title: Complement-mediated killing of Escherichia coli: dissipation of membrane potential by a C9-derived peptide.
abstract: The molecular mechanism of complement-mediated killing of Gram-negative bacteria has yet to be resolved, but it is generally accepted that assembly of the membrane attack complex (MAC) of complement on the outer bacterial membrane is a required step. We have now investigated the effect of the MAC and its precursor complex, C5b-8, on the membrane potential (delta Em) across the inner bacterial membrane. Delta Em of whole cells was measured directly by using a lipophilic cation (tetraphenylphosphonium) that equilibrates with the potential or indirectly by measuring transport of solutes (proline and galactoside), which is dependent on delta Em. Our results indicate that the C5b-8 complex caused a transient collapse of delta Em in the absence of cell killing. Addition of C9 to allow formation of the MAC dissipated delta Em irreversibly, and the cells were killed. Since delta Em is generated across the inner membrane in Gram-negative bacteria, inner membrane vesicles were prepared and membrane potentials were generated either by adding D-lactate to energize the electron-transport chain or by creating a K+ diffusion potential with valinomycin. C9 added in the absence of earlier acting complement proteins had no effect on delta Em of isolated, actively respiring vesicles or on K+ diffusion potentials. In contrast, its C-terminal thrombin fragment (C9b), which has been shown earlier to contain the membrane-active domain of C9, efficiently collapsed delta Em in such vesicles. C9b did not require a specific receptor since it was effective on "right-side-out" and "inside-out" vesicles. These results are interpreted to indicate that a C9-derived fragment deenergizes cells and may be the causative agent for cell death.
mesh_terms: D001692:Biological Transport; D002462:Cell Membrane / Q000276:immunology / Q000502:physiology; D003186:Complement C9 / Q000276:immunology* / Q000302:isolation & purification; D004926:Escherichia coli / Q000254:growth & development / Q000276:immunology*; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials; D010446:Peptide Fragments / Q000276:immunology; D011392:Proline / Q000378:metabolism; D013045:Species Specificity
chemical_list: D003186:Complement C9; D010446:Peptide Fragments; C048666:complement C9b; D011392:Proline