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A New Model for Pore Formation by Cholesterol-Dependent Cytolysins  [PDF]
Cyril F. Reboul,James C. Whisstock,Michelle A. Dunstone
PLOS Computational Biology , 2014, DOI: doi/10.1371/journal.pcbi.1003791
Abstract: Cholesterol Dependent Cytolysins (CDCs) are important bacterial virulence factors that form large (200–300 ?) membrane embedded pores in target cells. Currently, insights from X-ray crystallography, biophysical and single particle cryo-Electron Microscopy (cryo-EM) experiments suggest that soluble monomers first interact with the membrane surface via a C-terminal Immunoglobulin-like domain (Ig; Domain 4). Membrane bound oligomers then assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits. During this rearrangement it is proposed that Domain 2, a region comprising three β-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant yet currently undetermined, conformational change. Here we address this problem through a systematic molecular modeling and structural bioinformatics approach. Our work shows that simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Support for this idea comes from analysis of published cryo-EM maps of the pneumolysin pore, available crystal structures and molecular dynamics simulations. The latter data in particular reveal that Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other. Together, our data provide new and testable insights into the mechanism of pore formation by CDCs.
The Cholesterol-Dependent Cytolysin Signature Motif: A Critical Element in the Allosteric Pathway that Couples Membrane Binding to Pore Assembly  [PDF]
Kelley J. Dowd,Rodney K. Tweten
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002787
Abstract: The cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR). The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO), is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4) to distal structural changes in domain 3 (D3) that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY), a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.
Biliary cholesterol secretion: More than a simple ABC  [cached]
Arne Dikkers, Uwe JF Tietge
World Journal of Gastroenterology , 2010,
Abstract: Biliary cholesterol secretion is a process important for 2 major disease complexes, atherosclerotic cardiovascular disease and cholesterol gallstone disease. With respect to cardiovascular disease, biliary cholesterol secretion is regarded as the final step for the elimination of cholesterol originating from cholesterol-laden macrophage foam cells in the vessel wall in a pathway named reverse cholesterol transport. On the other hand, cholesterol hypersecretion into the bile is considered the main pathophysiological determinant of cholesterol gallstone formation. This review summarizes current knowledge on the origins of cholesterol secreted into the bile as well as the relevant processes and transporters involved. Next to the established ATP-binding cassette (ABC) transporters mediating the biliary secretion of bile acids (ABCB11), phospholipids (ABCB4) and cholesterol (ABCG5/G8), special attention is given to emerging proteins that modulate or mediate biliary cholesterol secretion. In this regard, the potential impact of the phosphatidylserine flippase ATPase class I type 8B member 1, the Niemann Pick C1-like protein 1 that mediates cholesterol absorption and the high density lipoprotein cholesterol uptake receptor, scavenger receptor class B type I, is discussed.
The Cytolytic Activity of Vaginolysin Strictly Depends on Cholesterol and Is Potentiated by Human CD59  [PDF]
Milda Zilnyte,eslovas Venclovas,Aurelija Zvirbliene,Milda Pleckaityte
Toxins , 2015, DOI: 10.3390/toxins7010110
Abstract: Gardnerella vaginalis produces cytolysin vaginolysin (VLY), which has been suggested to be a contributor to bacterial vaginosis pathogenesis. VLY along with intermedilysin (ILY) from Streptococcus intermedius have been attributed to a group of cholesterol-dependent cytolysins (CDCs) whose pore-forming activity depends on human CD59 (hCD59). Here, we show that different types of cells lacking hCD59 are susceptible to VLY-mediated lysis, albeit to different extents. We analyze the effects of both hCD59 and cholesterol on VLY cytolytic activity. We show that VLY binds to cholesterol-rich membranes of non-human cells, while VLY with an impaired cholesterol recognition site retains binding to the hCD59-containing cells. We further demonstrate that cholesterol binding by VLY is sufficient to trigger the formation of oligomeric complexes on cholesterol rich-liposomes lacking hCD59. Thus, VLY may induce cell lysis following two alternative pathways. One requires only cholesterol and does not depend on hCD59. The second pathway involves hCD59 contribution similarly to ILY . Apparently, under physiological conditions VLY acts in the most effective way by accepting the assistance of hCD59.
Cholesterol-dependent hemolytic activity of Passiflora quadrangularis leaves
Yuldasheva, L.N.;Carvalho, E.B.;Catanho, M.-T.J.A.;Krasilnikov, O.V.;
Brazilian Journal of Medical and Biological Research , 2005, DOI: 10.1590/S0100-879X2005000700009
Abstract: plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. the present study demonstrates for the first time the presence of a hemolysin in the leaves of passiflora quadrangularis l. this hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. the hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. in contrast, liposomes containing phosphatidylcholine were ineffective. the passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. the data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. the properties of the passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, hplc profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.
Cholesterol-dependent hemolytic activity of Passiflora quadrangularis leaves  [cached]
Yuldasheva L.N.,Carvalho E.B.,Catanho M.-T.J.A.,Krasilnikov O.V.
Brazilian Journal of Medical and Biological Research , 2005,
Abstract: Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.
