Passive administration of monoclonal antibodies to Anthrolysin O prolong survival in mice lethally infected with <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

Background  

Bacillus anthracis has two major virulence factors: a tripartite toxin that produces lethal and edema toxins and a polyglutamic acid capsule. A recent report suggested that a toxin belonging to the cholesterol dependant cytolysin (CDC) family, anthrolysin O (ALO) was a new virulence factor for B. anthracis but subsequent studies have questioned its relevance in pathogenesis. In this study, we examined the immunogenicity of recombinant anthrolysin O (rALO) in mice.     

Anthrolysin O and fermentation products mediate the toxicity of Bacillus anthracis to lung epithelial cells under microaerobic conditions

Bacillus anthracis generates virulence factors such as lethal and edema toxins, capsule, and hemolytic proteins under conditions of reduced oxygenation. Here, we report on the acute cytotoxicity of culture supernatants (Sups) of six nonencapsulated B. anthracis strains grown till the stationary phase under static microaerobic conditions. Human small airway epithelial, umbilical vein endothelial, Caco-2, and Hep-G2 cells were found to be susceptible. Sups displayed a reduction of pH to 5.3-5.5, indicating the onset of acid anaerobic fermentation; however, low pH itself was not a major factor of toxicity. The pore-forming hemolysin, anthrolysin O (ALO), contributed to the toxicity in a concentration-dependent manner. Its effect was found to be synergistic with a metabolic product of B. anthracis, succinic acid. Cells exposed to Sups demonstrated cytoplasmic membrane blebbing, increased permeability, loss of ATP, mitochondrial membrane potential collapse, and arrest of cell respiration. The toxicity was reduced by inhibition of ALO by cholesterol, decomposition of reactive oxygen species, and inhibition of mitochondrial succinate dehydrogenase. Cell death appears to be caused by an acute primary membrane permeabilization by ALO, followed by a burst of reactive radicals from the mitochondria fuelled by the succinate, which is generated by bacteria in the hypoxic environment. This mechanism of metabolic toxicity is relevant to the late-stage conditions of hypoxia and acidosis found in anthrax patients and might operate at anatomical locations of the host deprived from oxygen supply.     

A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins

Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1?, pXO2?), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1? A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10mg pure protein per liter of culture.     

Membrane cholesterol is required for activity of <Emphasis Type="Italic">Vibrio vulnificus </Emphasis>cytolysin

Vibrio vulnificus cytolysin (VVC) forms a pore in the plasma membrane and induces cytolysis of various cells including erythrocytes, neutrophil and endothelial cells. The cytolytic activity of VVC is inhibited by exogenously added cholesterol, suggesting that membrane cholesterol might be required for VVC cytolytic activity. However, there is no direct evidence that membrane cholesterol is involved in VVC-induced cytolysis. Herein we demonstrate that membrane cholesterol is required for binding of VVC to the plasma membrane. Membrane cholesterol depletion with methyl-β-cyclodextrin inhibited VVC-induced K⁺ release, 2-deoxy glucose release and Ca²⁺ influx, which are indicators of VVC pore formation. The cholesterol depletion-induced blockage of VVC cytolysis was due to the inhibition of VVC binding to membrane. These findings suggest that interaction with cholesterol is required for activity of VVC. Hong-Nu Yu, and Young-Rae Lee have equally contributed to this study.     

Hexadecylphosphocholine interaction with lipid monolayers

The phospholipid analogue miltefosine or hexadecylphosphocholine (HePC) is a drug of high interest in the treatment for fatal visceral leishmaniasis (VL) due to Leishmania donovani particularly because of its activity by oral route. In this study, the interaction of HePC with a monolayer of beta-palmitoyl-gamma-oleyl-phosphatidylcholine (POPC) as membrane model or sterol (ergosterol or cholesterol) was investigated. At a constant pressure of 25 mN/m, the adsorption kinetics of HePC into the monolayers showed that HePC molecules are inserted into the monolayer of lipids as monomers until the critical micellar concentration (CMC). At HePC concentrations superior to the CMC, the micelles of HePC are deployed at the interface as groups of monomers into the POPC or sterol monolayer. The study of mixture of HePC/(POPC or sterol), spread at the air-water interface, shows that a simple miscibility between HePC and POPC is observed, whereas a high condensation appears between HePC and sterols showing a high affinity between HePC and sterols. In addition, HePC does not act as detergent disturbing membrane integrity.     

