How complement kills E. coli. II. The apparent two-hit nature of the lethal event

We have studied the nature of complement (C) action on red blood cells and E. coli with respect to the number of "hits" required for membrane damage. Our method of analysis involves adding various amounts of purified C7 or C8 to serum preparations immunochemically depleted of C7 or C8, respectively, in order to construct dose-response curves for the action of C's terminal complex. The shape of the dose-response curves reflects the single or multiple-hit nature of C action. Our method confirms that C acts on red cells by a 1-hit mechanism, whether measured by lysis or by the permeation of a small molecule. In contrast, we find with E. coli that C-mediated outer membrane damage, inner membrane damage, and killing all appear to require more than 1 hit. We have also discovered a property of E. coli that displays a nonlethal 1-hit response to C that is particularly useful in the analysis of multiple-hit dose-response curves. Simultaneous measurements of this single-hit phenomenon and the multiple-hit killing of E. coli allow us to make direct comparisons of the amount of C needed for each response. On the basis of the midpoints of the single and multiple-hit curves, C-mediated membrane damage and killing of E. coli appear to be a 2-hit process.