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. JS 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 1311-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 <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 1251-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.

Interactions of complement proteins C1q and factor H with lipid A and Escherichia coli: further evidence that factor H regulates the classical complement pathway

“…In addition, C3b and iC3b opsonize both Gram-negative and Gram-positive bacteria (Joiner et al, 1986). The formation of the membrane attack complex and subsequent lysis of bacterial cells has been shown for a limited number of Gram-negative bacteria (Joiner et al, 1985). Opsonophagocytosis remains the major anti-bacterial defense mechanism.…”

Sub-minimal inhibitory concentrations of cefmetazole enhance serum bactericidal activity in vitro by amplifying poly-C9 deposition.

“…Earlier studies (8,9,27) demonstrated that for serum to exert bactericidal activity, C9 must be stably inserted into the bacterial outer membrane in the form of multimeric C9. We demonstrated the presence of multimeric or poly-C9 on CMZ-grown SH5770 by three different experimental approaches.…”

“…All panels are photographed at X 100. ment of C9 deposition on CMZ-grown organisms. More importantly, however, a significant portion ofbound C9 was present as poly-C9, which is thought to be critical for bacterial killing (8)(9)(10)(11). Thus we demonstrated at a molecular level the unique presence of polymeric C9 on genetically serum-resistant gram negative bacteria phenotypically converted to serum sensitivity by growth in subinhibitory concentrations of a ,-lactam antibiotic, CMZ.…”

“…Bacteri-cidal C9 must bind to a vulnerable spot in the cell wall, usually in the form of multimeric C9 (7)(8)(9)(10)(11)(12). The ultimate location of C9 is presumably influenced by the C3 binding site (13).…”

“…Negative controls were SH5770 incubated in HIS or in HBSS++ containing 5% goat serum before exposure to primary and secondary antibodies. Positive controls were serum-sensitive Escherichia coli J5, an organism shown by others (8) to bind multimeric C9 when incubated in serum.…”