It has been recognized since 1895 (1) that some gram-negative bacteria are sensitive to the lytic action of fresh serum, whereas others are highly serum resistant. In general, serum-resistant organisms are more pathogenic than serum-sensitive bacteria in animal models of infection, and serum-resistant organisms are more commonly isolated from the bloodstream of patients with gram-negative bacteremia (2). In attempts to define the basis of this important virulence factor, characteristics of the outer membrane of serum-sensitive and serum-resistant organisms have been analyzed and compared (3)(4)(5). The presence of a complete lipopolysaccharide (LPS) 1 (i.e., the smooth phenotype) is the characteristic most clearly associated with serum resistance. Rough bacteria lacking a complete LPS are almost invariably serum sensitive.The antibody and complement requirements for serum killing of bacteria also have been examined. It has been shown (6-8) that killing of gram-negative bacteria by serum requires the participation of terminal components of the complement system ((25-9). However, the mechanism of resistance of gram-negative bacteria to serum killing in the presence of adequate antibody is still unknown.Resistance to serum killing could involve the inability to form a membrane attack complex on the organism. An alternative hypothesis, however, is that a membrane attack complex that forms on the bacterial surface may be functionally impotent either because of failure to insert into the bacterial outer membrane or because the inserted complex does not cause damage to vital outer or inner membrane structures.Previous studies have examined a number of aspects of this issue. Studies have suggested that serum-sensitive and serum-resistant strains of Escherichia coli (9, 10) or Salmonella typhimurium (11) have equivalent amounts of C3 deposited. It is not resolved whether (25 is deposited on serum-resistant bacteria. Reynolds et al. (11) could not demonstrate deposition of functional C5 on serum-resistant S. typhimurium in Mg ++ saline after incubation in C6-deficient rabbit serum. On the other hand, Ogata and Levine (10) demonstrated equivalent (25 consumptior~ by strains of E. coli that varied in complement sensitivity; however, evidence for levels of cell-bound (25 was not 1 Abbreviations used in this paper: CFU, colony-forming unit; HBSS, Hanks' balanced salt solution; LPS, lipopolysaccharide; PNHS, pooled normal human serum; RT, room temperature. J. Exp. MED.

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.

To assess whether non-Ki, group 2 capsular serotypes are important in conferring serum resistance to extraintestinal isolates of Escherichia coli, a K54 blood isolate (CP9) was evaluated as a model pathogen. Transposon mutagenesis (TnphoA) was used to generate isogenic capsule-negative mutants. CP9 was resistant to the bactericidal effects of serum, growing in 80%v serum. In contrast, all of the capsule-negative mutants had an increased sensitivity to 80%o normal human serum, undergoing a 2to 3-log kill over 3 h when starting inocula of 104 to 107 CFU/ml were used. The killing of the capsule-negative strains was mediated through the alternative complement pathway and not by lysozyme or beta-lysins. The protective effect of the K54 capsule

Nucleated cells, unlike erythrocytes, are able to survive limited complement attack by eliminating potentially cytolytic complement channels from the plasma membrane (PM) by processes that involve, plasma membrane (PM) by processes that involve, but may not be limited to, endocytosis. The observation that C5b-9 channels, as well as C5b-8 and C5b-7 intermediates, are rapidly eliminated from the cell surface of nucleated cells has prompted us to examine whether terminal complement complexes stimulate membrane events that lead to accelerated elimination of these complexes. We have suggested previously that ion flux through terminal complement complexes might influence the rate of elimination on the basis of our finding that terminal complement complexes with larger functional channel sizes are more rapidly eliminated. In this study, we examined the role of Ca2+ on the elimination rate of terminal complement complexes in the PM of Ehrlich cells, because changes in Ca2+ flux across the PM are known to influence many metabolic activities including endocytosis. To determine the elimination rate for terminal complement complexes by functional analysis, cells bearing C5b-7 or C5b-8 complexes with or without a sublytic dose of C9 were incubated at 37 degrees C for various time intervals before converting the remaining complexes to lytic C5b-9 channels. The initial elimination rates for the terminal complement complexes were compared in the presence of 0.015, 0.15, and 1.5 mM CaCl2 in the medium. Sufficient lowering of the extracellular Ca2+ concentration, (Ca2+)o, resulted in prolonging the elimination of each of the terminal complement complexes to a different extent. The effect of (Ca2+)o on the elimination rate was most pronounced for C5b-8 in the presence of a sublytic number of C5b-9, with less of an effect on C5b-8 alone, and the least effect with C5b-7. The elimination rates for terminal complement complexes were also determined by measuring the persistence of C5b antigen on the cell surface at 37 degrees C in the presence of various (Ca2+)o by using fluorescence-activated cell sorter analysis and were comparable with that obtained by functional analysis. Examination of the effect of terminal complement complexes on the cellular Ca2+ concentration, (Ca2+)i, revealed that these complexes increased the (Ca2+)i in proportion with the known functional pore size of the terminal complement complex in the PM. In addition, Quin 2, which can buffer internal Ca2+ transients, was found to increase the susceptibility of Ehrlich cells to lysis by C5b-9, further suggesting a relationship between the (Ca2+)i and the elimination process.(ABSTRACT TRUNCATED AT 400 WORDS)

The mechanism of serum resistance for infective promastigotes of Leishmania major was investigated. Prior results suggested that the mechanism of resistance was mediated at a step after C3 deposition. Equivalent amounts of C3b were deposited on serum-susceptible, noninfective promastigotes harvested from log stage cultures (LOG) and on C-resistant, infective, metacyclic promastigotes (MP) purified from stationary stage cultures. Whereas binding of C9 to LOG was stable during incubation in serum, C9 binding to MP was minimal and unstable, because molecules bound initially to MP were released with continued incubation. Failure to bind C9 was not a result of inability to activate C; the kinetics of C3, C6, and C9 consumption were similar for LOG and MP. Deposition of C5b-7 on MP was stable, indicating that the initial steps in terminal complex formation were intact. Instead, the majority of C5b-9 formed on MP was spontaneously released into the serum as SC5b-9. Residual C5b-9 on MP was released with 1 M NaCl. These data show that developmental modification of the promastigote membrane during transition from a noninfective to an infective stage blocks insertion of lytic C5b-9 into the promastigote membrane.