Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1985 May;48(2):498–506. doi: 10.1128/iai.48.2.498-506.1985

Effects of Pseudomonas aeruginosa cytotoxin on human serum and granulocytes and their microbicidal, phagocytic, and chemotactic functions.

A L Baltch, M C Hammer, R P Smith, T G Obrig, J V Conroy, M B Bishop, M A Egy, F Lutz
PMCID: PMC261357  PMID: 3921465

Abstract

The effect of Pseudomonas aeruginosa cytotoxin on human granulocytes (PMNs) and pooled human serum was studied by hemacytometer counts, phagocytic, bactericidal, and chemotaxis assays, and by transmission electron microscopy. The optimal assay conditions for phagocytosis of 75Se-labeled P. aeruginosa 1348A included 20% pooled human serum and a ratio of one PMN to between 10 and 20 bacteria. For the bactericidal assay, 20% pooled human serum and a ratio of one PMN to between one and five bacteria were used. Chemotaxis of PMNs was studied by agarose gel technique with 10(-7) M f-Met-Leu-Phe or 0.01 to 35 micrograms of cytotoxin per ml as a chemoattractant. The degree of PMN destruction was dependent on cytotoxin concentrations and PMN exposure time to cytotoxin. Virtually complete PMN lysis was observed after a 2-h exposure to 6 to 10 micrograms of cytotoxin per ml. PMN exposure to 2 micrograms of cytotoxin per ml for as long as 2 h had no adverse effect on phagocytosis. PMN exposure to greater than or equal to 4 micrograms of cytotoxin per ml for 2 h demonstrated a significant decrease in the percentage of bacteria killed. The results of experiments designed to separate cytotoxin effect on PMN lysis from the effect on PMN bactericidal capacity showed that there is an effect of cytotoxin on PMN bactericidal function. PMN exposure to 4 micrograms of cytotoxin per ml for 30 min caused a significant decrease in PMN migration. Cytotoxin had no chemoattractant qualities or effect on pooled human serum as studied by chemotaxis and phagocytosis assays. Although a cytotoxin concentration of greater than or equal to 2 micrograms/ml was required to demonstrate PMN ultrastructural changes observed in transmission electron microscopy studies, at a concentration of 0.1 microgram/ml, cytotoxin caused an impairment in the integrity of the PMN membrane, allowing a low-molecular-weight substance (ruthenium red) to enter into the cytoplasm. Cytotoxin may be an important factor in the pathogenesis and in the high mortality rate of patients with P. aeruginosa infections.

Full text

PDF
498

Images in this article

502Image
on p.502
502Image
on p.502
503Image
on p.503
503Image
on p.503
504Image
on p.504
504Image
on p.504

