Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Sep;172(9):5382–5393. doi: 10.1128/jb.172.9.5382-5393.1990

Glycerophospholipid:cholesterol acyltransferase complexed with lipopolysaccharide (LPS) is a major lethal exotoxin and cytolysin of Aeromonas salmonicida: LPS stabilizes and enhances toxicity of the enzyme.

K K Lee 1, A E Ellis 1
PMCID: PMC213203  PMID: 2394687

Abstract

An extracellular lethal toxin produced by Aeromonas salmonicida was purified by fast-protein liquid ion-exchange chromatography. The toxin is composed of glycerophospholipid:cholesterol acyltransferase (GCAT) (molecular mass, 25 kilodaltons) aggregated with lipopolysaccharide (LPS), the GCAT/LPS complex having a molecular mass of about 2,000 kilodaltons, estimated by gel filtration chromatography. The toxin is lethal for Atlantic salmon (Salmo salar L.) at a concentration of 0.045 micrograms of protein per g of body weight. The toxin is a hemolysin (T-lysin, active on fish erythrocytes), leukocytolysin, and cytotoxin. Antiserum to the purified toxin neutralized the lethal toxicity of the crude extracellular toxins, indicating this toxin to be the major lethal factor produced by A. salmonicida. In the crude extracellular products, small amounts of free GCAT were also present. This has been purified, and its activities and properties have been compared with those of the GCAT/LPS complex. The presence of LPS did not influence the GCAT activity of the enzyme with egg yolk or phosphatidylcholine (lecithin) as a substrate, but the specific hemolytic activity and lethal toxicity was about eightfold higher in the complexed form. Furthermore, the free GCAT was more susceptible to proteolytic and heat inactivation than was the GCAT/LPS complex. Recombination of LPS (phenol extracted from extracellular products of A. salmonicida) with free GCAT enhanced the hemolytic activity, lethal toxicity, and heat stability of the latter but did not influence its lecithinase activity. In native polyacrylamide gel electrophoresis, the GCAT/LPS complex and the recombined GCAT-LPS both showed a high-molecular-mass band which did not enter the gel, while the free GCAT produced a single band with low molecular mass. In isoelectric focusing gels, the GCAT/LPS and recombined GCAT-LPS produced a nonfocusing smear with pIs from pI 5.0 to 5.8, while the free GCAT produced a single band with pI 4.3. These data show that free GCAT can combine with LPS to produce a high-molecular-mass complex with enhanced toxicity and heat stability compared with those of free GCAT, similar to the preexisting GCAT/LPS complex, and indicate that the LPS moiety of the toxin plays an active role in toxicity.

Full text

PDF
5382

Images in this article

5385Image
on p.5385
5385Image
on p.5385
5387Image
on p.5387
5387Image
on p.5387
5387Image
on p.5387
5387Image
on p.5387
5388Image
on p.5388
5388Image
on p.5388
5388Image
on p.5388
5389Image
on p.5389
5389Image
on p.5389

Selected References

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

  1. Bohach G. A., Snyder I. S. Chemical and immunological analysis of the complex structure of Escherichia coli alpha-hemolysin. J Bacteriol. 1985 Dec;164(3):1071–1080. doi: 10.1128/jb.164.3.1071-1080.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bohach G. A., Snyder I. S. Composition of affinity-purified alpha-hemolysin of Escherichia coli. Infect Immun. 1986 Aug;53(2):435–437. doi: 10.1128/iai.53.2.435-437.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Buckley J. T., Halasa L. N., MacIntyre S. Purification and partial characterization of a bacterial phospholipid: cholesterol acyltransferase. J Biol Chem. 1982 Mar 25;257(6):3320–3325. [PubMed] [Google Scholar]
  5. Buckley J. T. Substrate specificity of bacterial glycerophospholipid:cholesterol acyltransferase. Biochemistry. 1982 Dec 21;21(26):6699–6703. doi: 10.1021/bi00269a013. [DOI] [PubMed] [Google Scholar]
  6. Chart H., Shaw D. H., Ishiguro E. E., Trust T. J. Structural and immunochemical homogeneity of Aeromonas salmonicida lipopolysaccharide. J Bacteriol. 1984 Apr;158(1):16–22. doi: 10.1128/jb.158.1.16-22.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Evenberg D., Versluis R., Lugtenberg B. Biochemical and immunological characterization of the cell surface of the fish pathogenic bacterium Aeromonas salmonicida. Biochim Biophys Acta. 1985 May 14;815(2):233–244. doi: 10.1016/0005-2736(85)90294-9. [DOI] [PubMed] [Google Scholar]
  8. Fyfe L., Coleman G., Munro A. L. Identification of major common extracellular proteins secreted by Aeromonas salmonicida strains isolated from diseased fish. Appl Environ Microbiol. 1987 Apr;53(4):722–726. doi: 10.1128/aem.53.4.722-726.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hitchcock P. J., Brown T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol. 1983 Apr;154(1):269–277. doi: 10.1128/jb.154.1.269-277.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lee K. K., Ellis A. E. Rapid and sensitive silver-lipopolysaccharide staining using PhastSystem in fast horizontal polyacrylamide gel electrophoresis. Electrophoresis. 1989 Oct;10(10):729–731. doi: 10.1002/elps.1150101014. [DOI] [PubMed] [Google Scholar]
  11. Lee K. K., Ellis A. E. The quantitative relationship of lethality between extracellular protease and extracellular haemolysin of Aeromonas salmonicida in Atlantic salmon (Salmo salar L.). FEMS Microbiol Lett. 1989 Oct 1;52(1-2):127–131. doi: 10.1016/0378-1097(89)90183-3. [DOI] [PubMed] [Google Scholar]
  12. MacIntyre S., Trust T. J., Buckley J. T. Distribution of glycerophospholipid-cholesterol acyltransferase in selected bacterial species. J Bacteriol. 1979 Jul;139(1):132–136. doi: 10.1128/jb.139.1.132-136.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. MacIntyre S., Trust T. J., Buckley J. T. Identification and characterization of outer membrane fragments released by Aeromonas sp. Can J Biochem. 1980 Oct;58(10):1018–1025. doi: 10.1139/o80-138. [DOI] [PubMed] [Google Scholar]
  14. Nomura S., Fujino M., Yamakawa M., Kawahara E. Purification and characterization of salmolysin, an extracellular hemolytic toxin from Aeromonas salmonicida. J Bacteriol. 1988 Aug;170(8):3694–3702. doi: 10.1128/jb.170.8.3694-3702.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Poole K., Braun V. Influence of growth temperature and lipopolysaccharide on hemolytic activity of Serratia marcescens. J Bacteriol. 1988 Nov;170(11):5146–5152. doi: 10.1128/jb.170.11.5146-5152.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sakai D. K. Loss of virulence in a protease-deficient mutant of Aeromonas salmonicida. Infect Immun. 1985 Apr;48(1):146–152. doi: 10.1128/iai.48.1.146-152.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stuer W., Jaeger K. E., Winkler U. K. Purification of extracellular lipase from Pseudomonas aeruginosa. J Bacteriol. 1986 Dec;168(3):1070–1074. doi: 10.1128/jb.168.3.1070-1074.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  19. Ways P., Hanahan D. J. Characterization and quantification of red cell lipids in normal man. J Lipid Res. 1964 Jul;5(3):318–328. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES