The long-range objective of the proposed research is to understand the mechanisms through which Gram-negative bacteria escape complement-mediated killing. In order to achieve this goal it will be necessary to understand first how the membrane attack complex (MAC) of complement kills bacteria. Thus, our immediate objective is to elucidate the molecular mechanism of MAC induced killing of bacteria such as Escherichia coli in the absence of serum lysozyme. The reason for choosing this bacteria is the occurrence of different strains within this species that are resistant to complement-mediated killing, and therefore, pose a major threat to human health. The techniques that will be used to study mechanisms of killing include experiments on membrane potential-dependent bacterial transport by following proline transport with flow dialysis, lactose transport by filtration techniques, respiration using an oxygen electrode, and formation of ion diffusion potentials with fluorescent dyes or equilibration of lipophilic ions. The integrity of the outer and inner bacterial membrane will be studied by measuring release of periplasmic and cytoplasmic markers, integrity of the peptidoglycan layer will be followed by light scattering, and cell death will be assayed by comparing the assembly of the MAC on the outer membrane of sensitive and resistant cells and elucidating which step in the killing process, i.e., permeabilization of the outer or inner membrane, transport inhibition, protein synthesis, etc., is impaired in resistant bacteria.
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