This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: alphahemolysin (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 alpha 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 (LUV(1000)) were found to be more suitable for distinguishing between high alpha values whereas smaller ones (LUV(100)) were more appropriate for discriminating an all-or-none leakage (alpha=0) from the graded leakage with low values of alpha. While disrupting LUV(1000), CyaA caused a highly cation-selective leakage (alpha~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 (alpha approximately 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.

Department of Genetics Faculty of Science Charles University Prague Vinicná 5 CZ 128 44 Prague 2 Czech Republic

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