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. 1995 Oct;63(10):4115–4120. doi: 10.1128/iai.63.10.4115-4120.1995

Quantitation and biological properties of released and cell-bound lipooligosaccharides from nontypeable Haemophilus influenzae.

X X Gu 1, C M Tsai 1, M A Apicella 1, D J Lim 1
PMCID: PMC173578  PMID: 7558327

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media in children. NTHi releases lipooligosaccharide (LOS) as outer membrane fragments during its growth. The release of LOS may play an important role in the pathogenicity of otitis media caused by this organism. The amounts of LOS in bacterial cells and growth media for five NTHi strains were determined by quantitative silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These strains were estimated to have 1.6 x 10(6) to 4.8 x 10(6) LOS molecules per bacterium. During a 3-day growth period, these NTHi strains released variable but significant amounts of LOS into the growth medium. Cells started to release detectable amounts of LOS into the medium at 2 to 5 h and continued to do so for up to 48 or 72 h. The concentrations of LOS in the culture supernatants released by these five strains were 10 to 55 micrograms/ml at 24 h and 40 to 100 micrograms/ml at 72 h, which was 34 to 189% of the cell-bound LOS concentration. The biological properties of released and cell-bound LOSs from two representative strains were compared. Released LOS showed an approximately 10-fold increase in inducing human monocytes to produce tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6, a 13- to 28-fold increase in mouse lethal toxicity, and a 16- to 37-fold increase in the clotting of Limulus amebocyte lysate. These results suggested that released LOS or its inflammatory mediators play a more important role than the LOS in bacteria in the pathogenicity of otitis media caused by this organism.

