriction fragment length polymorphism analysis of five PCR-amplified genes, by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA, and …

Endotoxin accelerates atherosclerosis independent of complement activation

a Central Laboratory Animal Facility b Institute of Clinical Chemistry and Laboratory Medicine c Institute of Medical Microbiology and Hygiene, Johannes Gutenberg University, Mainz, Germany d Klinik fur Gefaschirurgie und Nierentransplantation, Heinrich Heine Universitatsklinik, Dusseldorf, Germany e Zentrum fur Medizin und Biowissenschaften, Forschungszentrum, Borstel, Germany f Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland g Department of Molecular Pathology, Graduate School of Medicine and Engineering, University of Yamanashi, Japan

Complement lysis: a hole is a hole.

recent experimental advances 21, it is now possible to produce MACs with a precise molecular composition 7 for better designed experiments. In my judgement, however, it will always be problematic to propose a single unifying mechanism for MAC action simply because MAC effects are not uniform. The reason for attempting to classify MACs as leaky patch or channel formers is a desire to wield Occam's razor and carve out the simplest unifying theory. But this razor often cuts one's throat, especially when it comes to immunological processes. A system that degranulates platelets, 'kills' such widely diverse targets as artificial liposomes, 'dead' viruses and erythrocytes, metabolically active cel…

Cysteine-Specific Radioiodination of Proteins with Fluorescein Maleimide

A protocol is described for coupling of carrier-free iodine to protein sulfhydryl groups via fluorescein maleimide. 125I is first coupled to fluorescein maleimide in the presence of chloramine T. Iodination is stopped with sodium thiosulfate, and the iodine-substituted fluorescein maleimide is reacted with free cysteines of the protein. Excess label is then removed by gel-permeation chromatography. The procedure avoids exposition of the protein to oxidative conditions and does not require purification of the labeled carrier reagent. Suitability of the method for a given protein can be evaluated spectrophotometrically without employing radioactivity. It can be applied under denaturing condit…

A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes

Depending on the size of the pores one wishes to produce in plasma membranes, the choice will probably fall on one of the three toxins discussed above. S. aureus alpha-toxin should be tried first when pores of 1-1.5 nm diameter are required. This is generally the case when Ca2+ and nucleotide dependence of a given process is being studied. If alpha-toxin does not work, this is probably due to the fact that the toxin either does not produce pores, or that the pores are too small. In this case, high concentrations of alpha-toxin should be tried. If this still does not work, we recommend the use of HlyA. When very large pores are to be created, e.g. for introduction of antibodies into the cell…

Role of C-reactive protein in atherogenesis: can the apolipoprotein E knockout mouse provide the answer?

Objective—Human C-reactive protein (CRP) was reported to accelerate atherosclerotic lesion development in male but not in female apolipoprotein E (apoE) knockout mice. Here, mice expressing rabbit CRP (rbCRP) were crossbred onto apoE knockout animals, and the effect on atherogenesis was studied.Methods and Results—Hemolytic complement activity could not be detected in apoE knockout mice. Furthermore, in contrast to human complement, neither rabbit nor human CRP complexed to modified low-density lipoprotein–activated murine complement. At 52 weeks, rbCRP levels were similar in male and female transgenic animals. Serum cholesterol levels were equivalent in female animals irrespective of rbCRP…

Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes.

We describe a novel flow cytometric method for quantifying opsonophagocytosis and killing of Staphylococcus aureus in cell-rich plasma obtained after dextran sedimentation of erythrocytes. To analyze opsonophagocytosis, phagocytes were labeled with a phycoerythrin-conjugated monoclonal antibody and were incubated with viable staphylococci containing carboxyfluorescein as a vital fluorescent dye. Phagocytosing cells assumed a dual, orange-green fluorescence. The relative numbers of bacteria associating with phagocytes could be determined by quantifying the decrease of free green fluorescent particles. A parallel incubation of fluorescent bacteria with unlabeled cell-rich plasma was performed…

Subcytocidal attack by staphylococcal alpha-toxin activates NF-kappaB and induces interleukin-8 production.

ABSTRACTFormation of transmembrane pores by staphylococcal alpha-toxin can provoke a spectrum of events depending on target cell species and toxin dose, and in certain cases, repair of the lesions has been observed. Here, we report that transcriptional processes are activated as a response of cells to low toxin doses. Exposure of monocytic (THP-1) or epithelial (ECV304) cells to 40 to 160 ng/ml alpha-toxin provoked a drop in cellular ATP level that was followed by secretion of substantial amounts of interleukin-8 (IL-8). Cells transfected with constructs comprising the proximal IL-8 promoter fused to luciferase or to green fluorescent protein cDNA exhibited enhanced reporter gene expression…

Kinetics of streptolysin O self-assembly.

Streptolysin O is a member of a family of membrane-damaging toxins that bind to cell membranes containing cholesterol and then polymerize to form large pores. We have examined the kinetics of toxin action using 125I-labelled streptolysin O. Binding of toxin monomers to membranes displays first-order kinetics and is reversible; the rate of desorption from red cells shows a marked dependence on temperature. To study oligomerization, toxin was bound to erythrocytes at 0 degrees C. Oligomer formation was then triggered by a sudden temperature shift and stopped by solubilization of membranes with deoxycholate. While at moderately high streptolysin O concentrations oligomerization behaves as a re…

Early albumin infusion improves global and local hemodynamics and reduces inflammatory response in hemorrhagic shock.

