Studies on the mechanism of bacterial resistance to complement-mediated killing. II. C8 and C9 release C5b67 from the surface of Salmonella minnesota S218 because the terminal complex does not insert into the bacterial outer membrane

The mechanism for consumption of terminal complement components and release of bound components from the surface of serum-resistant Salmonella minnesota S218 was studied. Consumption of C8 and C9 by S218 occurred through interaction with C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8-deficient serum and washed to remove all but cell-bound C5b67. Rapid release of ¹²⁵I C5 and ¹²⁵I C7 from the membrane of S218 was dependent on binding of C8 because ¹²⁵I C5 and ¹²⁵I C7 deposition in C8D serum was stable and was twofold higher in C8D than in PNHS, and addition of purified C8 or C8 and C9 to S218 previously incubated in C8D serum caused rapid release of ¹²⁵I C5 and ¹²⁵I C7 from the organism. Analysis by sucrose density gradient ultracentrifugation of the fluid phase from the reaction of S218 and 10% PNHS revealed a peak consistent with SC5b-9, in which the C9:C7 ratio was 3.3:1, but the NaDOC extracted bound C5b-9 complex sedimented as a broad peak with C9:C7 of <1.2:1. Progressive elution of C5b67 and C5b-9 from S218 but not serum-sensitive S. minnesota Re595 was observed with incubation in buffers of increasing ionic strength. Greater than 90% of the bound counts of ¹²⁵I C5 or ¹²⁵I C9 were released from S218 by incubation in 0.1% trypsin, but only 57% of ¹²⁵I C5 and 25% of ¹²⁵I C9 were released by treatment of Re595 with trypsin. These results are consistent with the concept that C5b-9 forms on the surface of the serum-sensitive S. minnesota Re595 and the serum-resistent S. minnesota S218 in normal human serum, but the formed complex is released and is not bactericidal for S218 because it fails to insert into hydrophobic outer membrane domains.