Hexadecylphosphocholine interaction with lipid monolayers

The phospholipid analogue miltefosine or hexadecylphosphocholine (HePC) is a drug of high interest in the treatment for fatal visceral leishmaniasis (VL) due to Leishmania donovani particularly because of its activity by oral route. In this study, the interaction of HePC with a monolayer of β-palmitoyl-γ-oleyl-phosphatidylcholine (POPC) as membrane model or sterol (ergosterol or cholesterol) was investigated. At a constant pressure of 25 mN/m, the adsorption kinetics of HePC into the monolayers showed that HePC molecules are inserted into the monolayer of lipids as monomers until the critical micellar concentration (CMC). At HePC concentrations superior to the CMC, the micelles of HePC are deployed at the interface as groups of monomers into the POPC or sterol monolayer. The study of mixture of HePC/(POPC or sterol), spread at the air-water interface, shows that a simple miscibility between HePC and POPC is observed, whereas a high condensation appears between HePC and sterols showing a high affinity between HePC and sterols. In addition, HePC does not act as detergent disturbing membrane integrity.     

Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach

Sterols are essential membrane components of eukaryotic cells and are important for membrane organization and function. Cholesterol is the most representative sterol present in higher eukaryotes. It is often found distributed non-randomly in domains or pools in biological and model membranes. Cholesterol-rich functional microdomains (lipid rafts) are often implicated in cell signaling and membrane traffic. Interestingly, lipid rafts have also recently been isolated from organisms such as yeast and Drosophila, which have ergosterol as their major sterol component. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is not very clear. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing the environment-sensitive fluorescent membrane probe DPH. Our results from steady state and time-resolved fluorescence measurements show, for the first time, differential effects of ergosterol and cholesterol toward membrane organization. These novel results are relevant in the context of lipid rafts in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

Influence of cholesterol and ergosterol on membrane dynamics using different fluorescent reporter probes

Ergosterol is an evolutionary precursor of cholesterol and is the major sterol present in lower eukaryotes. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is still at a very early stage. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing fluorescent reporter probes pyrene and TMA-DPH. These results show, for the first time, the important differences on the effect of cholesterol and ergosterol in short-range ordering (reported by TMA-DPH) and long-range dynamics (reported by pyrene). In addition, pyrene vibronic peak intensity ratio provides information on polarity of the microenvironment experienced by the probe. These novel results are relevant in the context of membrane domains in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

The role of sterols in the lipid vesicle response induced by the pore-forming agent nystatin

The influences of ergosterol and cholesterol on the activity of the nystatin were investigated experimentally in a POPC model membrane as well as theoretically. The behavior of giant unilamellar vesicles (GUVs) under osmotic stress due to the formation of transmembrane pores was observed on single vesicles at different nystatin concentrations using phase-contrast microscopy. A significant shift of the typical vesicle behavior, i.e., morphological alterations, membrane bursts, slow vesicle ruptures and explosions, towards lower nystatin concentrations was detected in the ergosterol-containing vesicles and a slight shift towards higher nystatin concentrations was detected in the cholesterol-containing membranes. In addition, the nystatin activity was shown to be significantly affected by the ergosterol membrane’s molar fraction in a non-proportional manner. The observed tension-pore behavior was interpreted using a theoretical model based on the osmotic phenomena induced by the occurrence of size-selective nystatin pores. The number of nystatin pores for different vesicle behavior was theoretically determined and the role of the different mechanical characteristics of the membrane, i.e., the membrane's expansivity and bending moduli, the line tension and the lysis tension, in the tension-pore formation process was quantified. The sterol-induced changes could not be explained adequately on the basis of the different mechanical characteristics, and were therefore interpreted mainly by the direct influences of the membrane sterols on the membrane binding, the partition and the pore-formation process of nystatin.     

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

Ostreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, beta-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3beta-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins.     