Objective To evaluate the effects of an early, short-term albumin infusion on mesenteric microcirculation and global hemodynamics in hemorrhagic shock. Design A prospective, randomized study. Setting Animal laboratory at a university medical clinic. Subjects Seventeen Sprague-Dawley rats weighing 250–400 g. Interventions The rats underwent median laparotomy and exteriorization of an ileal loop for intravital microscopy of the mesenteric microcirculation. Volume-controlled hemorrhagic shock was provoked by arterial blood withdrawal (2.5 mL/100 g body weight for 60 mins), followed by a 4-hr reperfusion period. Albumin (20%) or 0.9% NaCl was administered intravenously as a continuous infusion …

Antagonistic effects of fluconazole and 5-fluorocytosine on candidacidal action of amphotericin B in human serum

This study addressed the effects of fluconazole and 5-fluorocytosine on the candidacidal activity of amphotericin B in the presence of human serum. A Candida albicans isolate that was susceptible to all three agents according to standard testing procedures was employed. Fungicidal activity was estimated by using a flow cytometric procedure that exploited the fact that yeast cells killed by amphotericin B diminish in size and take up propidium iodide. The following findings were made. (i) Fluconazole and 5-fluorocytosine each failed to inhibit pseudohyphal formation and cell aggregation even when applied at 10 and 50 micrograms/ml, respectively, for up to 10 h. Hence, these agents were not f…

Pore formation by Vibrio cholerae cytolysin follows the same archetypical mode as beta-barrel toxins from gram-positive organisms.

Vibrio cholerae cytolysin (VCC) forms SDS-stable heptameric beta-barrel transmembrane pores in mammalian cell membranes. In contrast to structurally related pore formers of gram-positive organisms, no oligomeric prepore stage of assembly has been detected to date. In the present study, disulfide bonds were engineered to tie the pore-forming amino acid sequence to adjacent domains. In their nonreduced form, mutants were able to bind to rabbit erythrocytes and to native erythrocyte membranes suspended in PBS solution and form SDS-labile oligomers. These remained nonfunctional and represented the long-sought VCC prepores. Disulfide bond reduction in these oligomers released the pore-forming se…

Unsaturated Fatty Acids Drive Disintegrin and Metalloproteinase (ADAM)-dependent Cell Adhesion, Proliferation, and Migration by Modulating Membrane Fluidity*

The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA). Here, we show that unsaturated FFA increase ADAM-mediated substrate cleavage. We demonstrate that these alterations are not due to genuine ch…

Accumulation of Amphotericin B in Human Macrophages Enhances Activity against Aspergillus fumigatus Conidia: Quantification of Conidial Kill at the Single-Cell Level

ABSTRACT A cytofluorometric assay that allowed assessment of damage to phagocytosed Aspergillus fumigatus conidia at the single-cell level was developed. After ingestion by monocyte-derived macrophages (MDMs), conidia were reisolated by treatment of the cells with streptolysin O, a pore-forming toxin with lytic properties on mammalian cells but not on fungi. The counts obtained by staining of damaged conidia with propidium iodide and quantification by cytofluorometry correlated with colony counts. By the use of this method, we demonstrate that MDMs differentiated in vitro by low-dose granulocyte-macrophage colony-stimulating factor and gamma interferon have only a limited capacity to damage…

Oligomerization of Vibrio cholerae cytolysin yields a pentameric pore and has a dual specificity for cholesterol and sphingolipids in the target membrane.

Vibrio cholerae cytolysin permeabilizes animal cell membranes. Upon binding to the target lipid bilayer, the protein assembles into homo-oligomeric pores of an as yet unknown stoichiometry. Pore formation has been observed with model liposomes consisting of phosphatidylcholine and cholesterol, but the latter were much less susceptible to the cytolysin than were erythrocytes or intestinal epithelial cells. We here show that liposome permeabilization is strongly promoted if cholesterol is combined with sphingolipids, whereby the most pronounced effects are observed with monohexosylceramides and free ceramide. These two lipid species are prevalent in mammalian intestinal brush border membranes…

Correct oligomerization is a prerequisite for insertion of the central molecular domain of staphylococcal α-toxin into the lipid bilayer

Staphylococcal alpha-toxin is a primarily hydrophilic molecule that binds as a monomer to target membranes and then aggregates to form amphiphilic oligomers that represent water-filled transmembrane channels. Current evidence indicates that a region located in the center of the molecule inserts deeply into the bilayer. In the present study, we sought to determine whether membrane insertion was triggered by the oligomerization process, and whether insertion correlated with pore formation. Double mutants of alpha-toxin were prepared in which His-35 was replaced by Arg, and cysteine residues were introduced at positions 69, 130 and 186. Substitution of His-35 with Arg rendered the toxin molecu…

Vascular Leakage in Severe Dengue Virus Infections: A Potential Role for the Nonstructural Viral Protein NS1 and Complement

Background Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown. Methods The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patient…

Novel aspect of amphotericin B action: accumulation in human monocytes potentiates killing of phagocytosed Candida albicans.

