Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis |
| |
Authors: | Radovan Fi&scaron er |
| |
Affiliation: | Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vini?ná 5, CZ-128 44, Prague 2, Czech Republic |
| |
Abstract: | 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. |
| |
Keywords: | ANTS, 8-Aminonaphthalene-1,3,6-trisulfonic acid BLM, black lipid membranes CyaA, adenylate cyclase toxin from Bordetella pertussis DPX, p-xylene-bis-pyridinium bromide FITC, fluorescein-5-isothiocyanate HlyA, αhemolysin from Escherichia coli LUV100, LUV400, LUV1000, large unilamellar vesicles extruded through filters of 100, 400 and 1000  nm pore-size, respectively PolyB, Polymyxin B from Bacillus polymyxa |
本文献已被 ScienceDirect 等数据库收录! |
|