Is Almond Consumption More Effective Than Reduced Dietary Saturated Fat at Decreasing Plasma Total Cholesterol and LDL-c Levels? A Theoretical Approach  [PDF]
Rudy M. Ortiz,Steven Garcia,Arnold D. Kim
Journal of Nutrition and Metabolism , 2012, DOI: 10.1155/2012/265712
Abstract: Hypercholesterolemia can be a consequence of excessive dietary saturated fatty acid (SFA), while almond-supplemented diets can improve lipid profiles. However, the differential and independent impacts of dietary SFA and almondsupplemented diets on plasma total cholesterol (pTC) and low-density lipoprotein (pLDL-c) concentrations have not been directly compared and are not well described. We reviewed the available data to construct multiple regression analyses to theoretically assess the impact of relative almond intake (RAI) and dietary SFA on reducing pTC and pLDL-c concentrations. Strong, negative correlations between RAI and percent change in mean pTC and RAI and percent change in mean pLDL-c were detected. The relationships between percent change in mean dietary SFA, and percent change in mean pTC and mean pLDL-c were weaker and only significant for pLDL-c. The multiple regression analyses demonstrated modest improvements in the strength of the correlations for both pTC and pLDL-c . The models suggest that the increase in RAI contributes to the reduction in pTC and pLDL-c to a greater extent than a reduction in dietary SFA, but a simultaneous decrease in dietary SFA should further improve lipid profiles. 1. Introduction Chronic hypercholesterolemia is a critical risk factor in the development of cardiovascular disease (CVD), which is the most common cause of death worldwide [1–4]. Furthermore, low-density lipoprotein (LDL) is the major atherogenic lipoprotein and the primary target of cholesterol-lowering therapy because numerous clinical trials have demonstrated the efficacy of LDL-lowering therapy for reducing the risk of CVD [3, 4]. A recognized consequence of increased dietary saturated fatty acid (SFA) is hypercholesterolemia [5–11]. Conversely, diets supplemented with almonds or almond products (i.e., oil and butter) have been shown to produce a moderate, yet significant decrease in plasma total cholesterol (pTC) (3–11%) and plasma LDL cholesterol (pLDL-c) (3–18%) [12–23], which demonstrates a potential benefit from consuming almonds on improving cardiovascular health. For these reasons, studies of natural foods that have the potential to significantly improve circulating lipid profiles, especially reducing pLDL-c, are of particular importance. The nutriceutical benefits of nuts provide promise for taking a dietary approach to addressing the increasing prevalence of CVD globally. The mechanisms by which nuts and nut-supplemented diets contribute to reduced pTC and pLDL-c have not been revealed, and, given the nature of these types of studies,
Changes in Astrocyte Shape Induced by Sublytic Concentrations of the Cholesterol-Dependent Cytolysin Pneumolysin Still Require Pore-Forming Capacity  [PDF]
Christina F?rtsch,Sabrina Hupp,Jiangtao Ma,Timothy J. Mitchell,Elke Maier,Roland Benz,Asparouh I. Iliev
Toxins , 2011, DOI: 10.3390/toxins3010043
Abstract: Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin’s pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20–40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin’s lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.
Dietary cholesterol, female gender and n-3 fatty acid deficiency are more important factors in the development of non-alcoholic fatty liver disease than the saturation index of the fat
Tine M Comhair, Sonia C Garcia Caraballo, Cornelis HC Dejong, Wouter H Lamers, S Eleonore K?hler
Nutrition & Metabolism , 2011, DOI: 10.1186/1743-7075-8-4
Abstract: High-fat diets (42 en% fat), containing 0.2% cholesterol, were fed to male and female wild-type and hyperlipidemic APOE2ki C57BL/6J mice for three weeks. The fats were, in order of decreasing saturation, fractionated palm fat (fPF; ~95%), cocoa butter (CB; ~60%), olive oil (OO; ~15%), sunflower oil (SO; ~12%), and high-oleic-acid sunflower oil (hoSO; ~7%). Plasma and liver triglycerides (concentration and composition), liver inflammation (Ccl2, Cd68, Tnf-α mRNA), and infiltration of macrophages (Cd68, Cd11b immunohistochemistry) and neutrophils (Mpo) were quantified.Addition of cholesterol to a low-fat diet decreased plasma HDL and increased (V)LDL levels in APOE2ki mice. Plasma cholesterol levels in female, but not male APOE2ki mice correlated significantly with inflammation. Kupffer cells of inflamed livers were swollen. Wild-type mice refused the highly saturated fPF diet. The high-fat CB, OO, and SO diets induced hyperglycemia and a 2-fold increase in hepatic fat content in male, but not female wild-type mice (in females, hepatic fat content was similar to that in males fed a high-fat diet). All high-fat diets induced macrovesicular setatosis. APOE2ki mice were protected against high-fat diet-induced steatosis and hyperglycemia, except when fed a hoSO diet. This diet caused a 5-fold increase in liver triglyceride and mead-acid content, and an increased expression of lipogenic genes, suggesting a deficiency in poly-unsaturated fatty acids. Irrespective of the composition of the high-fat diet, oleic acid was the main triglyceride component of liver fat in wild-type and APOE2ki mouse livers. Liver inflammation was dependent on genotype (APOE2ki > wild type), gender (female > male), and cholesterol content (high > low) of the diet, but not on dietary fat composition.Dietary cholesterol plays a determining, independent role in inflammation, especially in female mice. The fatty-acid saturation of the diet hardly affected hepatic steatosis or inflammation.Non-alcoholic
Effect of cholesterol depletion on the pore dilation of TRPV1  [cached]
Jansson Erik T,Trkulja Carolina L,Ahemaiti Aikeremu,Millingen Maria
Molecular Pain , 2013, DOI: 10.1186/1744-8069-9-1
Abstract: The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.
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