Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli

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• 作者：Radovan Fiš ; er ; Ivo Konopá ; sek
• 关键词：CyaA ; HlyA ; ANTS ; DPX ; Fluorescence requenching method ; Liposome disruption
• 刊名：Biochimica et Biophysica Acta (BBA)/Biomembranes
• 出版年：2009
• 出版时间：June 2009
• 年：2009
• 卷：1788
• 期：6
• 页码：1249-1254
• 全文大小：587 K

文摘

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α  6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.