Effect of leukocyte hydrolases on bacteria. XIII. Role played by leukocyte extracts, lysolecithin, phospholipase a2, lysozyme, cationic proteins, and detergents in the solubilization of lipids from Staphylococcus aureus and group A streptococci: relation to bactericidal and bacteriolytic reactions in inflammatory sites.

The bactericidal and bacteriolytic effects of lysolecithin (LL) and egg-white lysozyme (LYZ) on Staph. aureus and group A streptococci and the solubilization of phospholipids from the bacterial membranes by these agents was studied. Low concentrations of lysolecithin (1--10 microgrames/ml) are highly bactericidal for Steph. aureus and group A streptococci, but induce neither bacteriolysis nor solubilization of a substantial amount of membrane phospholipids. On the other hand, while LL at greater than 50 micrograms/ml causes substantial lipid release, a combination of LL and LYZ is absolutely needed to solubilize lipids from streptococci. This combination is, however, not bacteriolytic for this microrganism. The solubilization of lipids from staphylococci by LL is much faster than that induced in streptococci by LL + LYZ. The solubilization of the bulk of membrane lipids from staphylococci can also be achieved by Triton X-100 and by sodium lauryl sulfate and from group A streptococci by Triton X-100 plus LYZ. A variety of other detergents (e.g., Cetavlon, sodium taurocholate, cetyl pyrdinium chloride) have no lipid-releasing properties even in the presence of LYZ. The release of lipids by LYZ (in the presence of LL) from group A streptococci is related to its enzymatic activity, on a still unknown substrate, but not to its cationic nature as this muramidase cannot be replaced by a variety of cation substances (histone, polylysin, leukocyte cationic proteins, polymyxin B, and spermidine). The release of lipids from staphylococci by LL is not inhibited by a variety of anionic and cationic polyelectrocytes (heparin, liquoid, chondroitin sulfate, DNA histone, and polylysine) which markedly inhibit the release of lipids from group A streptococci by LL and LYZ. Streptococci that had been cultivated in the presence of subinhibitory concentrations of penicillin G lose their membrane phospholipids to a larger extent and by much smaller concentrations of LL and LYZ, as compared to controls, suggesting that the interference with the synthesis of the peptidoglycan increases the accessibility of the cell membrane to the lipid-releasing agents. The mechanism by which LL collaborates with LYZ in lipid release is still not known. The possible role of bacterial lipids and lyso compounds in the control of bacterial survival in inflammatory sites is briefly discussed.

The effect of leukocyte hydrolases on bacteria VIII. The combined effect of leukocyte extracts, lysozyme, enzyme "cocktails," and penicillin on the lysis ofStaphylococcus aureus and group a streptococci in vitro.

Cultures ofStaphylococcus aureus, which are harvested from the stationary phase of growth, are extremely resistant to lysis by extracts of human blood leukocytes. Such bacteria are, however, rendered susceptible to bacteriolysis when cultivated in the presence of subinhibitory concentrations of penicillin G, nafcillin, or cloxacillin (0.05µg/ml). The lytic effect of the leukocyte extracts on the penicillin-grown bacteria is further augmented by the addition of egg-white lysozyme. Staphylococci, which are harvested from the logarithmic phase of growth in ordinary media, are susceptible to lysis by leukocyte extracts, maximal lysis being achieved with about 100µg/ml of leukocyte extracts. On the other hand, penicillin-grown staphylococci are lysed by much smaller amounts of leukocyte extracts (20µg/ml), and much shorter periods of incubation are needed to achieve maximal lysis. Similar results are obtained when the leukocyte extracts are substituted by a cocktail of lytic agents which contain crude trypsin, lysolecithin, and lysozyme. Lysis of the staphylococci by the leukocyte extracts, fortified by lysozyme, is optimal at pH 5.0 and is accompanied by the solubilization of the bulk of glucosamine, known to be mostly concentrated in the peptidoglycan of the cell wall. Penicillin-grown staphylococci are also more susceptible than controls to lysis by a mixture of histone and lysozyme. The lysis, by leukocyte extracts and by the cocktail of both regular and penicillin-grown staphylococci, is strongly inhibited to the same extent by heparin, liquoid, histone, protamine sulfate, IgG, and human serum. On the other hand, no inhibition of lysis is achieved by chloramphenicol, streptomycin, erythromycin, KCN, HgCl2, or by neutral polyelectrolytes. Group A streptococci, which are extremely resistant to degradation by leukocyte extracts or by the cocktail, when harvested from any phase of growth, also become susceptible to lysis by leukocyte extracts or by the cocktail when grown in the presence of small amounts of penicillin (0.004-0.008µ/ml). Bacteriolysis became even more pronounced when the reaction mixtures were incubated at 41 °C, a temperature likely to develop in patients with streptococcal infections. Electron-microscope examination of the staphylococci following treatment with leukocyte enzymes and penicillin revealed that both cell wall and cytoplasmic structures were severely damaged by the lytic agents. The mechanisms by which penicillin exposes the bacterial cell walls to cleavage by leukocyte extracts is discussed, and the phenomenon of enhanced susceptibility to lysis by leukocyte enzymes is related to the role played by undegraded bacterial constituents in the initiation of chronic inflammatory lesions.