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andersen B. R., Duncan J. L. Activation of human neutrophil metabolism by streptolysin O. J Infect Dis. 1980 May;141(5):680–685. doi: 10.1093/infdis/141.5.680. [DOI] [PubMed] [Google Scholar]
  2. Andriole V. T. Pseudomonas bacteremia: can antibiotic therapy improve survival? J Lab Clin Med. 1979 Aug;94(2):196–200. [PubMed] [Google Scholar]
  3. Baltch A. L., Griffin P. E., Hammer M. Pseudomonas aeruginosa bacteremia: relationship of bacterial enzyme production and pyocine types with clinical prognosis in 100 patients. J Lab Clin Med. 1979 Apr;93(4):600–606. [PubMed] [Google Scholar]
  4. Baltch A. L., Griffin P. E. Pseudomonas aeruginosa bacteremia: a clinical study of 75 patients. Am J Med Sci. 1977 Sep-Oct;274(2):119–129. [PubMed] [Google Scholar]
  5. Baltch A. L., Hammer M., Smith R. P., Sutphen N. Pseudomonas aeruginosa bacteremia: susceptibility of 100 blood culture isolates to seven antimicrobial agents and its clinical significance. J Lab Clin Med. 1979 Aug;94(2):201–214. [PubMed] [Google Scholar]
  6. Cavalieri S. J., Snyder I. S. Effect of Escherichia coli alpha-hemolysin on human peripheral leukocyte function in vitro. Infect Immun. 1982 Sep;37(3):966–974. doi: 10.1128/iai.37.3.966-974.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cross A. S., Sadoff J. C., Iglewski B. H., Sokol P. A. Evidence for the role of toxin A in the pathogenesis of infection with Pseudomonas aeruginosa in humans. J Infect Dis. 1980 Oct;142(4):538–546. doi: 10.1093/infdis/142.4.538. [DOI] [PubMed] [Google Scholar]
  8. Flick M. R., Cluff L. E. Pseudomonas bacteremia. Review of 108 cases. Am J Med. 1976 Apr;60(4):501–508. doi: 10.1016/0002-9343(76)90716-6. [DOI] [PubMed] [Google Scholar]
  9. Foley F. D., Greenawald K. A., Nash G., Pruitt B. A., Jr Pathology of Pseudomonas infection. Tex Med. 1969 Oct;65(10):36–39. [PubMed] [Google Scholar]
  10. Frimmer M., Homann J., Petzinger E., Rufeger U., Scharmann W. Comparative studies on isolated rat hepatocytes and AS-30D hepatoma cells with leucocidin from Pseudomonas aeruginosa. Naunyn Schmiedebergs Arch Pharmacol. 1976 Oct;295(1):63–69. doi: 10.1007/BF00509774. [DOI] [PubMed] [Google Scholar]
  11. Gemmell C. G., Peterson P. K., Schmeling D. J., Quie P. G. Effect of staphylococcal alpha-toxin on phagocytosis of staphylococci by human polymorphonuclear leukocytes. Infect Immun. 1982 Dec;38(3):975–980. doi: 10.1128/iai.38.3.975-980.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. HIRSCH J. G., STRAUSS B. STUDIES ON HEAT-LABILE OPSONIN IN RABBIT SERUM. J Immunol. 1964 Jan;92:145–154. [PubMed] [Google Scholar]
  13. Hammer M. C., Baltch A. L., Sutphen N. T., Smith R. P., Conroy J. V. Pseudomonas aeruginosa: quantitation of maximum phagocytic and bactericidal capabilities of normal human granulocytes. J Lab Clin Med. 1981 Dec;98(6):938–948. [PubMed] [Google Scholar]
  14. Harboe N., Ingild A. Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand J Immunol Suppl. 1973;1:161–164. doi: 10.1111/j.1365-3083.1973.tb03798.x. [DOI] [PubMed] [Google Scholar]
  15. Hegner D., Petter A., Kroker R., Anwer M. S., Scharmann W., Breuninger V. Leucocidin from Pseudomonas aeruginosa and membrane functions. Naunyn Schmiedebergs Arch Pharmacol. 1976;293(1):49–55. doi: 10.1007/BF00498870. [DOI] [PubMed] [Google Scholar]
  16. Helm E., Stille W. Klinik und Therapie der Sepsis durch Pseudomonas aeruginosa. Dtsch Med Wochenschr. 1972 Oct 20;97(42):1584–1589. doi: 10.1055/s-0028-1107608. [DOI] [PubMed] [Google Scholar]
  17. Hirayama T., Kato I. Mode of cytotoxic action of pseudomonal leukocidin on phosphatidylinositol metabolism and activation of lysosomal enzyme in rabbit leukocytes. Infect Immun. 1984 Jan;43(1):21–27. doi: 10.1128/iai.43.1.21-27.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johnson M. K., Boese-Marrazzo D., Pierce W. A., Jr Effects of pneumolysin on human polymorphonuclear leukocytes and platelets. Infect Immun. 1981 Oct;34(1):171–176. doi: 10.1128/iai.34.1.171-176.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. LIU P. V., ABE Y., BATES J. L. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. J Infect Dis. 1961 Mar-Apr;108:218–228. doi: 10.1093/infdis/108.2.218. [DOI] [PubMed] [Google Scholar]
