Serum Bactericidal Reaction Research Articles

Role of lipopolysaccharide and complement in susceptibility of Klebsiella pneumoniae to nonimmune serum.

The role of lipopolysaccharide (LPS) in the susceptibility of Klebsiella pneumoniae to serum and the mechanism of complement activation by serum-susceptible (SerS) strains were investigated. The classical and alternative complement pathways are involved in serum killing of susceptible K. pneumoniae strains. The LPS composition seems to play a very important role in the serum bactericidal reaction, while capsular polysaccharide from this bacterium does not play any role. High-molecular-weight LPS from serum-resistant (Serr) K. pneumoniae strains was able to inhibit completely the serum bactericidal activity. LPS from SerS K. pneumoniae strains was not able to inhibit completely the serum bactericidal activity; low-molecular-weight LPS from Serr K. pneumoniae strains could not either. All these findings suggested that LPS composition, especially the O-antigen polysaccharide chains, contributes to the susceptibility of K. pneumoniae strains to complement-mediated serum bactericidal activity.

Bactericidal activity for Neisseria meningitidis in properdin-deficient sera.

We investigated serum bactericidal reactions against Neisseria meningitidis (serogroups A, B, C, D, Y, W-135, 29E, X, and Z) in the sera of two healthy adults with properdin deficiency. Bactericidal reactions mediated via the classic complement pathway (unchelated system) were not impaired in properdin-deficient serum. The properdin-deficient sera supported alternative pathway-mediated killing (Mg++EGTA-chelated system) of some, but not all, of the strains investigated. Vaccination of the properdin-deficient individuals with serogroup A and C polysaccharide clearly increased the concentrations of antibody to meningococci. At least some of the antibodies induced by vaccination supported the bactericidal activity of properdin-deficient serum. Some antibodies to meningococci, probably of the IgM class, promoted alternative pathway-mediated bactericidal reactions in the absence of properdin. By contrast, presensitizing meningococci with IgG enhanced the alternative pathway-mediated reactions, but this was strictly a properdin-dependent effect.

Complement‐Dependent Bactericidal Activity for E. coli K12 in Serum of Preterm Newborn Infants

Complement-dependent serum bactericidal activity for E. coli K12 was assessed in 12 term infants and in 16 preterm infants. In both groups of newborns, at birth, bactericidal reaction by the classical pathway of complement activation was impaired with respect to normal controls at less than 0.001 level of significance (as estimated by Student's t-test). The serum bactericidal reaction by the alternative pathway of complement activation was significantly impaired only in preterm newborns, being normal in term infants. At a time corresponding to 40 weeks' gestational age also in preterm newborns alternative pathway mediated bactericidal activity for E. coli K12 was found normal. Classical pathway mediated bactericidal activity became normal only at an age corresponding to 52 weeks' gestational age.

IgG bearing covalently bound C3b has enhanced bactericidal activity for Escherichia coli 0111.

The mechanism was sought by which bactericidal IgG for E. coli 0111 (strain 12015) increases the bactericidal efficiency of C5b-9. IgG did not affect the distribution of C3 deposition on the O-Ag capsule and the outer membrane of 12015, suggesting that bactericidal IgG was not directing complement activation to different sites on the bacterial surface. However, one-fifth of the C3 that was deposited in the presence of IgG attached covalently to the antibody molecule. Covalent complexes between purified C3b and IgG were prepared in order to study the role of C3b-IgG in the bactericidal reaction. 8-10-fold less C3b-IgG than IgG was necessary to sensitize 12015 for serum killing. When aggregates were eliminated from the C3b-IgG and IgG preparations by sucrose density gradient ultracentrifugation, C3b-IgG remained three- to fourfold more effective than IgG on a molecule-for-molecule bound basis in mediating the serum bactericidal reaction. These results suggest that formation of C3b-IgG during the serum bactericidal reaction is critical for killing, and have important implications for the development of effective bactericidal vaccines.

Inhibition of serum bactericidal reaction by lipopolysaccharide.

