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

Abstract

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 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 al but cell bound C5b67. Rapid release of (125)I C5 and (125)I C7 from the membrane of S218 was dependent on binding of C8 because (125)I C5 and (125)I C7 deposition in C8D serum was stable and was twofold higher in C8D than in PNHA, and addition of purified C8 or C8 and C9 to S218 previously incubated in C8D serum caused rapid release of (125)I C5 and (125)I C7 from the organism. Analysis by sucrose density gradient ultracentrifugation of the fluid phase from the reaction of S218 and 10 percent 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 less than 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 percent of the bound counts of (125)I C5 or (125)I C9 were released from S218 by incubation in 0.1 percent trypsin, but only 57 percent of (125)I 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 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.

Antibodies against Tumor Cell Glycolipids and Proteins, but Not Mucins, Mediate Complement-Dependent Cytotoxicity

“…48 and 49). Comparing Salmonella minnesota with smooth (long LPS chains) and rough phenotype (short LPS chains), C3 binding and C5b-9 consumption after Ab binding were similar or increased with the smooth phenotypes but this was associated with "failure of complexes to bind hydrophobically in the outer membrane" of these smooth variants (50). Similar findings have been described for Salmonella montevideo (51) and Pseudomonas aeruginosa where serum resistance is associated with long LPS chains.…”

Lipopolysaccharide Size and Distribution Determine Serum Resistance in Salmonella montevideo

“…3, 4 2, and 3). Therefore, we postulate, based pn this and previous work (12,13,14) that a critical density (>20%) of long O-Ag side chains is required to protect the cells from serum killing and that the O-Ag protection is a result of steric hindrance of C5b-9 access to hydrophobic domains of the outer membrane.…”

“…Complement activation by smooth, enteric gram-negative bacteria is efficient, and the bactericidal C5b-9 complex is formed on the surface of such smooth cells (13). However, in contrast to the stable, hydrophobic interaction of C5b-9 with rough cells, the C5b-9 complex is bound by weak hydrophilic interactions to smooth cells and is released from the surface (14). Consequently, it is not bactericidal.…”

“…Consequently, it is not bactericidal. We and others (13,14,36) have suggested that C5b-9 complexes are shed because complement activation takes place on long O-Ag side chains and that C5b-9 complexes formed are sterically inhibited from inserting into hydrophobic membrane domains. In a recent paper (12), we provided evidence for this hypothesis by showing that C3, an earlier component in the cascade, binds preferentially to a minor subset of LPS molecules in Salmonella montevideo bearing the longest O-Ag side chains.…”

Activation of complement by serum-resistant Neisseria gonorrhoeae. Assembly of the membrane attack complex without subsequent cell death.

“…Studies of serum-resistant Salmonella minnesota have suggested that resistance in this bacterium is related to failure of the complement membrane attack complex (MAC) to insert into hydrophobic domains of the outer membrane (17,18). We undertook the following studies to assess the interaction of the complement system with resistant and sensitive _IV.. gonorrhoeae to characterize the relationship of the complement system to serum resistance.…”