  20. Leppla S. H. Large-scale purification and characterization of the exotoxin of Pseudomonas aeruginosa. Infect Immun. 1976 Oct;14(4):1077–1086. doi: 10.1128/iai.14.4.1077-1086.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lutz F., Grieshaber S., Schmidt K. Permeability changes of Ehrlich mouse ascites tumor cells induced by a cytotoxin from Pseudomonas aeruginosa. Naunyn Schmiedebergs Arch Pharmacol. 1982 Jul;320(1):78–80. doi: 10.1007/BF00499077. [DOI] [PubMed] [Google Scholar]
  22. Lutz F. Purification of a cytotoxic protein from Pseudomonas aeruginosa. Toxicon. 1979;17(5):467–475. doi: 10.1016/0041-0101(79)90280-0. [DOI] [PubMed] [Google Scholar]
  23. MONCRIEF J. A., TEPLITZ C. CHANGING CONCEPTS IN BURN SEPSIS. J Trauma. 1964 Mar;4:233–245. doi: 10.1097/00005373-196403000-00011. [DOI] [PubMed] [Google Scholar]
  24. Nelson R. D., Quie P. G., Simmons R. L. Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J Immunol. 1975 Dec;115(6):1650–1656. [PubMed] [Google Scholar]
  25. Obrig T. G., Baltch A. L., Moran T. P., Mudzinski S. P., Smith R. P., Lutz F. Effect of Pseudomonas aeruginosa cytotoxin on thymidine incorporation by murine splenocytes. Infect Immun. 1984 Sep;45(3):756–760. doi: 10.1128/iai.45.3.756-760.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Paton J. C., Ferrante A. Inhibition of human polymorphonuclear leukocyte respiratory burst, bactericidal activity, and migration by pneumolysin. Infect Immun. 1983 Sep;41(3):1212–1216. doi: 10.1128/iai.41.3.1212-1216.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Root R. K., Rosenthal A. S., Balestra D. J. Abnormal bactericidal, metabolic, and lysosomal functions of Chediak-Higashi Syndrome leukocytes. J Clin Invest. 1972 Mar;51(3):649–665. doi: 10.1172/JCI106854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Scharmann W. Cytotoxic effects of leukocidin from Pseudomonas aeruginosa on polymorphonuclear leukocytes from cattle. Infect Immun. 1976 Mar;13(3):836–843. doi: 10.1128/iai.13.3.836-843.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Scharmann W. Formation and isolation of leucocidin from Pseudomonas aeruginosa. J Gen Microbiol. 1976 Apr;93(2):283–291. doi: 10.1099/00221287-93-2-283. [DOI] [PubMed] [Google Scholar]
  30. Scharmann W., Jacob F., Porstendörfer J. The cytotoxic action of leucocidan from Pseudomonas aeruginosa on human polymorphonuclear leucocytes. J Gen Microbiol. 1976 Apr;93(2):303–308. doi: 10.1099/00221287-93-2-303. [DOI] [PubMed] [Google Scholar]
  31. Scharmann W. Purification and characterization of leucocidin from Pseudomonas aeruginosa. J Gen Microbiol. 1976 Apr;93(2):292–302. doi: 10.1099/00221287-93-2-292. [DOI] [PubMed] [Google Scholar]
  32. Van Epps D. E., Andersen B. R. Streptolysin O inhibition of neutrophil chemotaxis and mobility: nonimmune phenomenon with species specificity. Infect Immun. 1974 Jan;9(1):27–33. doi: 10.1128/iai.9.1.27-33.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Watanabe I., Donahue S., Hoggatt N. Method for electron microscopic studies of circulating human leukocytes and observations on their fine structure. J Ultrastruct Res. 1967 Oct 31;20(5):366–382. doi: 10.1016/s0022-5320(67)80106-0. [DOI] [PubMed] [Google Scholar]
  34. Weiner R. N., Reinacher M. Lower nephron toxicity of a highly purified cytotoxin from Pseudomonas aeruginosa in rats. Exp Mol Pathol. 1982 Oct;37(2):249–271. doi: 10.1016/0014-4800(82)90040-5. [DOI] [PubMed] [Google Scholar]
  35. Young L. S. The role of exotoxins in the pathogenesis of Pseudomonas aeruginosa infections. J Infect Dis. 1980 Oct;142(4):626–630. doi: 10.1093/infdis/142.4.626. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES