Tomlinson S, Taylor P W, Luzio J P
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
Biochemistry. 1989 Oct 17;28(21):8303-11. doi: 10.1021/bi00447a007.
A liposome-bacterial fusion system was developed in order to introduce preformed terminal complement complexes, C5b-9, into the outer membrane of Gram-negative bacteria. Liposomes were prepared from a total phospholipid extract of Salmonella minnesota Re595. Fusion between liposomes and Salmonella sp. or Escherichia coli 17 was dependent on time, temperature, pH, and Ca2+ and PO4- concentration. Only Salmonella sp. with attenuated LPS core regions were able to fuse efficiently with liposomes. It was demonstrated that fusion of liposomes with S. minnesota Re595 or E. coli 17 under optimum conditions resulted in (i) quantitative transfer of the self-quenching fluorescent membrane probe octadecyl rhodamine B chloride from the liposomal bilayer to the bacterial envelope, (ii) transfer of radiolabeled liposomal phospholipid to the bacterial outer membrane and its subsequent translocation to the cytoplasmic membrane, demonstrated by isolation of the bacterial membranes following fusion, and (iii) delivery of liposome-entrapped horseradish peroxidase (HRP) to the periplasmic space, confirmed by a chemiluminescent assay. Following fusion of liposomes incorporating C5b-9 complexes with S. minnesota Re595 or E. coli 17, immunological analysis of the isolated membranes revealed C5b-9 complexes located exclusively in the outer membrane.
为了将预先形成的末端补体复合物C5b - 9引入革兰氏阴性菌的外膜，开发了一种脂质体 - 细菌融合系统。脂质体由明尼苏达沙门氏菌Re595的总磷脂提取物制备。脂质体与沙门氏菌属或大肠杆菌17之间的融合取决于时间、温度、pH值以及Ca2 +和PO4 - 的浓度。只有具有减毒LPS核心区域的沙门氏菌属能够与脂质体有效融合。结果表明，在最佳条件下脂质体与明尼苏达沙门氏菌Re595或大肠杆菌17的融合导致：(i) 自猝灭荧光膜探针十八烷基罗丹明B氯化物从脂质体双层定量转移至细菌包膜；(ii) 放射性标记的脂质体磷脂转移至细菌外膜并随后转运至细胞质膜，这通过融合后分离细菌膜得以证明；(iii) 通过化学发光测定证实脂质体包裹的辣根过氧化物酶(HRP)递送至周质空间。将含有C5b - 9复合物的脂质体与明尼苏达沙门氏菌Re595或大肠杆菌17融合后，对分离膜的免疫分析显示C5b - 9复合物仅位于外膜。