An Rc-mutant of Escherichia coli that lacks UDPgalactose 4-epimerase grows normally without galactose but makes lipopolysaccharide lacking most of its carbohydrate. Exogenous galactose overrides the mutation and results in the formation of a complete lipopolysaccharide, thereby producing a smooth phenocopy. The smooth phenocopy was much more resistant to the bactericidal activity of normal human serum than was the rough phenotype. More complement was utilized by the rough mutant in the bactericidal process than by the smooth phenocopy. An antiserum was prepared in rabbits to a specific outer membrane protein in the mutant bacterium, the lambda receptor, whose expression could be suppressed by the addition of 10 mM maltose. The effect of the O-antigen in the lipopolysaccharide produced by the smooth phenocopy on the binding of antibody to the lambda receptor was determined. The smooth phenocopy exhibited significantly less binding of antibody than did the rough phenocopy. In addition, expression of the lambda receptor had little effect on the binding of antibody to the lambda receptor in the smooth phenocopy but caused significantly increased binding in the rough mutant. The results suggest that the increased resistance to the lethal action of normal human serum shown by the smooth phenocopy may be due to the blocking of antibody binding sites by the O-antigen of lipopolysaccharide, thereby preventing activation of the classical pathway of complement.

Serologic typing of Haemophilus paragallinarum based on serum bactericidal reactions.

Attempts were made to detect serologic specificities involved in the serum bactericidal (BC) reaction with Haemophilus paragallinarum (Hpg). On the basis of serum BC reactions, ten Hpg isolates were classified into two distinct serotypes. Specificity of the BC activity toward the encapsulated organisms of Hpg defined by the BC assay is similar to serotype specificity defined by the hemagglutination-inhibition or by the agglutination tests.

Human serum complement requirements for bacterial killing and protoplast lysis of Escherichia coli ML308 225.

Normal human serum kills Escherichia coli ML308 225 and lyses protoplasts derived from this organism. Human serum which is depleted of complement component C9 or deficient in component C8 is not bactericidal, but C9-depleted serum will lyse protoplasts whereas C8-deficient serum will not. Bacterial lipopolysaccharide, which can protect bacteria from the serum bactericidal reaction, does not protect protoplasts from lysis by serum.

Role of rabbit lysozyme in in vitro serum and plasma serum bactericidal reactions against Bacillus subtilis.

The antibacterial activity of purified rabbit lysozyme was kinetically investigated at concentrations comparable to those in normal rabbit serum and plasma serum. The bactericidal capability, lysozyme content, and electrophoretic composition of "purified beta-lysin," fractionated from normal rabbit serum, were also examined. In contrast to the extensive antibacterial activity of dilute normal rabbit serum observed in vitro, rabbit lysozyme was only weakly bactericidal for Bacillus subtilis. Inhibition of lysozyme enzymatic and bactericidal activities in normal rabbit serum by antilysozyme immunoglobulin G slightly reduced the initial rate of killing. The addition of neutralizing antibody or histamine (another lysozyme inhibitor) to partially purified bactericidal serum fractions had no effect on killing kinetics. Increasing the ionic strength of reaction mixtures containing normal serum or partially purified bactericidal fractions to levels which completely inhibited lysozyme activity resulted in stimulation of their respective killing kinetics. The addition of inhibitors to normal rabbit plasma serum completely eliminated its bactericidal activity. With regard to the killing of B. subtilis by rabbit and human blood fractions, these analyses clearly demonstrated that (i) although lysozyme is not a significant antibacterial component of normal rabbit serum, it represents the principal factor in normal rabbit plasma serum; (ii) different primary bactericidal mechanisms which are not detectable by singlepoint analyses operate in the sera of different species; and (iii) purified beta-lysin isolated from normal rabbit serum by the classical procedure is a heterogenous mixture of components.

Effect of pH and Haem Compounds on the Killing of Pasteurella septica by Specific Antiserum

The killing of Pasteurella septica by horse antiserum has features not previously associated with serum bactericidal reactions. The present work showed that lowering the pH from 7-4 to 6-8 abolished the action of antiserum. The bactericidal effect and the degradation of RNA seen when antiserum is added to P. septica growing in horse serum, were abolished at pH 6-8 in much the same way as when haem compounds were added to the system. Addition of chloramphenicol, rifampicin or puromycin to P. septica growing apparently normally in antiserum at pH 6-8 or in antiserum containing haem compounds led to rapid killing of the bacteria and to degration of their RNA. Addition of these antibiotics to P. septica growing in normal serum produced only bacteriostasis and did not induce RNA breakdown. In contrast, nalidixic acid, although inhibiting growth, did not induce rapid killing and RNA breakdown under the same conditions. These findings were unexpected and led to a reassessment of ideas concerning the mechanism of action of specific antiserum to P. septica. Although iron compounds clearly abolish the bactericidal based simply on an interference with bacterial iron supply is no longer sufficient. The process is more complex and must involve other factors.

