Multimeric complement component C9 is necessary for killing of Escherichia coli J5 by terminal attack complex C5b-9

Abstract

We studied the molecular composition of the complement C5b-9 complex required for optimal killing of Escherichia coli strain J5. J5 cells were incubated in 3.3%, 6.6%, or 10.0% C8-deficient serum previously absorbed to remove specific antibody and lysozyme. This resulted in the stable deposition after washing of 310, 560, and 890 C5b67 molecules per colony-forming unit, respectively, as determined by binding of 125I-labeled C7. Organisms were then incubated with excess C8 and various amounts of 131I-labeled C9. Plots of the logarithm (base 10) of E. coli J5 cells killed (log kill) vs. C9 input were sigmoidal, confirming the multihit nature of the lethal process. When C9 was supplied in excess, 3300, 5700, and 9600 molecules of C9 were bound per organism for cells bearing 310, 560, and 890 C5b-8 complexes, respectively, leading to C9-to-C7 ratios of 11.0:1, 10.8:1, and 11.4:1 and to log kill values of 1.3, 2.1, and 3.9. However, at low inputs of C9 that lead to C9-to-C7 ratios of less than 3.3:1, no killing occurred, and this was independent of the number of C5b-9 complexes bound. Formation of multimeric C9 at C9-to-C7 ratios permissive for killing was confirmed by electron microscopy and by binding of 125I-labeled antibody with specificity for multimeric but not monomeric C9. These experiments are the first to demonstrate a biological function for C9 polymerization and suggest that multimeric C9 is necessary for optimal killing of E. coli J5 cells by C5b-9.