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Cryptic peptidoglycan and the antiphagocytic effect of the Staphylococcus aureus capsule: model for the antiphagocytic effect of bacterial cell surface polymers.

The antiphagocytic effect of the Staphylococcus aureus capsule is known to be related to its ability to interfere with opsonization by normal human serum. In this study, evidence is presented with isolated cell surface components which indicates that the capsule hinders opsonization by masking cell wall peptidoglycan. In contrast to intact, encapsulated S. aureus M cells, peptidoglycan particles isolated from the organism were efficiently opsonized by normal human serum and phagocytized by human polymorphonuclear leukocytes. Cell wall particles retaining capsular material were opsonized less efficiently than peptidoglycan. Studies comparing the opsonic capacities of normal, C2-deficient, and heat-inactivated sera led to the conclusion that both the classical and the alternative complement pathways contribute to the opsonization of peptidoglycan in normal human serum. It appears that the capsule interferes with opsonization via both of these complement pathways. Serum from rabbits immunized with S. aureus M had significant heat-stable opsonic activity for the intact organism and cell walls retaining capsular material, but not for peptidoglycan. A general model is proposed to explain how antiphagocytic cell surface polymers may inhibit bacterial opsonization and thereby impede natural immunity.

Cell Wall

Phagocytosis and trypsin-resistant glass adhesion by osteoclasts in culture.

Osteoclasts were cultured in vitro and tested for the functions normally associated with cells of the mononuclear phagocyte system. They were found to ingest glutaraldehyde-fixed red cells and latex, but not opsonised or complement-coated sheep red cells. Osteoclasts are glass-adherent and resist removal by trypsin. The implications of these findings for the identity of the osteoclast precursor cell and its mechanism of fusion are discussed.

Animals

The characteristics of M proteins purified by column chromatography with hydroxyapatite from acid extracts of Streptococcus pyogenes of types 1, 3, 6, 12 and 17.

Purified M proteins were recovered from acid extracts of Streptococcus pyogenes, M-types 1, 3, 6, 12 and 17, by elution from columns of hydroxyapatite of the proteins precipitated with ammonium sulphate. M protein free from non-type-specific antigens was recovered only from M-type 12. Although similar fractions were not recovered from M-types 1, 3, 6 and 17, purified preparations containing a single cross-reactive antigen were obtained. In addition to the M proteins associated with cross-reactive antigens, type-specific antigens that did not stimulate opsonic antibodies were isolated from revealed molecular weights that ranged from 32,000 to 63,000 daltons, total amino acid compositions that were similar, and N-terminal amino acids that were variable.

Amino Acid Sequence

Sickle cell anemia and severe infections due to encapsulated bacteria.

Overwhelming infections caused by encapsulated bacteria are an important cause of morbidity and death in children with sickle cell anemia. The most important contributing factors to this increased susceptibility to infections are an opsonophagocytic defect due to an abnormality of the alternate pathway of complement activation, a state of functional hyposplenia, and a lack of specific circulating antibodies as a developmental phenomenon. If the inordinately high, early mortality rate associated with sickle cell anemia is to be prevented, early diagnosis of affected infants is crucial. Prophylactic therapy with penicillin has been advocated in recognition of the fact that a majority of the causative organisms are sensitive to penicillin. However, no controlled studies have proved the effectiveness of such therapy. Immunization with broadly polyvalent vaccines against Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitidis may ultimately represent the most effective way to reduce the incidence of catastrophic infections.

Anemia, Sickle Cell

Complement-mediated phagocytosis of Pseudomonas aeruginosa.

The nature of the opsonic factors in nonimmune human serum for six blood culture isolates of Pseudomonas aeruginosa was investigated by measuring uptake of [3H] adenine-labeled bacteria by human PMNs. Normal human serum, C2- and C4-deficient sera, zymosan-treated serum, and immunoglobulin-deficient sera were used as opsonic sources. Heat inactivation of each of these serum sources markedly reduced its opsonic capacity for all Pseudomonas strains, suggesting that the serum C system was essential for opsonization. Five strains were opsonized in the absence of the classical C pathway; however, kinetic studies revealed that opsonization proceeded at a faster rate when the classical pathway was present. In spite of markedly reduced factor B and C3 levels, zymosan-treated serum retained significant opsonic activity for one of the strains tested. Four strains were poorly opsonized by immunoglobulin-deficient serum, and C activation by these strains appeared to depend upon the presence of antibodies. Two strains, however, were effectively opsonized in a relative absence of antibodies. Thus, in the nonimmune state, phagocytosis of P. aeruginosa is mediated primarily via the C system, and antibodies appear to play a role in the opsonization of some but perhaps not all Pseudomonas strains.

Complement Factor B

Physiochemical consequences of opsonization of Salmonella typhimurium with hyperimmune IgG and complement.

Partition in an aqueous, two-polymer phase system containing dextran and polyethylene glycol was employed to investigate the physico-chemical changes inflicted upon the cell surface of a smooth strain of Salmonella typhimurium by the binding of antibody IgG and complement. The minimum antibody concentration for increased phagocytosis in vitro was approximately the same as that for a significant change in two-phase partition, ca 8000 mol/bacterium, whereas a lower concentration, less than 4000 mol/bacterium, was sufficient to increase clearance in vivo. After pepsin digestion of IgG, larger quantities, ca 35,000 mol/bacterium, was required for opsonization and to influence two-phase partition. Addition of normal rabbit or guinea-pig serum to bacteria sensitized with a low concentration of antibody IgG conspicuously enhanced phagocytosis and affinity for the dextran-rich phase. The results show that binding of 8000 IgG antibody molecules or more to smooth S. typhimurium generates physicochemical changes of the bacterial surface which from studies on S leads to R mutations are known to correlate with hydrophobicity, negative charge and phagocytosis. Such results support the view that one important function of IgG antibody and complement is to decrease the hydrophilic properties of the bacteria which is thought to be a prerequisite for phagocytosis.

Binding Sites, Antibody

Streptococcal M protein: an antiphagocytic molecule assembled on the cell wall.

After extraction with nonionic detergent, type 6 streptococcal M protein was found to be composed of multiple proteins ranging in molecular weight from 35,000 to 6,000 daltons. The antiphagocytic proteins, however, were found to be limited to three species having molecular weights of 28,000, 31,000, and 35,000 daltons. These molecules which removed opsonic antibodies from immune serum could be separated from those proteins that had only type specificity. Pulse chase experiments supported by chemical and immunological data suggest that the smaller, type-specific molecules are used to assemble the larger, antiphagocytic proteins. Type 6 M protein was radialabeled and used in a binding assay for the measurement of opsonic antibodies in human serum. Good correlation was observed between binding and the presence of opsonic antibodies in both systems. However, certain sera did exhibit binding but lacked type-specific opsonic activity. Results of competitive inhibition experiments demonstrated that the nonopsonic serum was deficient in certain antibodies that were present in opsonics serum and that the anitphagocytic molecules contained the sites necessary to bind these antibodies.

Amino Acids

A defect of the alternative pathway of complement.

The sera of patients defective at opsonization for yeast phagocytosis fix haemolytic complement poorly following incubation with the alternative pathway complement activators, inulin and bacterial endotoxin. This suggests that the defect lies early in the alternative pathway of complement.

Adult

Serum complement-like opsonic activities in human, animal, vegetable, and proprietary milks.

Human, animal, proprietary, and soy milks are comparable to human serum C5 in opsonization of baker's yeast. Bovine milk and human serum opsonically reconstitute C5-deficient mouse serum. Such reconstitution is selectively inhibited by antiserum to human C5. Further characterization suggests that bovine milk contains material structurally and functionally similar, but not identical, to human C5.

Animals

Antigenicity of the M proteins of group A hemolytic streptococci: further evidence for shared determinants among serotypes.