Metabolically controlled killing of Pasteurella septica by antibody and complement

Recent work on the killing of Pasteurella septica by antibody and complement has shown that the process has features not previously associated with serum bactericidal reactions. In the present work it is shown that uncouplers and inhibitors of oxidative phosphorylation protect the bacteria against the action of antibody and complement. The results suggest that the bactericidal reaction is dependent upon bacterial oxidative energy metabolism.

Cross-Reactivity of Neisseria gonorrhoeae and Neisseria meningitidis and the Nature of Antigens Involved in the Bactericidal Reaction

Antigenic differences and similarities among strains of Neisseria gonorrhoeae were demonstrated in the serum bactericidal reaction with the use of hyperimmune rabbit antisera to N. gonorrhoeae. Antisera to Neisseria meningitidis were also capable of killing gonococci in vitro and provided evidence for differences among gonococcal strains. Bactericidal activity of rabbit antisera was unaffected by the type of gonococcal colony used in the assay. The principal antigens involved in the bactericidal reaction appeared to be surface proteins and lipopolysaccharides.

Protective role of smooth lipopolysaccharide in the serum bactericidal reaction.

The mechanism by which smooth lipopolysaccharide (LPS) protects Salmonella typhimurium C5 from the lethal action of specific antibody and complement has been investigated. Tris(hydroxymethyl)aminomethane (Tris) and ethylenediaminetetraacetate (EDTA), which sensitize S. typhimurium C5 to the bactericidal action of immune serum, were shown to cause appreciable structural modification of the smooth LPS associated with these cells. Mg(2+) was shown to reverse the sensitization of C5 by Tris, but this divalent cation was unable to reverse the sensitizing effect of Tris plus EDTA, which, unlike Tris, caused relatively large amounts of O-antigenic components to be released from the C5 cell wall. The possibility that smooth LPS blocks the interaction of activated complement components with receptors on the cell wall is discussed.

Nature and Heterogeneity of the Antigens of Neisseria gonorrhoeae Involved in the Serum Bactericidal Reaction

Sixty strains of Neisseria gonorrhoeae have been classified into four main groups according to their resistance to killing by human complement together with either normal human or immune rabbit antibodies. The rabbit antisera had been raised against 10 of the strains tested. The normal human antibodies had probably been formed against cross-reacting organisms since they could be removed by absorption with N. pharyngis sicca, N. pharyngis flavus, N. catarrhalis, or Escherichia coli. Bactericidal antibodies could be absorbed from both normal and immune sera by N. gonorrhoeae which had been autoclaved or trypsinized, and by red cells coated with gonococcal lipopolysaccharides. The results suggest that the antigens involved in the bactericidal reaction are lipopolysaccharides of several distinct specificities. Since individual sera always reacted more widely in hemagglutination than in bactericidal tests, it is postulated that surface-blocking antigens may restrict access to the lipopolysaccharides in the intact organisms.

The serum bactericidal reaction. IV. Phenotypic conversion of Escherichia coli from serum-resistance to serum-sensitivity by diphenylamine.

The serum bactericidal reaction. IV. Phenotypic conversion of Escherichia coli from serum-resistance to serum-sensitivity by diphenylamine.

