Bacterial killing by complement. C9-mediated killing in the absence of C5b-8

Abstract

The ability of serum complement to kill Gram-negative bacteria requires assembly of the membrane attack complex (MAC) on the cell surface. The molecular events that lead to cell killing after MAC assembly are unknown. We have investigated the effect of C9 on bacterial survival in the presence and absence of its receptor, the C5b-8 complex, on the outer membrane. A fluorescence assay of the membrane potential across the inner bacterial membrane revealed that addition of C9 to cells bearing the performed C5b-8 complex caused a rapid and complete dissipation of the membrane potential. No fluorescence change was observed in serum-resistant strains of Escherichia coli. Addition of trypsin, after C9 was bound to C5b-8, did not rescue the cells from the lethal effects of C9. Furthermore, assays of cell killing kinetics and C9 binding indicate that formation of tubular poly(C9) is not required for killing. When C9 was introduced into the periplasmic space in the absence of its receptor by means of an osmotic shock procedure, cell killing occurred. Other proteins, such as C8 or serum albumin, were not toxic, and C9 was ineffective against two resistant strains. The results presented here and previously [Dankert & Esser (1986) Biochemistry 25, 1094-1100], when considered together, indicate that the 'lethal unit' in complement killing of some Gram-negative bacteria is a C9-derived product that acts by dissipation of cellular energy.

Molecular Aspects of Complement-mediated Bacterial Killing

“…Finally, Dankert and Esser (7) showed that the C-terminal half of C9, the C9b fragment, dissipated the membrane potential across respiring inner membrane (IM) vesicles, although the complete molecule had no effect. In addition, the requirement for a C9 receptor (that is, C5b-8 assembly) on the OM could be bypassed if the C9 molecule was osmotically shocked into the periplasmic space (13). This report firmly established that the bactericidal activity of complement is dependent upon C9 because none of the other terminal proteins when shocked into the periplasm elicited any cytotoxic effects.…”

“…Effect of C9 on Viability of E. coli Spheroplasts-We previously discovered that when C9 was shocked into the periplasm it killed sensitive E. coli, whereas it had no effect on respiring IM vesicles in contrast to the C9b fragment (7,13). This prompted us to ask whether periplasmic processing was required for cytotoxicity.…”

“…Serum or C9-mediated bacterial killing of bacteria was measured as published (13). Viable spheroplasts were prepared by the method of Marvin and Witholt (22) and assayed for killing as described for whole cells.…”

Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck.

“…Although some studies have suggested that CSb-8 alone may produce slow bacteriolysis (5 1 ), the binding ofmultiple molecules of C9 to the surface C5b-8 complex appear to be required for rapid and efficient killing of both E. coli and S. typhimurium (44-46, 52, 53). Sequential C9 polymerization is associated with expression ofhydrophobic binding sites (54), stable insertion in the outer membrane (55), increased outer membrane permeability (46,53,56), dissipation ofthe cytoplasmic transmembrane potential (45,53,57,58 ), and membranolysis ( 55,59). However, it is uncertain whether bacterial killing requires the formation of circularly polymerized C9 resistant to SDS (the classical ring structure of the MAC).…”

“…However, it is uncertain whether bacterial killing requires the formation of circularly polymerized C9 resistant to SDS (the classical ring structure of the MAC). Thrombin-cleaved C9, which can form linear but not circular C9 polymers, is capable of killing rough E. coli strains in vitro (58). However, mutationally altered regions of S. typhimurium outer membrane LPS, regions that render these bacteria susceptible to complement-mediated killing, show evidence of preferential deposition of tubular poly C9 (60).…”