The influence of low doses of amphotericin B on the capacity of human monocytes to kill Candida albicans was investigated. Killing rates were quantified by a novel flow cytometric assay and were found to be 37% +/- 3% (standard error of the mean) after 3 h. Preincubation of monocytes for 6 to 20 h with low concentrations of amphotericin B (0.2 microgram/ml) resulted in a markedly augmented fungicidal capacity. Enhancement of killing was 80% +/- 11% (standard error of the mean) over that by the controls. This effect did not appear to be due to amphotericin B-dependent monocyte activation; the respiratory burst and expression of human leukocyte antigen-DR were unaltered, and no stimulation of…

Possible protective role for C-reactive protein in atherogenesis: complement activation by modified lipoproteins halts before detrimental terminal sequence.

Background—Previous work indicated that enzymatically remodeled LDL (E-LDL) might activate complement in atherosclerotic lesions via a C-reactive protein (CRP)–dependent and CRP-independent pathway. We sought to substantiate this contention and determine whether both pathways drive the sequence to completion.Methods and Results—E-LDL was prepared by sequential treatment of LDL with a protease and cholesteryl esterase. Trypsin, proteinase K, cathepsin H, or plasmin was used with similar results. Functional tests were used to assess total complement hemolytic activity, and immunoassays were used to demonstrate C3 cleavage and to quantify C3a, C4a, C5a, and C5b-9. E-LDL preparations activated …

Electrophysiological evidence for heptameric stoichiometry of ion channels formed by Staphylococcus aureus alpha-toxin in planar lipid bilayers.

Staphylococcal alpha-toxin forms homo-oligomeric channels in lipid bilayers and cell membranes. Here, we report that electrophysiological monitoring of single-channel function using a derivatized cysteine substitution mutant allows accurate determination of the subunit stoichiometry of the oligomer in situ. The electrophysiological phenotype of channels formed in planar lipid bilayers with the cysteine replacement mutant I7C is equal to that of the wild type. When pores were formed with I7C, alterations of several channel properties were observed upon modification with SH reagents. Decreases in conductance then occurred that were seen only as negative voltage was applied. At the level of si…

Flow cytometric assay for estimating fungicidal activity of Amphotericin B in human serum

We describe a simple and rapid bioassay for estimating fungicidal activity of Amphotericin B in human serum using flow cytometry. The method exploits the fact that Candida albicans damaged by Amphotericin B show a decrease in size and take up propidium iodide to exhibit a red fluorescence after deoxycholate treatment. These phenomena display characteristic dose dependencies, and their assessment permits serum fungicidal activity to be broadly grouped into three categories: (1) subfungicidal; (2) fungicidal; and (3) strongly fungicidal. In normal human serum, these three categories correspond to Amphotericin B concentrations of 0 less than or equal to 0.5 micrograms/ml, 0.75-1.5 micrograms/m…

vacA Genotypes and Genetic Diversity in Clinical Isolates of Helicobacter pylori

ABSTRACT Genetic diversity in Helicobacter pylori strains may affect the function and antigenicity of virulence factors associated with bacterial infection and, ultimately, disease outcome. In this study, DNA diversity of H. pylori isolates was examined by analysis of vacA genotypes and by restriction fragment length polymorphism (RFLP) analysis of H. pylori -associated genes ( vacA , cagA , flaA , ureAB , and ureCD ). Thirty-seven H. pylori isolates from 26 patients were successfully classified into distinct vacA allelic genotypes. The signal sequence allele s1 (31 of 37) predominated over the s2 allele (6 of 37) and was significantly associated with the occurrence (past or present) of gas…

Effects of Escherichia coli hemolysin on human monocytes. Cytocidal action and stimulation of interleukin 1 release.

Abstract This study reports on the potent cytocidal and interleukin-1 releasing properties of Escherichia coli hemolysin (ECH) on human monocytes. Nanomolar concentrations of purified ECH (250-2,000 ng/ml) caused rapid and irreversible depletion of cellular ATP to levels below 20% of controls within 60 min. Subcytocidal doses (10-200 ng/ml) of ECH induced rapid release within 60-120 min of large amounts of interleukin 1 beta (IL-1 beta) from cultured monocytes. IL-1 beta release occurred in the presence of actinomycin D and cycloheximide, and was thus probably due to processing and export of intracellular IL-1 beta precursor. Incubation of toxin-producing E. coli at ratios of only 0.3-3 col…

Enzymatically hydrolyzed low-density lipoprotein modulates inflammatory responses in endothelial cells

SummaryThere is evidence that low-density lipoprotein (LDL) is modified by hydrolytic enzymes,and that the product (E-LDL) induces selective production of interleukin 8 (IL-8) in endothelial cells. Since nuclear factor-kappaB (NF-κB) is a major regulator of IL-8 transcription, we studied its activation in endothelial cells treated with E-LDL. Unexpectedly,the modified lipoprotein not only failed to activate NF-κB, but completely blocked its activation by tumour necrosis factor-alpha (TNF-α) in EA.hy926-cells, as assessed by electrophoretic mobility shift assays and immunofluorescence. Inhibition occurred upstream of NF-κB translocation, as inhibitor of NF-κB- (IκB)-phosphorylation was suppr…

Bacterial Cytolysin Perturbs Round Window Membrane Permeability Barrier In Vivo: Possible Cause of Sensorineural Hearing Loss in Acute Otitis Media

ABSTRACT The passage of radioiodinated streptolysin-O (SLO) and albumin through the round window membrane (RWM) was studied in vivo. When applied to the middle ear, SLO became quantitatively entrapped in this compartment and no passage to the cochlea occurred. However, flux of radioiodinated albumin through the toxin-damaged RWM was observed. We propose that the passage of noxious macromolecules, such as proteases, from a purulent middle-ear effusion may be facilitated by pore-forming toxins, resulting in cochlear damage and sensorineural hearing loss.