The Serum Bactericidal Reaction. III. Antibody and Complement Requirements for Killing a Rough Escherichia Coli

Summary The serum bactericidal reaction was studied using the rough E. coli 200P and a system of serum reagents made selectively deficient in various complement components. In replacement experiments functionally pure individual components were added and requirements for C1, C4 and C2, and C9 as well as antibody were shown. Cellular intermediates BAC1 and BAC142 were demonstrated. The results suggest that the entire complement system is required to kill bacteria. Note Added in Proof. Inoue et al. (Inoue, K., Yonemasu, K., Takamizawa, A. and Amano, T. Biken J., 11: 203, 1968) have recently reported that BAC142 cells, C3, 5, 6, 7, 8, 9 and lysozyme were required for immune bacteriolysis of a rough strain of E. coli. The complement components used were purified from guinea pig serum. Results were based on observations of spheroplast formation: with the complete system about 25% of the rod-shaped bacteria were converted to spheroplasts.

Locus of the lethal event in the serum bactericidal reaction.

Hypertonic sucrose inhibited the bactericidal activity of lysozyme-free serum against a rough strain of Escherichia coli. The duration of the inhibition correlated with the duration of plasmolysis caused by the sucrose. Although the lethal action of the serum was delayed, the prompt release of alkaline phosphatase by the cells suggested that nonlethal damage to the cell wall had taken place under these conditions. In contrast, the crypticity of the cells for beta-galactosidase did not deteriorate until the viability of the bacteria began to decrease. It is concluded that the primary site of action of serum is at the bacterial cell wall; however, in the absence of lysozyme, the lethal event was subsequent damage to the bacterial cell membrane.

Locus of the action of serum and the role of lysozyme in the serum bactericidal reaction.

The mechanism of the lethal action of human serum on a rough strain of Escherichia coli was investigated by use of serum with and without lysozyme, in medium of low and high osmotic pressure, with cells radioactively labeled in the peptidoglycan polymer, and by electron microscopy. The results suggested that there are two separate components in the bacterial cell wall that afford structural support for the cell. Lysozyme attacked one of these, the peptidoglycan polymer. Serum damaged the other, which is probably the peripherally located lipopolysaccharide-phospholipid complex. The cell wall damage caused by lysozyme-free serum promptly resulted in cell death under usual conditions. In plasmolyzed cells, however, the wall damage was not lethal, presumably because the membrane of the plasmolyzed cell was protected from secondary lethal changes which otherwise occur.

Reversal of the bactericidal reaction of serum by magnesium ion.

Muschel, Louis H. (University of Minnesota, Minneapolis), and Jean E. Jackson. Reversal of the bactericidal reaction of serum by magnesium ion. J. Bacteriol. 91:1399-1402. 1966.-Magnesium ion was found to reverse the bactericidal action of the antibody-complement system. Concentrations of 0.03 to 0.11 m MgSO(4) or MgCl(2) were effective, provided that the reaction of the antibody-complement system did not proceed longer than 50 min. Salts of other monovalent and divalent cations, the polyamine, spermine, and several anticomplementary substances were inactive in reversing the serum bactericidal reaction. Thus, the bactericidal reaction of serum may be bacteriostatic under certain conditions, and this finding is compatible with the persistence of infection in certain diseases despite high levels of antibody.

Analysis of sequential stages in serum bactericidal reactions.

Michael, J. Gabriel (House of the Good Samaritan, Children's Hospital Medical Center, Boston, Mass.), and Werner Braun. Analysis of sequential stages in serum bactericidal reaction. J. Bacteriol. 87:1067-1072. 1964.-The bactericidal reaction of "normal" human serum against Escherichia coli was found to be separable into two distinctive stages. The early (first) stage of the reaction lasts for a relatively short period of time, and involves factors that are present in sufficient amounts only in slightly diluted serum. The later (second) stage needs more time and requires factors present in highly diluted serum. The first stage depends on the presence of Ca(++) and Mg(++) and on the activity of all components of complement; the second stage does not require divalent cations and C'1, C'2, and C'4, but requires factors that can be removed by zymosan. Under our conditions, removal of lysozyme did not influence either stage of the reaction. Bacteria exposed to concentrated serum for a short time, during the first stage, are essentially unaffected as far as their potential for subsequent multiplication is concerned; the actual damage to cellular integrity occurs only during the second stage of the reaction. In the absence of cell division, the "sensitization" produced during the first stage can be preserved for prolonged periods, and the bactericidal reaction can be completed later by exposure to antibody-free, highly diluted serum (second stage). Cell multiplication abolishes the sensitizing effects of the first stage.