Shared antigenic determinants between M proteins of group A streptococci appear to be widespread among serotypes. This is demonstrated by the ability of purified M proteins to absorb opsonic antibody from a variety of heterologous antisera prepared against whole cells or purified M proteins. This absorption procedure has the capacity to separate passive mouse protecting and passive hemagglutinating antibodies from opsonic antibodies measured in vitro. When whole cells or M proteins are used as heterologous absorbents, immunoglobulin may be recovered from the cells or precipitates. The recovered antibody has most of the opsonic and some of the precipitating qualities of the original unabsorbed serum, but hemagglutinating titers are significantly lower. These data provide additional evidence that shared antigenicity among M proteins can be the result of common antigenic determinants. Arguments are presented that these cross-reactions are not the result of a nonspecific protein fraction associated with purified M proteins.

Animals

Interaction of Mycoplasma pneumoniae with alveolar macrophages: viability of adherent and ingested mycoplasmas.

Guinea pig peritoneal or alveolar macrophages were inoculated with Mycoplasma pneumoniae cells. Extracellular mycoplasms were killed by complement treatment, and the effect of macrophage action on the number of the remaining viable mycoplasmas was observed. The complement killing was to some extent inhibited by the presence of the macrophages, but the mechanism of this protection remains unknown. Opsonized mycoplasmas were ingested, and approximately 98% were killed within 4 h. The killing rate was somewhat lower than comparable data for bacteria, but lack of cell wall and high lipid content of the membrane apparently do not cause a significant delay in intracellular destruction.

Animals

Alternate complement pathway activation by group A streptococci: role of M-protein.

Avirulent strains of group A streptococci readily activate the complement system in normal human serum via the alternate complement pathway (ACP). Virulent M-positive group A streptococci are much less potent as activators of the ACP. The ability of M-positive streptococci to activate the ACP is enhanced by trypsinization or mild peptic digestion. The latter treatment removes the serologically active and antiphagocytic type-specific moieties of M protein, but retains the surface fuzzy layer. The phagocytosis of avirulent streptococci is markedly enhanced by preopsonization in serum chelated with Mg-ethylene glycol tetraacetic acid (classic complement pathway blocked) but not in serum devoid of heat-labile factors. These studies suggest that the function of M protein as a virulence factor may be mediated, at least in part, by its ability to retard interaction of ACP components with structures present on the streptococcal cell surface.

Bacterial Proteins

Decreased heat-labile opsonic activity and complement levels associated with evidence of C3 breakdown products in infected pleural effusions.

Heat-labile opsonic activity was measured simultaneously in serum and pleural fluid of patients with transudates, infectious exudates (with positive or negative bacterial culture) and neoplastic exudates, using two different complement-dependent phagocytic tests: the killing of Staphylococcus aureus Wood 46 variant strain (K50 opsonic titers) and the assessment of ingestion rate of endotoxin-coated paraffin particles (Oil Red 0 uptake test). K50 opsonic titers were lower in culture-positive pleural effusions as compared to culture-negative (P < 0.002) or neoplastic effusions (P < 0.002). These results were corroborated by the Oil Red 0 uptake test. The data obtained with the two assays showed a significant correlation (P < 0.001). The hemolytic activity of complement (CH50) as well as the levels of C3 breakdown product, C3d, were measured in the same sera and pleural fluid samples and in an additional group of patients with pleural effusions of the same etiology. Effusions with positive cultures showed lower CH50 values (P < 0.01) and higher C3d values (P < 0.05) when compared to culture-negative pleural fluids. Finally, evidence for immune complexes in pleural effusions and sera was looked for by determination of Clq binding activity. Levels were higher in culture-positive effusions when compared to culture-negative fluids (P = 0.005).K50 opsonic titers showed a positive correlation with CH50 values (P < 0.001) for all fluids tested. Similarly Clq binding activity correlated with C3d levels in effusions of infectious origin (P = 0.05). Recovery experiments using the various bacterial species isolated from culture-positive pleural effusions showed evidence of complement inactivation upon incubation with pooled sera at concentrations of 10(7)-10(8) microorganisms/ml. These results indicate that one important reason for bacterial persistence in empyema may be decreased opsonization secondary to local consumption of complement.

Adult