Characterization of a catalase-negative methicillin-resistant Staphylococcus aureus strain.

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.

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) as the triggers of this pathway. Levels of FFA in native LDL are low and the lipoprotein is therefore not cytotoxic; enzymatic cleavage of cholesterylesters liberates FFA that can rapidly trigger an apoptosis signaling cascade in neighboring cells. Blockade of this pathway can rescue cells from death.

An hypothesis for the immunopathogenesis of atherosclerosis.

According to the textbooks, oxidative processes transform low density lipoprotein (LDL) to an atherogenic moiety. Oxidized LDL contains potentially harmful constituents that induce inflammatory responses in endothelial cells, smooth muscle cells and macrophages. The oxidation hypothesis, born 20 years ago through the work of Steinberg and colleagues [Steinberg et al. 1989], forms the basis for current discussions on the pathogenesis of atherosclerosis [Glass and Witztum 2000, Lusis 2000]. In the clinical setting, however, a puzzle relates to the failure of antioxidants to prevent and cure coronary heart disease; we here allude to just the most recent study [Brown et al. 2001]. More unobtrusively, experimental observations accumulating over the past years have laid the foundation for an alternative view on atherogenesis that will be outlined here.

Application of C1-esterase inhibitor during reperfusion of ischemic myocardium: dose-related beneficial versus detrimental effects.

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 dose of 40, 100, and 200 IU/kg body wt. Compared with the NaCl controls, C1-INH 40 IU/kg reduced myocardial injury (44.1+/-13.8% versus 76.7+/-4.6% necrosis of area at risk, P/=100 IU/kg) of C1-INH will provoke detrimental side effects, probably via its procoagulatory action.

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 coli lipopolysaccharide 1.25 to 2.5 microg, once per week) or a self-limiting cutaneous Staphylococcus aureus infection 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 administration. The extent of atherosclerosis was evaluated by computer-assisted morphometry in the aortas en face (Sudan IV) and by histology at 8 weeks after start of the experiments. Endotoxin-treated animals exhibited significantly accelerated atherosclerosis compared with control animals (141+/-38 versus 45+/-16 mm(3) total lesion volume, n=7 to 9 rabbits each, P<0.001). CONCLUSIONS: Nonspecific stimulation of the innate immune system accelerates cholesterol-induced atherosclerosis. These data support the concept that atherosclerosis has an immunopathological component and render it improbable that a single infectious agent should assume particular importance in its initiation or progression.

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 during the first 2 h of surgery, and the other 20% over the following 5 h of intravital microscopy. The control group received a continuous infusion of normal saline. Plasma protein loss was mainly due to loss of albumin. A significant increase in adherent and rolling leukocytes was observed during the course of mesenteric exteriorization, which was almost entirely reversed by albumin replacement. Albumin substitution led to stabilisation of mean arterial pressure and abdominal blood flow and also attenuated reductions in arterial base excess. Albumin infusions to replace plasma protein loss may be a simple and effective measure to attenuate microcirculatory disturbances and may be of benefit in patients undergoing abdominal surgery.

Lumen geometry of ion channels formed by Vibrio cholerae EL Tor cytolysin elucidated by nonelectrolyte exclusion.

Vibrio cholerae EL Tor cytolysin, a water-soluble protein with a molecular mass of 63 kDa, forms small pores in target cell membranes. In this communication, planar lipid bilayers under voltage clamp conditions were used to investigate the geometric properties of the pores. It was established that all cytolysin channels were inserted into membranes with the same orientation. Sharp asymmetry in the I-V curve of fully open cytolysin channels persisting at high electrolyte concentrations indicated asymmetry in the geometry of the channel lumen. Using the nonelectrolyte exclusion method, evidence was obtained that the cis opening of the channel had a larger diameter (< or = 1.9 nm) than the trans opening (< or = 1.6 nm). The channel lumen appeared constricted, with a diameter of < or = 1.2 nm. Cup-shaped lumen geometry was deduced for both channel openings, which appeared to be connected to each other via a central narrow part. The latter contributed significantly to the total electrical resistance and determined the discontinuous character of channel filling with nonelectrolytes. Comparisons of the properties of pores formed by cytolysins of two V. cholerae biotypes (EL Tor and non-O1) indicated that the two ion channels possessed a similar geometry.

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

Formation 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 following toxin treatment. Electrophoretic mobility shift and immunofluorescence assays demonstrated that IL-8 secretion was preceded by activation of NF-kappaB. Transfection experiments conducted with p65/p50 double-deficient cells showed that activation of the IL-8 promoter/reporter by toxin was absolutely dependent on NF-kappaB. In contrast, this transcription factor was not required for lesion repair. Attack of cells by low doses of a pore-forming toxin can lead to transcriptional gene activation, which is followed by production of mediators that may contribute to the initiation and propagation of inflammatory lesions.

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 Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.