Case of fatal systemic infection with an Aureobacterium sp.: identification of isolate by 16S rRNA gene analysis

The case of a 75-year-old man who succumbed to a disseminated infection most likely caused by a species of the genus Aureobacterium is reported. Identification of the isolate was achieved by comparative 16S rRNA gene analysis. Aureobacteria are commonly found in the environment. However, only recently have they been recognized as a cause of infections including septicemia and soft tissue infections. To our knowledge, this is the first documentation of a fatal infection caused by an Aureobacterium sp.

Possible reason for preferential damage to renal tubular epithelial cells evoked by amphotericin B

An important determinant of nephrotoxicity, which is the major complication of long-term amphotericin B treatment, is dysfunction of distal tubular epithelial cells. The underlying cause for this rather selective damage to the cells is unknown. In the present investigation, it was shown that kidney epithelial cells were initially damaged by amphotericin B at concentrations of 2.5 to 10 micrograms/ml, as demonstrable by a dramatic drop in cellular K+ levels. Cells could recover from the initial toxic action of the polyene if they were kept in medium of neutral pH, and cellular K+ levels returned to normal after 6 h. However, the recovery mechanisms failed at lower pHs of 5.6 to 6.0. At low p…

Putative identification of an amphipathic alpha-helical sequence in hemolysin of Escherichia coli (HlyA) involved in transmembrane pore formation.

Abstract Escherichia coli hemolysin is a pore-forming protein belonging to the RTX toxin family. Cysteine scanning mutagenesis was performed to characterize the putative pore-forming domain of the molecule. A single cysteine residue was introduced at 48 positions within the sequence spanning residues 170–400 and labeled with the polarity-sensitive dye badan. Spectrofluorimetric analyses indicated that several amino acids in this domain are inserted into the lipid bilayer during pore formation. An amphipathic α-helix spanning residues 272–298 was identified that may line the aqueous pore. The importance of this sequence was highlighted by the introduction of two prolines at positions 284 and…

Enzymatically modified, nonoxidized LDL induces selective adhesion and transmigration of monocytes and T-lymphocytes through human endothelial cell monolayers.

Abstract —Circulating monocytes and T lymphocytes extravasate through the endothelium at sites of developing atheromatous lesions, where they tend to accumulate and mediate the progression of the disease. We have previously demonstrated the presence of an enzymatically degraded, nonoxidized form of LDL (E-LDL) in early human fatty streaks, which possesses major biological properties of an atherogenic lipoprotein. The effects of E-LDL on human endothelial cells have now been studied with respect to adhesion and transmigration of monocytes and T lymphocytes. E-LDL induced a rapid and dose-dependent selective adhesion of monocytes and T lymphocytes to endothelial cell monolayers within 30 min…

Functional size of complement and perforin pores compared by confocal laser scanning microscopy and fluorescence microphotolysis

Abstract Confocal laser scanning microscopy and fluorescence microphotolysis (also referred to as fluorescence photobleaching recovery) were employed to study the transport of hydrophilic fluorescent tracers through complement and perforin pores. By optimizing the confocal effect it was possible to determine the exclusion limit of the pores in situ, i.e. without separation of cells and tracer solution. Single-cell flux measurements by fluorescence microphotolysis yielded information on the sample population distribution of flux rates. By these means a direct comparison of complement and perforin pores was made in sheep erythrocyte membranes. In accordance with previous studies employing a v…

Presence of immunoglobulins, C3 and cytolytic C5b-9 complement components on the surface of erythrocytes from patients with β-thalassaemia/HbE disease

The occurrence of IgG, IgM, IgA, C3 and C5b-9 complement complexes on erythrocytes from 43 patients with beta-thalassaemia HbE disease was investigated. Indirect immunoradiometric assays using radioiodinated protein A were employed to quantify the individual components. We confirmed that circulating erythrocytes from thalassaemic patients contained elevated amounts of IgG, and small but significant amounts of C3. In addition, small but significant amounts of C5b-9 were detected. Levels of cell-bound IgG, C3 and C5b-9 were higher in splenectomized versus non-splenectomized patients. The presence of C5b-9 on circulating cells from five splenectomized patients was confirmed by an ELISA employi…

Alpha-toxin of Staphylococcus aureus

Alpha-toxin, the major cytotoxic agent elaborated by Staphylococcus aureus, was the first bacterial exotoxin to be identified as a pore former. The protein is secreted as a single-chain, water-soluble molecule of Mr 33,000. At low concentrations (less than 100 nM), the toxin binds to as yet unidentified, high-affinity acceptor sites that have been detected on a variety of cells including rabbit erythrocytes, human platelets, monocytes and endothelial cells. At high concentrations, the toxin additionally binds via nonspecific absorption to lipid bilayers; it can thus damage both cells lacking significant numbers of the acceptor and protein-free artificial lipid bilayers. Membrane damage occu…

Enzymatic modification of low-density lipoprotein in the arterial wall: a new role for plasmin and matrix metalloproteinases in atherogenesis.

Objective— Functionally interactive proteases of the plasminogen/plasmin and the matrix metalloproteinase (MMP) system degrade and reorganize the extracellular matrix of the vessel wall in atherosclerosis. Here we investigated whether such proteases are able to confer atherogenic properties onto low density lipoprotein by nonoxidative modification. Methods and Results— Similar to the recently described enzymatically-modified low-density lipoprotein (E-LDL), native LDL exposed to plasmin or matrix MMP-2 or MMP-9 and cholesterylester-hydrolase (CEH) showed extensive deesterification, with ratios of free cholesterol to total cholesterol rising to 0.8 compared with 0.2 in native LDL. When the …

Characterization of a Catalase-Negative Methicillin-Resistant Staphylococcus aureus Strain

ABSTRACT We describe an unusual clinical strain of catalase-negative methicillin-resistant Staphylococcus aureus sensu stricto. Sequence analysis of its catalase gene showed 99.60% identities to the catalase genes of the reference strains. A 5-base deletion, however, led to a shift of the nucleotide reading frame and a loss of the enzymatic activity.

The Participation of the Complement System in Atherosclerotic Vascular Disease

Atherosclerosis is a vascular disease of large and medium-sized arteries wherein the tunica intima becomes thickened due to lipid accumulation, mostly cholesterol and its esters, smooth muscle cell proliferation, and increased deposition of connective tissue matrix. A major risk factor in the development of this disease is hypercholesterolemia arising from elevated levels of low density lipoproteins (LDL). The earliest recognizable lesion, which may be a precursor to the fibrofatty plaque, is the fatty streak. It is predominantly composed of monocyte-derived macrophage foam cells, i.e. cells ladened with intracellular lipid droplets. Hence, a fundamental aspect of atherogenesis is the insud…

Pore-forming toxins trigger shedding of receptors for interleukin 6 and lipopolysaccharide.

Cleavage of membrane-associated proteins with the release of biologically active macromolecules is an emerging theme in biology. However, little is known about the nature and regulation of the involved proteases or about the physiological inducers of the shedding process. We here report that rapid and massive shedding of the interleukin 6 receptor (IL-6R) and the lipopolysaccharide receptor (CD14) occurs from primary and transfected cells attacked by two prototypes of pore-forming bacterial toxins, streptolysin O and Escherichia coli hemolysin. Shedding is not induced by an streptolysin O toxin mutant which retains cell binding capacity but lacks pore-forming activity. The toxin-dependent c…

Resealing of large transmembrane pores produced by streptolysin O in nucleated cells is accompanied by NF‐κB activation and downstream events

Streptolysin O (SLO), archetype of a cholesterol-binding bacterial cytolysin, forms large pores in the plasma membrane of mammalian cells. We have recently reported that when a limited number of pores are generated in a cell, they can be sealed in a Ca++-dependent process. Here, we show that resealing is followed by the release of IL-6 and IL-8 from keratinocytes and from endothelial cells, both relevant targets for SLO attack. Production of cytokines by these cells was preceded by activation of transcription factor nuclear factor kappaB, which thus emerges as a common denominator of stress responses to various pore-forming agents, including alpha-toxin of Staphylococcus aureus and compleme…

Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions

Incubation of human keratinocytes with nanomolar concentrations of Staphylococcus aureus alpha-toxin leads to irreversible depletion of cellular ATP. The toxin forms hexamers in the target cell membranes, and rapid transmembrane flux of K+, Na+, and 86Rb+ is observed. Unexpectedly, pores formed in keratinocytes through application of low but lethal doses of alpha-toxin appeared to be considerably smaller than those formed in erythrocyte membranes. They permitted neither rapid influx of Ca2+ or propidium iodide, nor efflux of carboxyfluorescein. Larger pores allowing flux of all three markers did form when the toxin was applied at high concentrations. Flux of monovalent ions and reduction in…

Enzymatically modified LDL induces cathepsin H in human monocytes: potential relevance in early atherogenesis.

Objective—Modification with proteases and cholesterylesterase transforms LDL to a moiety that resembles lipoproteins isolated from atherosclerotic lesions and possesses atherogenic properties. To identify changes in monocyte-derived foam cells laden with enzymatically modified LDL (E-LDL), we compared patterns of the most abundant transcripts in these cells after incubation with LDL or E-LDL.Methods and Results—Serial analyses of gene expression (SAGE) libraries were constructed from human monocytes after treatment with LDL or E-LDL. Several tags were differentially expressed in LDL-treated versus E-LDL–treated cells, whereby marked selective induction by E-LDL of cathepsin H was conspicuou…

Vibrio cholerae cytolysin: assembly and membrane insertion of the oligomeric pore are tightly linked and are not detectably restricted by membrane fluidity

AbstractHemolytic strains of Vibrio cholerae secrete a cytolysin that, upon binding as a monomer, forms pentameric pores in animal cell membranes. Pore formation is inhibited at low temperature and in the absence of cholesterol. We here posed the following questions: firstly, can oligomerization be observed in the absence of pore formation? Secondly, is membrane fluidity responsible for the effect of temperature or of cholesterol upon pore formation? The first issue was approached by chemical cross-linking, by electrophoretic heteromer analysis, and by electron microscopy. None of these methods yielded any evidence of a non-lytic pre-pore oligomer. The second question was addressed by the u…

