Inflammation

, Volume 1, Issue 4, pp 371–407 | Cite as

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

  • C. Efrati
  • T. Sacks
  • N. Ne'eman
  • M. Lahav
  • I. Ginsburg
Article

Abstract

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.

Keywords

Lysozyme Glucosamine Protamine HgCl2 Streptococcal Infection 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Lahav, M., N. Ne'eman, E. Adler, andI. Ginsburg. 1974. The effect of leukocyte hydrolases on bacteria. I. Degradation of14C-labeled streptococci and staphylococci by leukocyte lysates in vitro.J. Infect. Dis. 129:528.Google Scholar
  2. 2.
    Ne'eman, N., M. Lahav, andI. Ginsburg. 1974. The effect of leukocyte hydrolases on bacteria. II. The synergistic action of lysozyme and extracts of PMN, macrophages, lymphocytes and platelets in bacteriolysis.Proc. Soc. Exp. Biol. Med. 146:1137.Google Scholar
  3. 3.
    Lahav, M., N. Ne'eman, J. James, andI. Ginsburg. 1975. The effect of leukocyte hydrolases on bacteria. III. Bacteriolysis induced by extracts of different leukocyte populations and the inhibition of lysis by macromolecular substances.J. Infect. Dis. 131:149.Google Scholar
  4. 4.
    Ginsburg, I., N. Ne'eman, Z. Duchan, M.N. Sela, J. James, andM. Lahav. 1975. The effect of leukocyte hydrolases on bacteria. IV. The role played by artificial enzyme “cocktails” and tissue enzymes in bacteriolysis.Inflammation 1:41.Google Scholar
  5. 5.
    Sela, M.N., M. Lahav, N. Ne'eman, Z. Duchan, andI. Ginsburg. 1975. The effect of leukocyte hydrolases on bacteria. V. Modification of bacteriolysis by antiinflammatory agents and by cationic and anionic polyelectrolytes.Inflammation 1:57.Google Scholar
  6. 6.
    Ginsburg, I., M. Lahav, N. Ne'eman, Z. Duchan, S. Chanes, andM.N. Sela. 1975. 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 antiinflammatory agents.In Future Trends in Inflammation. II. Agents and Actions. Vol. 6, p. 292.Google Scholar
  7. 7.
    Warren, G.H., andJ. Gray. 1965. Effect of sublethal concentrations of penicillins on the lysis of bacteria by lysozyme and trypsin.Proc. Soc. Exp. Biol Med. 120:504.Google Scholar
  8. 8.
    Myrvik, Q.N., E.S. Leake, andB. Fariss. 1961. Studies on the pulmonary alveolar macrophages from the normal rabbit: A technique to produce them in a high state of purity.J. Immunol. 86:128.Google Scholar
  9. 9.
    Ginsburg, I., M.Lahav, N.Ne'eman, and J. M.James. 1974. The inhibition by byasic and acidic polyelectroytes of the degradation of bacteria by leukocyte enzymes: Relation to the persistence of microbial constituents in inflammatory sites.In Activation of Macrophages, Second Workshop Conferences Hoechst. W.H. Wagner and H. Hahn, editors. Excerpta Medica Publications 162.Google Scholar
  10. 10.
    Gatt, R., andE.R. Berman. 1966. A rapid procedure for the estimation of amino sugars or a micro scale.Anal. Biochem. 15:167.Google Scholar
  11. 11.
    Fischetti, V.A., E.C. Gotschlich, andA.W. Bernheimer. 1971. Purification and physical properties of group C phage-associated lysin.J. Exp. Med. 133:1105.Google Scholar
  12. 12.
    McCarty, M. 1952. The lysis of group A hemolytic streptococci by extracellular enzymes ofStreptomyces albus. I. Production and fractionation of the lytic enzymes.J. Exp. Med. 96:555.Google Scholar
  13. 13.
    Schwab, J.H., andS.H. Ohanian. 1967. Regulation of streptococcal cell wall antigens in vitro.J. Bacteriol. 94:1346.Google Scholar
  14. 14.
    Ginsburg, I. 1972. Mechanisms of cell and tissue injury induced by group A streptococci: Relation to poststreptococcal sequelae.J. Infect. Dis. 126:294.Google Scholar
  15. 15.
    Gallis, H.A., and R.W.Wheat. 1974. Degradation of 14C-labeled cell walls of group A streptococci by lysozyme and lysosomal enzymes.Am. Sci. (abstract).Google Scholar
  16. 16.
    Hayashi, H., Y. Araki, andE. Ito. 1973. Occurrence of glucosamine residues with free amino groups in cell wall peptidoglycan from bacilli as a factor responsible for resistance to lysozyme.J. Bacteriol. 113:592.Google Scholar
  17. 17.
    Wagner, B., andM. Wagner. 1975. Immunoelectron microscopic localization of cell wall antigens in streptococci. II. Localization of the group-specific polysaccharide of group C streptococci with ferittin and peroxidase-labeled Helix pomatia agglutinin.261 Bakt. Hyg. I. Abt. Orig. A. 231:81.Google Scholar
  18. 18.
    Park, J.T., J.R. Edwards, andE.M. Wise, Jr. 1974. In vivo studies on the uptake and binding of β-lactam antibiotics in relation to inhibition of wall synthesis and cell death.Ann. N. Y. Acad. Sci. 235:300.Google Scholar
  19. 19.
    Noller, E.C., andS.E. Hartsell. 1961. Bacteriolysis of enterobacteriaceae. I. Lysis by four lytic systems utilizing lysozyme.J. Bacteriol. 81:482.Google Scholar
  20. 20.
    Zeya, H.I., andJ.K. Spitznagel. 1966. Cationic proteins of polymorphonuclear leukocyte lysosomes. II. Composition properties and mechanisms of antibacterial action.J. Bacteriol. 91:755.Google Scholar
  21. 21.
    Hirsch, J.G. 1958. Bactericidal action of histone.J. Exp. Med. 108:925.Google Scholar
  22. 22.
    Brumfitt, W., A.C. Wardlaw, andJ.T. Park. 1958. Development of lysozyme-resistance inMicrococcus lysodeikticus and its association with an increasedO-acetyl content of the cell wall.Nature 181:1783.Google Scholar
  23. 23.
    Mandelstam, M., andJ.L. Strominger. 1961. On the structure of the cell wall ofStaphylococcus aureus (Copenhagen).Biochem. Biophys. Res. Commun. 5:466.Google Scholar
  24. 24.
    Ginsburg, I., andM.N. Sela. 1976. The role of leukocytes and their hydrolases in the persistence, degradation and transport of bacterial constituents in tissues: Relation to chronic inflammatory processes in staphylococcal, streptococcal and mycobacterial infections and in chronic periodontal disease.Crit. Rev. Microbiol. 4:249.Google Scholar
  25. 25.
    Morse, S.I. 1965. Biological attributes of staphylococcal cell walls.Ann. N.Y. Acad. Sci. 128:191.Google Scholar
  26. 26.
    Krause, R.M., andM. McCarty. 1961. Studies on the chemical structure of the streptococcal cell wall.J. Exp. Med. 114:127.Google Scholar
  27. 27.
    Perkins, H.R. 1965. The action of hot formamide on bacterial cell walls.Biochem. J. 95:876.Google Scholar
  28. 28.
    Strominger, J.L., andJ.M. Ghusen. 1968. Mechanism of enzymatic bacteriolysis.Science 156:213.Google Scholar
  29. 29.
    Fleck, J., J.P. Martin, andM. Mock. 1974. Action of lysozyme on penicillin-induced filaments ofProteus vulgaris.J. Bacteriol. 120:929.Google Scholar
  30. 30.
    Suganuma, A. 1972. Fine structure of staphylococci: Electron microscopy.In The Staphylococci, J.O. Cohen, editor. Interscience, New York.21.Google Scholar
  31. 31.
    Makao, M., E. Kitanaka, K. Ochiai, andS. Nakazawa. 1972. Cell wall synthesis inStaphylococcus aureus in the presence of protein synthesis inhibitory agents. I. Lincomycin, clindomycin, macrolide antibiotics.Jpn. J. Microbiol. 16:403.Google Scholar
  32. 32.
    Schwab, J.H., W.J. Cromartie, J.H. Ohanian, andJ.E. Craddock. 1967. Association of experimental chronic arthritis with the persistence of group A streptococcal cell walls in the articular tissue.J. Bacteriol. 94:1728.Google Scholar
  33. 33.
    Stein, H., R. Yarom, S. Levine, T. Dishon, andI. Ginsburg. 1973. Chronic and self-perpetuating arthritis induced in the rabbit by cell-free extracts of group A streptococci. I. Description of the model.Proc. Soc. Exp. Biol. Med. 143:1106.Google Scholar
  34. 34.
    Page, C.R., P. Davies, andA.C. Allison. 1974. Participation of mononuclear phagocytes in chronic inflammatory diseases.J. Reticuloendothel. Soc. 15:413.Google Scholar
  35. 35.
    Ginsburg, I., andR. Trost. 1971. Localization of group A streptococci and titanium dioxide particles in arthritic and hepatic lesions in the rabbit.J. Infect. Dis. 123:792.Google Scholar
  36. 36.
    Ginsburg, I., S. Mitrani, N. Ne'eman, andM. Lahav. 1975. Granulomata in streptococcal inflammation: Mechanisms of localization transport and degradation of streptococci in inflammatory sites.In Mononuclear Phagocytes in Immunity, Infection and Pathology. R. van Furth, editor. Blackwell Scientific, Oxford. 981.Google Scholar

Copyright information

© Plenum Publishing Corporation 1976

Authors and Affiliations

  • C. Efrati
    • 1
    • 2
  • T. Sacks
    • 1
    • 2
  • N. Ne'eman
    • 1
    • 2
  • M. Lahav
    • 1
    • 2
  • I. Ginsburg
    • 1
    • 2
  1. 1.Department of Oral Biology, Faculty of Dental MedicineHebrew University-Hadassah Medical CenterJerusalemIsrael
  2. 2.Department of Clinical Microbiology, Faculty of MedicineHebrew University-Hadassah Medical CenterJerusalemIsrael

Personalised recommendations