Abstract
Granule contents from rat polymorphonuclear neutrophils were prepared by extraction with 0.2 M acetate (pH 4), dialyzed against phosphate-buffered saline (pH 7), and tested for bactericidal activity. Bactericidal assays consisted of mixing rat granule extract with 1 × 103 to 3 × 103 bacterial cells per ml at 37°C for 1 h in a medium suited for bacterial growth. The granule extract demonstrated a distinctive dose-dependent bactericidal activity against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT-2, independent of added hydrogen peroxide or other active oxygen derivatives. The rough bacterial mutants showed an ordered increase in sensitivity to the rat lysosomal extracts inversely related to the length of their lipopolysaccharide carbohydrate side chains. Fractionation of the rat polymorphonuclear neutrophil granule extract with Sephadex G-100 column chromatography revealed an elution profile containing three major areas (peaks) of protein. Polyacrylamide gel electrophoresis and examination of enzymatic activity showed that these peaks contained myeloperoxidase (peak A), neutral protease (peak B), and lysozyme (peak C) activities. Also observed in peak C were cationic protein species whose cathodal electrophoretic migration was faster than that for lysozyme. Only peak C exhibited a bactericidal activity against the rough mutants of S. typhimurium LT-2 similar to that obtained for the unfractionated granule extract, with susceptibility of the bacterial mutants increasing with a progressive loss of carbohydrate residues in the lipopolysaccharide of the cell wall. The bactericidal activity of the peak C protein fraction was dose dependent. Boiling the unfractionated granule extract or peak C for 30 min had little affect on their antimicrobial activity when reacted against a deep-rough lipopolysaccharide mutant. However, trypsin pretreatment of these fractions significantly reduced their antimicrobial activity for the same mutant chemotype.
Full text
PDF







Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Brune K., Spitznagel J. K. Peroxidaseless chicken leukocytes: isolation and characterization of antibacterial granules. J Infect Dis. 1973 Jan;127(1):84–94. doi: 10.1093/infdis/127.1.84. [DOI] [PubMed] [Google Scholar]
- Buck P., Rest R. F. Effects of human neutrophil granule extracts on macromolecular synthesis in Neisseria gonorrhoeae. Infect Immun. 1981 Aug;33(2):426–433. doi: 10.1128/iai.33.2.426-433.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Calamai E. G., Spitznagel J. K. Characterization of rat polymorphonuclear leukocyte subcellular granules. Lab Invest. 1982 Jun;46(6):597–604. [PubMed] [Google Scholar]
- Friedberg D., Shilo M. Interaction of Gram-Negative Bacteria with the Lysosomal Fraction of Polymorphonuclear Leukocytes I. Role of Cell Wall Composition of Salmonella typhimurium. Infect Immun. 1970 Mar;1(3):305–310. doi: 10.1128/iai.1.3.305-310.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goren M. B. Phagocyte lysosomes: interactions with infectious agents, phagosomes, and experimental perturbations in function. Annu Rev Microbiol. 1977;31:507–533. doi: 10.1146/annurev.mi.31.100177.002451. [DOI] [PubMed] [Google Scholar]
- Klebanoff S. J. Myeloperoxidase-halide-hydrogen peroxide antibacterial system. J Bacteriol. 1968 Jun;95(6):2131–2138. doi: 10.1128/jb.95.6.2131-2138.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klebanoff S. J. Oxygen metabolism and the toxic properties of phagocytes. Ann Intern Med. 1980 Sep;93(3):480–489. doi: 10.7326/0003-4819-93-3-480. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Lyman M. B., Steward J. P., Roantree R. J. Characterization of the virulence and antigenic structure of Salmonella typhimurium strains with lipopolysaccharide core defects. Infect Immun. 1976 Jun;13(6):1539–1542. doi: 10.1128/iai.13.6.1539-1542.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Modrzakowski M. C., Cooney M. H., Martin L. E., Spitznagel J. K. Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes. Infect Immun. 1979 Mar;23(3):587–591. doi: 10.1128/iai.23.3.587-591.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Modrzakowski M. C., Paranavitana C. M. Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes: role of bacterial membrane lipid. Infect Immun. 1981 May;32(2):668–674. doi: 10.1128/iai.32.2.668-674.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Modrzakowski M. C., Spitznagel J. K. Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes: antagonism of granule cationic proteins by lipopolysaccharide. Infect Immun. 1979 Aug;25(2):597–602. doi: 10.1128/iai.25.2.597-602.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Odeberg H., Olsson I. Antibacterial activity of cationic proteins from human granulocytes. J Clin Invest. 1975 Nov;56(5):1118–1124. doi: 10.1172/JCI108186. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Odeberg H., Olsson I. Mechanisms for the microbicidal activity of cationic proteins of human granulocytes. Infect Immun. 1976 Dec;14(6):1269–1275. doi: 10.1128/iai.14.6.1269-1275.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Osserman E. F., Lawlor D. P. Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J Exp Med. 1966 Nov 1;124(5):921–952. doi: 10.1084/jem.124.5.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rest R. F., Cooney M. H., Spitznagel J. K. Bactericidal activity of specific and azurophil granules from human neutrophils: studies with outer-membrane mutants of Salmonella typhimurium LT-2. Infect Immun. 1978 Jan;19(1):131–137. doi: 10.1128/iai.19.1.131-137.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rest R. F., Cooney M. H., Spitznagel J. K. Susceptibility of lipopolysaccharide mutants to the bactericidal action of human neutrophil lysosomal fractions. Infect Immun. 1977 Apr;16(1):145–151. doi: 10.1128/iai.16.1.145-151.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rest R. F., Spitznagel J. K. Myeloperoxidase-Cl--H2O2 bactericidal system: effect of bacterial membrane structure and growth conditions. Infect Immun. 1978 Mar;19(3):1110–1112. doi: 10.1128/iai.19.3.1110-1112.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Root R. K., Cohen M. S. The microbicidal mechanisms of human neutrophils and eosinophils. Rev Infect Dis. 1981 May-Jun;3(3):565–598. doi: 10.1093/clinids/3.3.565. [DOI] [PubMed] [Google Scholar]
- SHUGAR D. The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochim Biophys Acta. 1952 Mar;8(3):302–309. doi: 10.1016/0006-3002(52)90045-0. [DOI] [PubMed] [Google Scholar]
- Smit J., Kamio Y., Nikaido H. Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J Bacteriol. 1975 Nov;124(2):942–958. doi: 10.1128/jb.124.2.942-958.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Starkey P. M., Barrett A. J. Neutral proteinases of human spleen. Purification and criteria for homogeneity of elastase and cathepsin G. Biochem J. 1976 May 1;155(2):255–263. doi: 10.1042/bj1550255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walton E. The preparation, properties and action on Staphylococcus aureus of purified fractions from the cationic proteins of rabbit polymorphonuclear leucocytes. Br J Exp Pathol. 1978 Aug;59(4):416–431. [PMC free article] [PubMed] [Google Scholar]
- Zeya H. I., Spitznagel J. K. Isolation of polymorphonuclear leukocyte granules from rabbit bone marrow. Lab Invest. 1971 Mar;24(3):237–245. [PubMed] [Google Scholar]