Effect of Membrane Structure on the Action of Polyenes II: Nystatin Activity along the Phase Diagram of Ergosterol- and Cholesterol-Containing POPC Membranes

Pores formed by the polyene antibiotic nystatin were studied in solvent-free lipid membranes. The membranes were formed by the tip-dip technique using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with different mol fractions (0–50%) of cholesterol or ergosterol. The effects of the mol fraction of sterol and of temperature variation (15–35°C) on the activity of the pores, their unitary conductances, lifetimes and time average conductances were studied. The results were used to analyze the behavior of nystatin channels along the phase diagrams previously reported for these lipid mixtures and to propose that membrane structure is the determinant factor for the known ergosterol/cholesterol selectivity.     

Effect of Membrane Structure on the Action of Polyenes: I. Nystatin Action in Cholesterol- and Ergosterol-Containing Membranes

A detailed and thorough characterization of nystatin-induced permeability on lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-containing ergosterol or cholesterol is presented. The results show that the same collection of transmembrane pores appears in membranes containing either sterol. The concentration range for the appearance of these pores is sterol-dependent. Another mechanism of action, membrane disruption, is also observed in ergosterol-POPC membranes. The greater potency of nystatin present in ergosterol-containing membranes cannot be explained simply by the longer opening times of its pores, as has been suggested; it is also due to an increased number of events in these membranes. The present results and those of a companion paper lead us to propose that membrane structure is the determining factor for drug selectivity in membranes with different sterols.     

Elasticity and phase behavior of DPPC membrane modulated by cholesterol, ergosterol, and ethanol

Giant vesicles formed of 1,2-dipalmitoylphosphatidylcholine (DPPC) and sterols (cholesterol or ergosterol) in water and water/ethanol solutions have been used to examine the effect of sterol composition and ethanol concentration on the area compressibility modulus (K(a)), overall mechanical behavior, vesicle morphology, and induction of lipid alkyl chain interdigitation. Our results from micropipette aspiration suggest that cholesterol and ergosterol impact the order and microstructure of the gel (L(beta)') phase DPPC membrane. At low concentration (10-15 mol%) these sterols disrupt the long-range lateral order and fluidize the membrane (K(a) approximately 300 mN/m). Then at 18 mol%, these sterols participate in the formation of a continuous cohesive liquid-ordered (L(o)) phase with a sterol-dependent membrane density (K(a) approximately 750 for DPPC/ergosterol and K(a) approximately 1100 mN/m for DPPC/cholesterol). Finally at approximately 40 mol% both cholesterol and ergosterol impart similar condensation to the membrane (K(a) approximately 1200 mN/m). Introduction of ethanol (5-25 vol%) results in drops in the magnitude of K(a), which can be substantial, and sometimes individual vesicles with lowered K(a) reveal two slopes of tension versus apparent area strain. We postulate that this behavior represents disruption of lipid-sterol intermolecular interactions and therefore the membrane becomes interdigitation prone. We find that for DPPC vesicles with sterol concentrations of 20-25 mol%, significantly more ethanol is required to induce interdigitation compared to pure DPPC vesicles; approximately 7 vol% more for ergosterol and approximately 10 vol% more for cholesterol. For lower sterol concentrations (10-15 mol%), interdigitation is offset, but by <5 vol%. These data support the idea that ergosterol and cholesterol do enhance survivability for cells exposed to high concentrations of ethanol and provide evidence that the appearance of the interdigitated (L(beta)I) phase bilayer is a major factor in the disruption of cellular activity, which typically occurs between approximately 12 and approximately 16 vol% ethanol in yeast fermentations. We summarize our findings by producing, for the first time, "elasticity/phase diagrams" over a wide range of sterol (cholesterol and ergosterol) and ethanol concentrations.     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.     

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

Ostreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins.     

