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. 1995 Aug;61(8):2845–2851. doi: 10.1128/aem.61.8.2845-2851.1995

Characterization of lipopolysaccharide heterogeneity in Salmonella enteritidis by an improved gel electrophoresis method.

J Guard-Petter 1, B Lakshmi 1, R Carlson 1, K Ingram 1
PMCID: PMC167560  PMID: 7487016

Abstract

Salmonella enteritidis field isolates of different phage types and pathogenicities were assessed for changes in lipopolysaccharide (LPS) structure, using an improved method of polyacrylamide gel electrophoresis (PAGE) that revealed the same degree of structural detail as mass spectroscopy. The method allowed characterization of an LPS chemotype that may be associated, regardless of phage type, with increased virulence of S. enteritidis. The virulent variant SE6-E21, which efficiently contaminates eggs and yields high numbers of organisms from chick spleens, had an O-antigen/core ratio of 2.8, as determined from gels by densitometry, and 1.67 micrograms of mannose per microgram of 2-keto-3-deoxy-octulosonic acid (KDO), while the avirulent variant SE6-E5 had O-antigen/core ratios of 1.2 and 1.00. The association between O antigen and virulence was also seen on analysis of five new field isolates. One of the new field isolates generated a mixed population of smooth and semismooth variants in agreement with its mixed virulence in chicks. When LPS was purified from large-volume cultures, only the most virulent isolate yielded high amounts of O antigen (1.6 micrograms of mannose per microgram of KDO), while the other isolates had ratios characteristic of semismooth variants (< or = 1.0 microgram of mannose per microgram of KDO), including the isolate of mixed virulence. These results indicate that the improved PAGE method might provide a rapid, sensitive, in vitro assessment of field isolate virulence prior to the performance of definitive infectivity trials.

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Selected References

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  1. Brade H., Brade L., Schade U., Zähringer U., Holst O., Kuhn H. M., Rozalski A., Röhrscheidt E., Rietschel E. T. Structure, endotoxicity, immunogenicity and antigenicity of bacterial lipopolysaccharides (endotoxins, O-antigens). Prog Clin Biol Res. 1988;272:17–45. [PubMed] [Google Scholar]
  2. Darveau R. P., Hancock R. E. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol. 1983 Aug;155(2):831–838. doi: 10.1128/jb.155.2.831-838.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dubray G., Bezard G. A highly sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 15;119(2):325–329. doi: 10.1016/0003-2697(82)90593-0. [DOI] [PubMed] [Google Scholar]
  4. Fomsgaard A., Freudenberg M. A., Galanos C. Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels. J Clin Microbiol. 1990 Dec;28(12):2627–2631. doi: 10.1128/jcm.28.12.2627-2631.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Helander I. M., Moran A. P., Mäkelä P. H. Separation of two lipopolysaccharide populations with different contents of O-antigen factor 122 in Salmonella enterica serovar typhimurium. Mol Microbiol. 1992 Oct;6(19):2857–2862. doi: 10.1111/j.1365-2958.1992.tb01465.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Karibian D., Deprun C., Caroff M. Comparison of lipids A of several Salmonella and Escherichia strains by 252Cf plasma desorption mass spectrometry. J Bacteriol. 1993 May;175(10):2988–2993. doi: 10.1128/jb.175.10.2988-2993.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kido N., Ohta M., Kato N. Detection of lipopolysaccharides by ethidium bromide staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Bacteriol. 1990 Feb;172(2):1145–1147. doi: 10.1128/jb.172.2.1145-1147.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Klena J. D., Ashford R. S., 2nd, Schnaitman C. A. Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen. J Bacteriol. 1992 Nov;174(22):7297–7307. doi: 10.1128/jb.174.22.7297-7307.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Komuro T., Galanos C. Analysis of Salmonella lipopolysaccharides by sodium deoxycholate-polyacrylamide gel electrophoresis. J Chromatogr. 1988 Oct 26;450(3):381–387. doi: 10.1016/s0021-9673(01)83593-7. [DOI] [PubMed] [Google Scholar]
  11. MacLachlan P. R., Kadam S. K., Sanderson K. E. Cloning, characterization, and DNA sequence of the rfaLK region for lipopolysaccharide synthesis in Salmonella typhimurium LT2. J Bacteriol. 1991 Nov;173(22):7151–7163. doi: 10.1128/jb.173.22.7151-7163.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Palva E. T., Mäkelä P. H. Lipopolysaccharide heterogeneity in Salmonella typhimurium analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eur J Biochem. 1980;107(1):137–143. doi: 10.1111/j.1432-1033.1980.tb04634.x. [DOI] [PubMed] [Google Scholar]
  13. Petter J. G. Detection of two smooth colony phenotypes in a Salmonella enteritidis isolate which vary in their ability to contaminate eggs. Appl Environ Microbiol. 1993 Sep;59(9):2884–2890. doi: 10.1128/aem.59.9.2884-2890.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Raetz C. R., Ulevitch R. J., Wright S. D., Sibley C. H., Ding A., Nathan C. F. Gram-negative endotoxin: an extraordinary lipid with profound effects on eukaryotic signal transduction. FASEB J. 1991 Sep;5(12):2652–2660. doi: 10.1096/fasebj.5.12.1916089. [DOI] [PubMed] [Google Scholar]
  15. Ryan J. M., Conrad H. E. Structural heterogeneity in the lipopolysaccharide of Salmonella newington. Arch Biochem Biophys. 1974 Jun;162(2):530–535. doi: 10.1016/0003-9861(74)90213-6. [DOI] [PubMed] [Google Scholar]
  16. Schnaitman C. A., Klena J. D. Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol Rev. 1993 Sep;57(3):655–682. doi: 10.1128/mr.57.3.655-682.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. St Louis M. E., Morse D. L., Potter M. E., DeMelfi T. M., Guzewich J. J., Tauxe R. V., Blake P. A. The emergence of grade A eggs as a major source of Salmonella enteritidis infections. New implications for the control of salmonellosis. JAMA. 1988 Apr 8;259(14):2103–2107. [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]

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