Staphylococcus aureus alpha-toxin attack on human platelets promotes assembly of the prothrombinase complex.

alpha-Toxin, the major cytolysin of Staphylococcus aureus, promotes blood coagulation by its attack on human platelets (Bhakdi S., Muhly, M., Mannhardt, U., Hugo, F., Klapettek, K., Mueller-Eckhardt, C., and Roka, L. (1988) J. Exp. Med. 168, 527-542). In the present study we demonstrate that toxin attack on gel-filtered human platelets initiates the assembly of prothrombinase complexes at rates up to 10-fold of controls. Treatment of platelets with 0.1 microgram/ml alpha-toxin resulted in generation of 1.4 units of thrombin/10(8) platelets. A similar rate of thrombin generation was noted when platelets were subjected to three cycles of freezing and thawing. However, the alpha-toxin-induced …

Selective killing of human monocytes and cytokine release provoked by sphingomyelinase (beta-toxin) of Staphylococcus aureus.

The best-known activity of Staphylococcus aureus sphingomyelinase C, alias beta-toxin, is as a hemolysin that provokes hot-cold lysis of erythrocytes which contain substantial amounts of sphingomyelin in the plasma membrane. Sheep erythrocytes are most susceptible, and we found that one hemolytic unit, representing the toxin concentration that elicits 50% hemolysis of 2.5 X 10(8) erythrocytes per ml, corresponds to 0.05 enzyme units or to approximately 0.25 microg of sphingomyelinase per ml. The cytotoxic action of beta-toxin on nucleated cells has not been described in any detail before, and the present investigation was undertaken to fill this information gap. We now identify beta-toxin a…

Stimulation of monokine production by lipoteichoic acids

Lipoteichoic acids (LTAs) isolated from bacterial species, including Staphylococcus aureus, Streptococcus pyogenes A, Enterococcus faecalis, Streptococcus pneumoniae, and Listeria monocytogenes, were tested for their ability to stimulate the production of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha in cultured human monocytes. LTAs from S. aureus and S. pneumoniae failed to induce monokine production when applied in the concentration range of 0.05 to 5.0 micrograms/ml. However, LTAs from several enterococcal species (0.5 to 5 micrograms/ml) induced the release of all three monokines at levels similar to those observed after lipopolysaccharide stimulation. The kinet…

Helicobacter sp. strain Mainz isolated from an AIDS patient with septic arthritis: case report and nonradioactive analysis of 16S rRNA sequence

A campylobacter-like organism was isolated from an effusion of the left knee joint of an AIDS patient 2 weeks after bacteremia with a morphologically identical organism. Amplified genomic 16S rRNA sequences were analyzed by a nonradioactive blotting technique. The closest match was found with Helicobacter fenelliae (97.7% homology). Sequence data and phenotype suggest that the isolate may represent a so far unrecognized species of the genus Helicobacter.

Pore-forming toxins activate MAPK p38 by causing loss of cellular potassium.

Mitogen activated protein kinase (MAPK) p38 has emerged as a survival protein in cells that are attacked by bacterial toxins forming small membrane pores. Activation of p38 by pore forming toxins (PFT) has been attributed to osmotic stress, but here we show that loss of K+ is likely to be the critical parameter. Several lines of evidence support this conclusion: first, osmoprotection did not prevent p38-phosphorylation in alpha-toxin-loaded cells. Second, treatment of cells with a K+ ionophore, or simple incubation in K+-free medium sufficed to cause robust p38-phosphorylation. Third, media containing high [K+] prevented p38-activation by Staphylococcus aureus alpha-toxin, Vibrio cholerae c…

Complement activation by oxidatively modified low-density lipoproteins

Background Oxidatively modified low-density lipoproteins (LDLs) have been implicated in the pathogenesis of atherosclerosis and are found in human vascular lesions. There is increasing evidence that complement activation may also play a role in atherogenesis. Activated complement proteins have been demonstrated to be present in early atherosclerotic lesions, and lipids isolated from lesions have been shown to activate complement, hence their designation as lesion complement activator (LCA). The question now arose whether oxidized LDLs would also activate complement. Material and methods The complement-activating capacity of a lesion complement activator preparation and of minimally as well …

On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety.

Combined treatment with trypsin, cholesterol esterase, and neuraminidase transforms LDL, but not HDL or VLDL, to particles with properties akin to those of lipid extracted from atherosclerotic lesions. Single or double enzyme modifications, or treatment with phospholipase C, or simple vortexing are ineffective. Triple enzyme treatment disrupts the ordered and uniform structure of LDL particles, and gives rise to the formation of inhomogeneous lipid droplets 10-200 nm in diameter with a pronounced net negative charge, but lacking significant amounts of oxidized lipid. Enzymatically modified LDL (E-LDL), but not oxidatively modified LDL (ox-LDL), is endowed with potent complement-activating c…

Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2.