Coupling of cholesterol and cone-shaped lipids in bilayers augments membrane permeabilization by the cholesterol-specific toxins streptolysin O and Vibrio cholerae cytolysin.

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 as D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal alpha-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.

Membrane insertion of the heptameric staphylococcal alpha-toxin pore. A domino-like structural transition that is allosterically modulated by the target cell membrane.

Staphylococcal alpha-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 beta-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 results in cooperative membrane penetration. This cooperation of the central loops commences while these are still remote from the lipid bilayer. Nevertheless, it is subject to modulation by the target membrane, which therefore acts across a distance much like an allosteric effector. However, while allosteric transitions usually are reversible, membrane insertion of alpha-toxin is an irreversible event, and we show here that it can proceed to completion in a domino-like fashion when triggered by as little as a single foreign atom within the entire heptamer.

Interaction of Escherichia coli hemolysin with biological membranes. A study using cysteine scanning mutagenesis.

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 insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca2(+), suggesting that Ca2(+) is not required for the toxin to enter membranes. However, binding of Ca2(+) to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca2(+) to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.

C1-esterase-inhibitor treatment at early reperfusion of hemorrhagic shock reduces mesentry 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 mimicked a "pre-hospital" phase of 30 minutes followed by a quasi "in-hospital" phase of 3.5 hours. The "in-hospital" phase was initiated by substitution of blood followed by fluid resuscitation with normal saline. RESULTS: Application of C1-INH markedly reduced rolling and adherent leukocytes to numbers approaching baseline values. Vmax and shear rate of the mesenteric microcirculation improved in both groups after reperfusion with a trend to higher values in the C1-INH group (n.s. p = 0.08). CONCLUSION: C1-INH applied in a bolus dose of 100 IU/kg body wt. i.v. abrogated enhanced leukocyte adhesion and rolling in the mesenteric microcirculation after hemorrhagic shock. Single bolus treatment with a complement inhibitor may provide clinical benefit when applied at an early stage of reperfusion during hemorrhagic shock.

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

Hemolytic 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 use of two susceptible liposome models, consisting of cholesterol admixed to bovine brain lipids and to asolectin, respectively. The two liposome species clearly differed in membrane fluidity as judged by diphenylhexatriene fluorescence polarization. Nevertheless, their permeabilization by the cytolysin decreased with temperature in a closely parallel fashion, virtually vanishing at 5 degrees C. Omission of cholesterol from the liposomes uniformly led to an increase in membrane fluidity but prevented permeabilization by the cytolysin. The effects of temperature and of cholesterol upon cytolysin activity are thus not mediated by fluidization of the target membrane. The findings of our study distinguish V. cholerae cytolysin from several previously characterized pore-forming toxins.

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

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 ceramide analog. Our data identify a novel path to the shedding process and show that activation of neutral sphingomyelinase with the generation of ceramide is an important event underlying enhanced sheddase function in cells permeabilized by a pore-forming toxin.

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 isolates were resistant to quinolones in vitro and this correlated with dissence of mutations in the QRDR region. The data show that comparative sequence analysis of a short fragment of the gyrA gene is a potentially useful tool for typing of Legionella beyond the serogroup level. It is anticipated that mutations of the QRDR may arise in Legionella as a consequence of the introduction of quinolones as the agents of choice for the treatment of infections with this agent in immunocompromised patients. The employment of QRDR-typing maybe helpful in uncovering such mutations.

Plastic foil technique attenuates inflammation in mesenteric intravital microscopy.

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 rates decreased significantly in the organ bath technique, and leukocyte adhesion to the endothelium was significantly increased. Likewise, abdominal blood flow decreased significantly by 35% and base excess declined (-10.0+/-0.4 mmol/L) in the organ bath, with reduced pco(2) (26.4+/-2.5 mm Hg vs. 33.7+/-1.1 mm Hg in plastic foil technique) due to respiratory pH compensation. CONCLUSIONS: The plastic foil technique was found clearly superior to the organ bath technique for maintenance of stable baseline metabolic, hemodynamic, and microcirculatory conditions in mesenteric intravital microscopy.

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

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 by vacA and cagA genotyping. The 16S rRNA gene exhibited little allelic variation, as expected for a unique bacterial species. In contrast, the vacA, flaA, ureAB, and lspA-glmM genes were highly polymorphic, with a mean genetic diversity of 0.83, which exceeds the levels recorded for all other bacterial species. In conjunction with PFGE, 59 H. pylori isolates could be differentiated into 21 clonal types. Each individual harbored only one clone, occasionally with a clonal variant. Identical strains were always found either between siblings or between a mother and her children. Statistical analysis revealed clonality of population structure in all isolates. The results of this study suggest the possible coexistence of a large array of clonal lineages that are evolving in each individual in isolation from one another. Transmission appears to occur primarily from mother to child and perhaps between siblings.