Activation of the Alternative Complement Pathway by Escherichia Coli: Resistance of Bound C3b to Inactivation by C3bINA and β1H

Abstract The surfaces of activators of the alternative pathway, such as zymosan and rabbit erythrocytes, provide sites which protect bound C3b from inactivation by C3b inactivator (C3bINA) and β1H, and P,C3b,Bb from decay-dissociation by β1H. This circumstance permits surface-associated amplified generation and deposition of C3b. The capacity of a bacterial surface, if analogous to zymosan and rabbit erythrocytes, to protect and thereby accumulate increasing amounts of C3b by local circumvention of the regulatory proteins would be compatible with a unique role of the alternative pathway in host defense. The susceptibility of C3b bound to Escherichia coli to C3bINA was compared with that of C3b bound to the nonactivator, sheep erythrocytes, in the state, EAC4b,3b. E. coli W3110, killed by heating at 60°C for 60 min, activated the alternative pathway in human serum diluted 1:5 in GVB containing 2 mM Mg++ and 8 mM EGTA as demonstrated by 82% C3 and 87% B consumption. The killed E. coli were coated with C3b by incubation with purified C3, B and D, washed in GVB containing 0.04 M EDTA (GVB-EDTA) and resuspended in GVB with 0.15 mM Ca++ and 0.5 M Mg++ (GVB++). The dose responses of particle-bound C3b were established by incubating dilutions of EAC4b,3b and E. coli-C3b, respectively, with B and D in GVB++ for 60 min at 37°C, followed by centrifugation and hemolytic assay of residual fluid phase B. The relative susceptibilities of C3b bound to sheep erythrocytes and E. coli to inactivation by purified C3MNA in the presence of β1H were studied in a time-dependent fashion with amounts of particle-bound C3b that gave comparable inactivation of B by D. The purified control proteins inactivated 76% C3b on EAC4b,3b in 60 min at 37°C but reduced C3b activity on E. coli by only 10%. Similarly, when human serum diluted in GVB-EDTA was used as the source of control proteins, 96% of C3b activity on sheep erythrocytes was abolished in 60 min, whereas only 18% of C3b on E. coli was inactivated in the same time interval. Thus, the surface of E. coli, like that of two other activators of the alternative pathway, zymosan and rabbit erythrocytes, protects bound C3b from inactivation and promotes alternative pathway-dependent opsonic recognition.