The effect of leukocyte hydrolases on bacteria : V. Modification of bacteriolysis by antiinflammatory agents and by cationic and anionic polyelectrolytes.

Lysis of(14)C-labeledStaph. aureus by human blood leukocyte lysates, by extracts of rabbit small intestines and pancreas, and by the "cocktail" of enzymes (containing trypsin, lysolecithin, and lysozyme) is strongly inhibited by anionic polyelectrolytes (e.g., heparin, chondroitin sulfate, liquoid (polyanethole sulfonic acid), and DNA). Most of the lytic agents employed were inhibited by cationic polyelectrolytes (e.g., histone, protamin sulfate and polylysin), as well as by gold thiomalate, normal human serum, synovial fluids obtained from patients with knee-joint trauma, extracts of coffee, tea, and cocoa, Ultracorten- and Dexamethasone. On the other hand, some antiinflammatory agents tested (e.g., indomethacin, aspirin, hydrocortisone acetate and succinate, and prednisolone acetate and tributyl acetate) were not inhibitory. All the cationic polyelectrolytes employed and liquoid were also strong inhibitors of lysozyme. Since mixtures of cationic and anionic polyelectrolytes at equimolar concentrations failed to inhibit bacteriolysis, it is postulated that the balance between charged macromolecular substances, which are likely to accumulate in inflammatory foci, may determine the fate of cellular components of bacteria in inflamed tissues. The possible role played by lysosomal enzymes and by tissue inhibitors in tissue damage and in the survival of bacteria in chronic inflammatory lesions is discussed.

Effect of leukocyte hydrolases on bacteria. XIV. Bacteriolytic effects of human sera, synovial fluids, and purulent exudates on Staphylococcus aureus and Streptococcus faecalis: modulation by Cohn's fraction II and by polyelectrolytes.

Normal sera and plasma, derived from humans, calves, rats, rabbits, horses, human synovial fluids, inflammatory exudates, and leukocyte extracts, when sufficiently diluted are highly bacteriolytic for Staph, aureus, Strep. faecalis, B. sutilis and to a variety of gram-negative rods. On the other hand, concentrated serum or the other body fluids are usually not bacteriolytic for these bacterial species. While the lysis of Staph, aureus and B. subtilis by diluted serum is not lysozyme dependent, lysis of Strep. faecalis is absolutely dependent on the concentration of lysozyme. The lytic factor in human serum is present in Cohn's fractions III, IV, and V. It is nondialyzable, resistant to heating for 75 degrees C and 20 min, and acts optimally at pH 5.0. Like leukocyte extracts, synovial fluids, and inflammatory exudates, it lyses only young staphylococci. The inability of concentrated serum to lyse Staph. aureus and Strep. faecalis is due to the presence in the gamma globulin fraction of a potent inhibitor, which can be partly removed by dilution of by adsorption upon the homologous bacteria. Lysis of the bacteria is also strongly inhibited by Cohn's fraction II (gamma globulin) by high-molecular-weight DNA, heparin, liquoid, and histone. The possible role played by serum globulin in the protection of bacteria against degradation by leukocyte is discussed.

The effect of leukocyte hydrolases on bacteria : VII. Bactericidal and bacteriolytic reactions mediated by leukocyte and tissue extracts and their modifications by polyelectrolytes.