Abstract We report that potassium leakage from cells leads to activation of the Ca2+-independent phospholipase A2 (iPLA2), and the latter plays a pivotal role in regulating the cleavage of pro-IL-1β by the IL-converting enzyme caspase-1 in human monocytes. K+ efflux led to increases of cellular levels of glycerophosphocholine, an unambiguous indicator of phospholipase A2 activation. Both maturation of IL-1β and formation of glycerophosphocholine were blocked by bromoenol lactone, the specific iPLA2 inhibitor. Bromoenol lactone-dependent inhibition of IL-1β processing was not due to perturbation of the export machinery for pro-IL-1β and IL-1β or to caspase-1 suppression. Conspicuously, activ…

SPHINGOLIPID TRANSPORT FROM THE TRANSGOLGI NETWORK TO THE APICAL SURFACE IN PERMEABILIZED MDCK CELLS

AbstractWe have measured the transport of de novo synthesized fluorescent analogs of sphingomyelin and glucosylceramide from the trans-Golgi network (TGN) to the apical membrane in basolaterally permeabilized Madin-Darby canine kidney (MDCK) cells. Sphingolipid transport was temperature, ATP and cytosol dependent. Introduction of bovine serum albumin (BSA), which binds fluorescent sphingolipid monomer, into the permeabilized cells, did not affect lipid transport to the apical membrane. Both fluorescent sphingomyelin and glucosylceramide analogs were localized to the lumenal bilayer leaflet of isolated TGN-derived vesicles. These results strongly suggest that both sphingolipids are transport…

Coupling of Cholesterol and Cone-shaped Lipids in Bilayers Augments Membrane Permeabilization by the Cholesterol-specific Toxins Streptolysin O and Vibrio cholerae Cytolysin

Abstract Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective asd-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide …

Identification of a putative membrane-inserted segment in the alpha-toxin of Staphylococcus aureus.

To gain a fuller understanding of the regions of the Staphylococcus aureus alpha-toxin important in pore formation, we have used Forster dipole-dipole energy transfer to demonstrate that a central glycine-rich region of alpha-toxin (the so-called "hinge" region) inserts deeply into the bilayer on association of toxin with liposomes. Mutant alpha-toxins with unique cysteine (C) residues at positions 69 and 130 [Palmer, M., et al. (1993) J. Biol. Chem. 268, 11959) were reacted with the C-specific fluorophore acrylodan, which acted as an energy donor. The chosen acceptor was N-(7-nitrobenz-2-oxa-13- diazol-4-yl)-1,2-bis(hexadecanoyl)-sn-glycero-3-phosphoethanolamin e (NBD-PE). Measurement of t…

Cytocidal effects of Escherichia coli hemolysin on human T lymphocytes.

Escherichia coli hemolysin is the prototype of a large family of pore-forming toxins produced by gram-negative organisms. Besides its known cytotoxic activities against granulocytes, monocytes, endothelial cells, and renal epithelial cells, we now demonstrate that the toxin potently kills human T lymphocytes. Evidence based on different and independent approaches indicates that lymphocidal activity is due to formation of transmembrane pores. Additionally, cells prestimulated with phytohemagglutinin respond to low doses of E. coli hemolysin with DNA fragmentation similar to that observed in cells undergoing programmed cell death. Kinetic considerations lead us to conclude that DNA degradatio…

Application of C1-Esterase Inhibitor During Reperfusion of Ischemic Myocardium

Background—Complement activation during reperfusion of ischemic myocardium augments myocardial injury, and complement inhibition with C1-esterase inhibitor (C1-INH) at the time of reperfusion exerts marked cardioprotective effects in experimental studies. Application of C1-INH in newborns, however, was recently reported to have dangerous and even lethal side effects. This study addresses the essential role of dosage in studies using C1-INH.Methods and Results—Cardioprotection by C1-INH was examined in a pig model with 60 minutes of coronary occlusion followed by 120 minutes of reperfusion. C1-INH was administered intravenously 5 to 10 minutes before coronary reperfusion without heparin at a…

Staphylococcal alpha-toxin: repair of a calcium-impermeable pore in the target cell membrane

Staphylococcal alpha-toxin forms heptameric pores that render membranes permeable for monovalent cations. The pore is formed by an amphipathic beta-barrel encompassing amino acid residues 118-140 of each subunit of the oligomer. Human fibroblasts are susceptible to alpha-toxin but are able to repair the membrane lesions. Thereby, toxin oligomers remain embedded in the plasma membrane and exposed to the extracellular medium. In this study, we sought to detect structural changes occurring in the pore-forming sequence during lesion repair. Single cysteine substitution mutants were labelled with the environmentally sensitive fluorochrome acrylodan and, after mixing with wild-type toxin, incorpo…

Endotoxin accelerates atherosclerosis in rabbits on hypercholesterolemic diet

Analysis of complement C3 activation products in human atherosclerotic lesions.

Abstract Cleavage of the complement C3 protein is essential for complement activation. Saline extracts of human atherosclerotic lesions were examined by various techniques for the presence of C3 cleavage fragments. Crossed intermediate gel immunoelectrophoresis revealed that native C3 was the predominate C3 protein in extracts and that the C3dg fragment was also detected. SDS-PAGE/ Western blot analyses of lesion extracts employing monoclonal antibodies directed at C3c and C3dg fragment determinants demonstrated molecular weight bands corresponding to the known molecular weights of all the physiologic C3 cleavage fragments, except Cab which is known to have a short half-life. After C3, the …

Interaction ofEscherichia colihemolysin with biological membranes

Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca2(+) to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating inserti…

Prions, mad cow disease, and preventive measures: a critical appraisal

In 1996 the first key epidemiological study on bovine spongiform encephalitis (BSE) appeared in the renowned journal Nature [1]. In that article it was estimated that by the year 1996, some 750,000 cows with BSE had entered the food chain in Great Britain. Accordingly, millions of people in GB must have consumed contaminated meat. That same year the first report on a new form of Creutzfeldt-Jakob disease [variant (v) CJD] manifesting in young patients appeared [26]. A connection between this disease and BSE was assumed. In view of the suspicion that the use of meat and bone meal (MBM) had led to the outbreak of BSE, feeding with MBM was banned in the year 1988. The number of new BSE infecti…

Transmembrane beta-barrel of staphylococcal alpha-toxin forms in sensitive but not in resistant cells.

