title: Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O.
abstract: Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3beta-hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Andrej
authors.initials: A
authors.lastname: Bavdek
authors.forename: Nelson O
authors.initials: NO
authors.lastname: Gekara
authors.forename: Dragan
authors.initials: D
authors.lastname: Priselac
authors.forename: Ion
authors.initials: I
authors.lastname: Gutiérrez Aguirre
authors.forename: Ayub
authors.initials: A
authors.lastname: Darji
authors.forename: Trinad
authors.initials: T
authors.lastname: Chakraborty
authors.forename: Peter
authors.initials: P
authors.lastname: Macek
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Lakey
authors.forename: Siegfried
authors.initials: S
authors.lastname: Weiss
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000502:physiology; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006388:Hemagglutinins / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008089:Listeria monocytogenes / Q000378:metabolism; D021962:Membrane Microdomains / Q000502:physiology; D015394:Molecular Structure; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D016062:Porosity; D011485:Protein Binding; D013261:Sterols / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D037521:Virulence Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; D006388:Hemagglutinins; D006460:Hemolysin Proteins; D008081:Liposomes; D010713:Phosphatidylcholines; D013261:Sterols; D037521:Virulence Factors; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine; C038705:hlyA protein, Listeria monocytogenes; C007740:fluorexon