Crude extracts of human blood leukocytes were employed as a source of bactericidal and bacteriolytic agents againstStaphylococcus aureus. While the bactericidal action of the extracts was a very rapid process, bacteriolysis is a very slow process. Both the killing and the lysis of staphylococci depended on the age of the culture, maximal effects being obtained only with young cells. The killing of staphylococci by the extracts was absolutely dependent on the density of bacteria employed. On the other hand, bacteriolysis was only very slightly affected when large numbers of bacteria were employed. Both the bactericidal and bacteriolytic reactions were optimal at pH 5.0. Under similar conditions, extracts of pus and the "cocktail" of enzymes were both bactericidal and bacteriolytic, but extracts of small intestine and of platelets were not significantly bactericidal. Experiments, designed to differentiate between the bactericidal and bacteriolytic properties of the extracts showed that both properties were preserved following heating in acid solutions but were completely destroyed following heating in alkaline solutions. The bactericidal factor in the lysates could be readily adsorbed on large numbers of viableStaph. aureus andE. coli, but the bacteriolytic properties of the extracts could not be removed by adsorption. The bactericidal effect of the extracts could not be inhibited by a variety of anionic polyelectrolytes, but all these agents strongly inhibited the bacteriolytic effect. Moreover, several of the anionic substances potentiated the bactericidal effects mediated by the extracts. Potentiation of these effects was also caused by protamine sulfate and by polylysine, which were highly bactericidal by themselves. The only substance that was found to abolish the bactericidal effects of the extracts is ultracorten H. Historie and polylysine (which are highly bactericidal) lost their effects when mixed with certain concentrations of heparin or polyglutamic acid, which by themselves are not bactericidal, indicating that an appropriate balance between cationic and anionic substances may determine the bactericidal effects of cationic substances. Since the bactericidal properties of the lysates could not be abolished by any of the anionic macromolecular substances employed; it is suggested that the bactericidal agents present in crude whole lysates of leukocytes comprise a complex mixture of agents, some of which are not identical with cationic substances. Thus, the data suggest that the employment of highly purified cationic proteins of leukocytes and tissues to study bactericidal models may not reflect the actual conditions that prevail in inflammatory exudates. The possible role played by cationic and anionic polyelectrolytes in the control of bacterial survival and lysis in inflammatory exudates is discussed.

The effect of leukocyte hydrolases on bacteria. III. Bacteriolysis induced by extracts of different leukocyte populations and the inhibition of lysis by macromolecular substances.

The lysis of 14C-labeled bacteria by hydrolases of human and rabbit leukocytes was studied in vitro. While Staphylococcus albus, Streptococcus faecalis, and Streptococcus mutans were highly susceptible to lysis, Staphylococcus auresus was intermediate in its susecptibility to lysis by the leukocyte enzymes. Group A Streptococcus, Listeria monocytogenes, Shigella flexneri, Escherichia coli, and Mycobacterium smegmatis were very resistant to degradation by these enzymes. The lytic activity of leukocyte lysates from human and rabbit blood was probably due to acid hydrolases of polymorphonuclear leukocytes. Extracts of human blood monocytes and of rabbit peritoneal and lung macrophages were less lytic for the bacteria tested. Lymphocytes and platelet extracts were not bacteriolytic. The lytic effect of the leukocyte lysates was not inhibited by KCN or sodium azide, but was abolished to a large extent by cationic polyelectrolytes such as protamine sulfate, histone and leukocyte cationic proteins, and poly-lysine, as well as by the anionic polyelectrolytes such as heparin, chondroitin sulfate, DNA, carrageenin, alginate sulfate, dextran sulfate, and ploy-L-glutamic acid. Other potent inhibitors of bacteriolysis were trypan blue, congo red, phosphatidic acid, normal immunoglobulins, and components of streptococcal cell wall.

The interaction of leukocytes and their hydrolases with bacteria in vitro and in vivo: the modification of the bactericidal and bacteriolytic reactions by cationic and anionic macromolecular substances and by anti-inflammatory agents.

Acid hydrolases from extracts of human blood leucocytes lyse Staph.aureus, Staph.albus and Strep.faecalis in vitro. The leucocyte enzymes can be substituted by a lytic mixture which contains crude trypsin, lysolecithin, phospholipase C and lysozyme, which lyse other bacterial species, e.g. E.coli and Listeria which are resistant to leucocyte enzymes. Bacteriolysis by the lytic agents is strongly inhibited by the anionic polyelectrolytes, heparin, chondroitin sulphate, DNA, dextran sulphate and other sulphated mucopolysaccharides, by the cationic materials, histone, protamine sulphate, leucocyte cationic proteins and polylysine. Other strong inhibitors are trypsan blue and congo red, the phospholipids phosphatidyl serine and ethanolamine, gold thiomalate, extracts of coffee and tea and the anti-inflammatory agents, ultracorten-H, and ultracortenol. Bacteriolysis is also strongly inhibited by normal human serum and by synovial fluids from patients with a variety of joint diseases. The inhibitors in these body fluids are associated with the globulin fractions. Since mixtures of anionic and cationic polyelectrolytes, at equimolar concentrations, failed to inhibit bacteriolysis by leucocyte enzymes, it is postulated that a delicate balance between positively and negatively charged inhibitors control the degradation of cell wall components of bacteria in inflamed areas. Such bacterial components, induce 'storage type' granulomas. The possible role played by polyelectrolytes in the control of the inflammatory process induced by leucocyte hydrolases will be discussed.