Transfer of phospholipid and protein into the envelope of gram-negative bacteria by liposome fusion.
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.
Tomlinson
S
S
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
Taylor
P W
PW
Luzio
J P
JP
D001692:Biological Transport; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D015938:Complement Membrane Attack Complex / Q000378:metabolism; D004755:Enterobacteriaceae / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism*; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D010710:Phosphates / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012475:Salmonella / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D015938:Complement Membrane Attack Complex; D008081:Liposomes; D010710:Phosphates; D010743:Phospholipids; D011506:Proteins; D006735:Horseradish Peroxidase; D002118:Calcium