Effects of the detergent sucrose monolaurate on binding of amphotericin B to sterols and its toxicity for cells

Amphotericin B (AmB) is a potent antifungal agent used to treat patients with systemic mycoses. The cytotoxicity of AmB is related to its binding to membrane sterols and its clinical usefulness is based on its greater affinity to ergosterol, the fungal sterol, compared to the mammalian cell sterol, cholesterol (1-3). Here we report that sucrose monolaurate (L.S.) decreased the binding of AmB to cholesterol without interfering with its binding to ergosterol. Furthermore, the toxicity of AmB for mouse erythrocytes (RBC) and cultured mouse fibroblasts, L-929, cells was significantly decreased by low concentrations of L.S., whereas under the same conditions, its toxicity for Candida albicans was unaffected. We observed a very good correlation between the spectroscopic and cell studies. The results reported here on the effects of L.S. on the selectivity of AmB toxicity for fungal cells compared to animal cells and the relative nontoxic nature of sugar esters suggest a potential for compounds of this type to enhance the therapeutic index of AmB.     

Cholesterol, lanosterol, and ergosterol attenuate the membrane association of LL-37(W27F) and temporin L

Sterols impart significant changes to the biophysical properties of lipid bilayers. In this regard the impact of cholesterol on membrane organization and dynamics is particularly well documented and serves for comparison with other sterols. However, the factors underlying the molecular evolution of cholesterol remain enigmatic. To this end, cholesterol attenuates membrane perturbation by the so-called antimicrobial peptides (AMPs), produced ubiquitously by eukaryotic cells to combat bacterial infections by compromising the permeability barrier function of the microbial target membranes. In the present study, we addressed the effects of cholesterol, ergosterol, and lanosterol on the membrane association of two structurally and functionally diverse AMPs viz. LL-37(F27W) and temporin L (TemL) using fluorescence spectroscopy. Interestingly, sterol concentration dependent effects on the membrane association of these peptides were observed. At X(Sterol)=0.5 cholesterol was most effective in reducing the membrane intercalation of both LL-37(F27W) and TemL, the corresponding efficiencies of the three sterols decreasing as cholesterol>lanosterol> or =ergosterol, and cholesterol>lanosterol>ergosterol. It is conceivable that part of the selection pressure for the chemical evolution of cholesterol may have derived from the ability to protect the AMP-secreting host cell from the membrane damaging action of the antimicrobial peptides.     

Bilayer thickness and thermal response of dimyristoylphosphatidylcholine unilamellar vesicles containing cholesterol, ergosterol and lanosterol: a small-angle neutron scattering study

Small-angle neutron scattering (SANS) measurements are performed on pure dimyristoyl phosphatidylcholine (DMPC) unilamellar vesicles (ULV) and those containing either 20 or 47 mol% cholesterol, ergosterol or lanosterol. From the SANS data, we were able to determine the influence of these sterols on ULV bilayer thickness and vesicle area expansion coefficients. While these parameters have been determined previously for membranes containing cholesterol, to the best of our knowledge, this is the first time such results have been presented for membranes containing the structurally related sterols, ergosterol and lanosterol. At both molar concentrations and at temperatures ranging from 10 to 45 degrees C, the addition of the different sterols leads to increases in bilayer thickness, relative to pure DMPC. We observe large differences in the influence of these sterols on the membrane thermal area expansion coefficient. All three sterols, however, produce very similar changes to membrane thickness.     

Susceptibilities of phospholipid membranes containing cholesterol or ergosterol to gramicidin and its derivative incorporated in lysophospholipid micelles

Complex formation of gramicidin (GA) and desformylgramicidin (des-GA) with sterols was investigated by measuring the intrinsic Trp fluorescence. In organic solvents, the Trp fluorescence of momeric GA was quenched upon binding either cholesterol or ergosterol, but that of monomeric des-GA was not quenched by adding cholesterol. Both dimeric GA and des-GA bound highly to ergosterol, but not to cholesterol, determined by quenching of Trp fluorescence. Furthermore, GA- and des-GA-loaded lysophosphatidylcholine micelles were incubated with phosphatidylcholine vesicles containing cholesterol or ergosterol. The results showed that both monomeric and dimeric peptides hardly bound to cholesterol incorporated into phospholipid vesicles, but markedly bound to ergosterol incorporated into the bilayer membranes. Interestingly, des-GA bound more specifically to the two sterols than GA. In addition, fluorescence resonance energy transfer analysis showed that des-GA bound more specifically to the two sterol than GA.