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 3 -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.

Detergent-activated BAX Protein Is a Monomer

“…2). SPR has been applied to the study of membrane binding and integration of a number of pore-forming proteins and peptides (27,28), including BAX (22). Using this technique it is not necessary to label or chemically modify the protein under study.…”

IFN-β Increases Listeriolysin O-Induced Membrane Permeabilization and Death of Macrophages

“…These trigger rapid and irreversible denaturation of the structure at neutral pH at temperatures above 30°C. However, it has been recently shown that binding and permeabilization of cells is possible at neutral pH, provided the cholesterol content of the membrane is sufficiently high (10).…”

Fluxes of Ca 2+ and K + Are Required for the Listeriolysin O-Dependent Internalization Pathway of Listeria monocytogenes

“…Initially identified as a major virulence factor that is critical for intracellular survival (18,19), LLO mediates the escape of L. monocytogenes from the endocytic vacuole to the cytosol, where the bacterium replicates (18,20). While LLO displays optimal activity at an acidic pH, which facilitates the disruption of the endocytic vacuole, it is still active at neutral pH in the extracellular environment (3,21). Indeed, LLO is released by L. monocytogenes in the extracellular environment (22,23), eliciting various host cell responses.…”