Transfer of preformed terminal C5b-9 complement complexes into the outer membrane of viable gram-negative bacteria: effect on viability and integrity

Abstract

An efficient fusion system between Gram-negative bacteria and liposomes incorporating detergent-extracted C5b-9 complexes has been developed that allows delivery of preformed terminal complexes to the cell envelope (Tomlinson et al., 1989b). Fusion of Salmonella minnesota Re595 and Escherichia coli 17 with C5b-9-incorporated liposomes resulted in the transfer of 1900 C5b-9 complexes to each target bacterial cell. No loss in viability of bacteria was observed following fusion, even though the deposotion of 900 complexes onto the envelope following exposure to lysozyme-free serum effected a greater than 99% loss of viability. Increased sensitivity to antibiotics normally excluded from the cell by an integral outer membrane (OM), as well as the ability of the chromogenic substrate PADAC to gain access to periplasmically located beta-lactamase, indicated that transferred C5b-9 complexes functioned as water-filled channels through the OM. A similar conclusion was drawn from measurements demonstrating the uptake by cells of the lipophilic cation tetraphenylphosphonium (bromide), a result further indicating that the membrane potential across the cytoplasmic membrane was maintained following C5b-9 transfer to the OM. Examination of S. minnesota Re595 by electron microscopy revealed no obvious difference between cells exposed to lethal concentrations of lysozyme-free serum and cells following fusion with C5b-9-incorporated liposomes. These data suggest either that there are critical sites in the OM to which liposome-delivered C5b-9 complexes are unable to gain access or that bacterial cell death is related to events occurring during polymerization of C9 on the cell surface.