Bavdek Andrej, Gekara Nelson O, Priselac Dragan, Gutiérrez Aguirre Ion, Darji Ayub, Chakraborty Trinad, Macek Peter, Lakey Jeremy H, Weiss Siegfried, Anderluh Gregor
Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia.
Biochemistry. 2007 Apr 10;46(14):4425-37. doi: 10.1021/bi602497g. Epub 2007 Mar 15.
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.
溶血素O（LLO）是细胞内病原体单核细胞增生李斯特菌最重要的毒力因子。其主要任务是使细菌从吞噬体空泡逃逸到细胞质中。LLO属于胆固醇依赖细胞溶素（CDC）家族，但与其他成员不同，因为它在低pH值下表现出最佳活性。在李斯特菌发病机制中，其在较高pH值下形成孔道的能力在很大程度上被忽视了。在此我们表明，膜中高胆固醇浓度可恢复LLO在高pH值下的低活性。在生理或甚至略呈碱性的pH值下，LLO以胆固醇依赖的方式与脂质膜结合。结合、插入脂质单层以及使负载钙黄绿素的脂质体通透化在胆固醇含量约高于35 mol%时达到最大值，这是脂质筏中典型的浓度范围。分隔低活性和高活性的胆固醇浓度的狭窄过渡区域表明，胆固醇不仅允许LLO与膜结合，还影响孔道形成的其他步骤。我们能够通过基于表面等离子体共振的测定检测到其中一些步骤。特别是，我们表明LLO对胆固醇的识别由胆固醇最暴露的3β-羟基决定。此外，在生理或略呈碱性的pH值下，LLO以胆固醇依赖的方式与J774细胞和人红细胞结合并使其通透化。结果清楚地表明，生理pH值下LLO的活性不可忽视，并且其在细胞进入位点远端的作用可能对李斯特菌发病机制具有重要的生理影响。