Staphylococcal α-toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118–140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity coul…

Proteomic Profiling of Secreted Proteins for the Hematopoietic Support of Interleukin-Stimulated Human Umbilical Vein Endothelial Cells

Human umbilical cord vein endothelial cells (HUVECs) secrete a number of factors that greatly impact the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). These factors remain largely unknown. Here, we report on the most comprehensive proteomic profiling of the HUVEC secretome and identified 827 different secreted proteins. Two hundred and thirty-one proteins were found in all conditions, whereas 369 proteins were identified only under proinflammatory conditions following IL-1β, IL-3, and IL-6 stimulation. Thirteen proteins including complement factor b (CFb) were identified only under IL-1β and IL-3 conditions and may potentially represent HSPC prolifer…

Quantitative analysis of opsonophagocytosis and of killing of Candida albicans by human peripheral blood leukocytes by using flow cytometry

We describe a simple, rapid, automated procedure for measuring opsonophagocytosis and killing of Candida albicans by human peripheral blood leukocytes. Yeast cells are labelled by allowing uptake and cleavage of membrane-permeable bis-carboxyethyl-carboxyfluorescein pentaacetoxymethylester to its membrane-impermeable fluorescent derivative bis-carboxyethyl-carboxyfluorescein. The yeast cells are added to cell-rich plasma obtained after dextran sedimentation of erythrocytes. Opsonophagocytosis and killing are quantified by using automated fluorescent cell analysis, and the following parameters can be obtained: (i) relative percentage of phagocytes that participate in opsonophagocytosis, (ii)…

Digestive vacuoles of Plasmodium falciparum are selectively phagocytosed by and impair killing function of polymorphonuclear leukocytes.

AbstractSequestration of parasitized erythrocytes and dysregulation of the coagulation and complement system are hallmarks of severe Plasmodium falciparum malaria. A link between these events emerged through the discovery that the parasite digestive vacuole (DV), which is released together with infective merozoites into the bloodstream, dually activates the intrinsic clotting and alternative complement pathway. Complement attack occurs exclusively on the membrane of the DVs, and the question followed whether DVs might be marked for uptake by polymorphonuclear granulocytes (PMNs). We report that DVs are indeed rapidly phagocytosed by PMNs after schizont rupture in active human serum. Uptake …

Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation.

Peripheral-blood human T lymphocytes were treated with Staphylococcus aureus alpha-toxin. Membrane permeabilization was assessed by measuring efflux of K+ and Rb+ and influx of Na+, Ca2+, and propidium iodide. Cellular ATP and [3H]thymidine incorporation following lectin stimulation were measured as parameters for cell viability. Internucleosomal cleavage characteristic of programmed cell death was assessed by agarose gel electrophoresis and by quantifying low-molecular-weight, [3H]thymidine-labeled DNA fragments. Nanomolar concentrations of alpha-toxin evoked protracted, irreversible ATP depletion in both activated and resting T lymphocytes. Toxin-damaged cells also lost their ability to i…

The Streptococcal Exotoxin Streptolysin O Activates Mast Cells To Produce Tumor Necrosis Factor Alpha by p38 Mitogen-Activated Protein Kinase- and Protein Kinase C-Dependent Pathways

ABSTRACTStreptolysin O (SLO), a major virulence factor of pyogenic streptococci, binds to cholesterol in the membranes of eukaryotic cells and oligomerizes to form large transmembrane pores. While high toxin doses are rapidly cytocidal, low doses are tolerated because a limited number of lesions can be resealed. Here, we report that at sublethal doses, SLO activates primary murine bone marrow-derived mast cells to degranulate and to rapidly induce or enhance the production of several cytokine mRNAs, including tumor necrosis factor alpha (TNF-α). Mast cell-derived TNF-α plays an important protective role in murine models of acute inflammation, and the production of this cytokine was analyzed…

Isolation and characterization of a pigmentless-conidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence.

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. The factors contributing to the predominance of A. fumigatus as an opportunistic pathogen are largely unknown. Since the survival of conidia in the host is a prerequisite for establishing disease, we have been attempting to identify factors which are associated with conidia and, simultaneously, important for infection. Therefore, an A. fumigatus mutant strain (white [W]) lacking conidial pigmentation was isolated. Scanning electron microscopy revealed that conidia of the W mutant also differed in their surface morphology from those of the wild t…

Colorimetric susceptibility testing for Aspergillus fumigatus: comparison of menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and Alamar blue tests

Two colorimetric methods that use Alamar Blue or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for assaying the in vitro activities of antifungal agents have been described. We report that both tests performed similarly when the antifungal activity of amphotericin B against Candida albicans was determined. However, only the MTT test generated interpretable data when Aspergillus fumigatus was used.