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Selected References

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  1. Abrahamsen T. G., Carter C. S., Read E. J., Rubin M., Goetzman H. G., Lizzio E. F., Lee Y. L., Hanson M., Pizzo P. A., Hoffman T. Stimulatory effect of counterflow centrifugal elutriation in large-scale separation of peripheral blood monocytes can be reversed by storing the cells at 37 degrees C. J Clin Apher. 1991;6(1):48–53. doi: 10.1002/jca.2920060110. [DOI] [PubMed] [Google Scholar]
  2. Andersen B. M. Endotoxin release from neisseria meningitidis. Relationship between key bacterial characteristics and meningococcal disease. Scand J Infect Dis Suppl. 1989;64:1–43. doi: 10.3109/inf.1989.21.suppl-64.01. [DOI] [PubMed] [Google Scholar]
  3. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartková G., Majtán V., Ciznár I. Properties of free and bound Citrobacter freundii lipopolysaccharides. J Hyg Epidemiol Microbiol Immunol. 1986;30(4):455–463. [PubMed] [Google Scholar]
  5. Bishop D. G., Work E. An extracellular glycolipid produced by Escherichia coli grown under lysine-limiting conditions. Biochem J. 1965 Aug;96(2):567–576. doi: 10.1042/bj0960567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cadieux J. E., Kuzio J., Milazzo F. H., Kropinski A. M. Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa. J Bacteriol. 1983 Aug;155(2):817–825. doi: 10.1128/jb.155.2.817-825.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cerami A., Beutler B. The role of cachectin/TNF in endotoxic shock and cachexia. Immunol Today. 1988 Jan;9(1):28–31. doi: 10.1016/0167-5699(88)91353-9. [DOI] [PubMed] [Google Scholar]
  8. Crutchley M. J., Marsh D. G., Cameron J. Free Endotoxin. Nature. 1967 Jun 3;214(5092):1052–1052. doi: 10.1038/2141052a0. [DOI] [PubMed] [Google Scholar]
  9. DeMaria T. F., Briggs B. R., Lim D. J., Okazaki N. Experimental otitis media with effusion following middle ear inoculation of nonviable H influenzae. Ann Otol Rhinol Laryngol. 1984 Jan-Feb;93(1 Pt 1):52–56. doi: 10.1177/000348948409300113. [DOI] [PubMed] [Google Scholar]
  10. DeMaria T. F., Prior R. B., Briggs B. R., Lim D. J., Birck H. G. Endotoxin in middle-ear effusions from patients with chronic otitis media with effusion. J Clin Microbiol. 1984 Jul;20(1):15–17. doi: 10.1128/jcm.20.1.15-17.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Denny F. W. Effect of a toxin produced by Haemophilus influenzae on ciliated respiratory epithelium. J Infect Dis. 1974 Feb;129(2):93–100. doi: 10.1093/infdis/129.2.93. [DOI] [PubMed] [Google Scholar]
  12. Dinarello C. A. Biology of interleukin 1. FASEB J. 1988 Feb;2(2):108–115. [PubMed] [Google Scholar]
  13. Evans M. E., Pollack M. Effect of antibiotic class and concentration on the release of lipopolysaccharide from Escherichia coli. J Infect Dis. 1993 Jun;167(6):1336–1343. doi: 10.1093/infdis/167.6.1336. [DOI] [PubMed] [Google Scholar]
  14. Galanos C., Freudenberg M. A., Reutter W. Galactosamine-induced sensitization to the lethal effects of endotoxin. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5939–5943. doi: 10.1073/pnas.76.11.5939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gibson B. W., Melaugh W., Phillips N. J., Apicella M. A., Campagnari A. A., Griffiss J. M. Investigation of the structural heterogeneity of lipooligosaccharides from pathogenic Haemophilus and Neisseria species and of R-type lipopolysaccharides from Salmonella typhimurium by electrospray mass spectrometry. J Bacteriol. 1993 May;175(9):2702–2712. doi: 10.1128/jb.175.9.2702-2712.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goldstein J., Hoffman T., Frasch C., Lizzio E. F., Beining P. R., Hochstein D., Lee Y. L., Angus R. D., Golding B. Lipopolysaccharide (LPS) from Brucella abortus is less toxic than that from Escherichia coli, suggesting the possible use of B. abortus or LPS from B. abortus as a carrier in vaccines. Infect Immun. 1992 Apr;60(4):1385–1389. doi: 10.1128/iai.60.4.1385-1389.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gu X. X., Tsai C. M. Purification of rough-type lipopolysaccharides of Neisseria meningitidis from cells and outer membrane vesicles in spent media. Anal Biochem. 1991 Aug 1;196(2):311–318. doi: 10.1016/0003-2697(91)90472-6. [DOI] [PubMed] [Google Scholar]
  18. Herriott R. M., Meyer E. Y., Vogt M., Modan M. Defined medium for growth of Haemophilus influenzae. J Bacteriol. 1970 Feb;101(2):513–516. doi: 10.1128/jb.101.2.513-516.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hochstein H. D., Elfin R. J., Cooper J. F., Seligmann E. B., Jr, Wolff S. M. Further developments of Limulus Amebocyte Lysate test. Bull Parenter Drug Assoc. 1973 May-Jun;27(3):139–148. [PubMed] [Google Scholar]
  21. Hozbor D., Rodriguez M. E., Samo A., Lagares A., Yantorno O. Release of lipopolysaccharide during Bordetella pertussis growth. Res Microbiol. 1993 Mar-Apr;144(3):201–209. doi: 10.1016/0923-2508(93)90045-4. [DOI] [PubMed] [Google Scholar]
  22. Iino Y., Kaneko Y., Takasaka T. Endotoxin in middle ear effusions tested with Limulus assay. Acta Otolaryngol. 1985 Jul-Aug;100(1-2):42–50. doi: 10.3109/00016488509108586. [DOI] [PubMed] [Google Scholar]
  23. Iino Y., Yuasa R., Kaneko Y., Takasaka T., Kawamoto K. Prognosis and endotoxin contents in middle ear effusions in cases after acute otitis media. Acta Otolaryngol Suppl. 1987;435:85–89. doi: 10.3109/00016488709107355. [DOI] [PubMed] [Google Scholar]
  24. Johnson A. P., Inzana T. J. Loss of ciliary activity in organ cultures of rat trachea treated with lipo-oligosaccharide from Haemophilus influenzae. J Med Microbiol. 1986 Nov;22(3):265–268. doi: 10.1099/00222615-22-3-265. [DOI] [PubMed] [Google Scholar]
  25. Kawauchi H., DeMaria T. F., Lim D. J. Endotoxin permeability through the round window. Acta Otolaryngol Suppl. 1989;457:100–115. doi: 10.3109/00016488809138892. [DOI] [PubMed] [Google Scholar]
  26. Kowalewska D., Romanowska E. Comparative studies on the immunologic properties of free endotoxin and lipopolysaccharide from Shigella sonnei. Arch Immunol Ther Exp (Warsz) 1977;25(3):323–330. [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Lim D. J., Kawauchi H., DeMaria T. F. Role of middle ear endotoxin in inner ear inflammatory response and hydrops: long-term study. Ann Otol Rhinol Laryngol Suppl. 1990 Jun;148:33–34. doi: 10.1177/00034894900990s609. [DOI] [PubMed] [Google Scholar]
  29. MASTER R. W. POSSIBLE SYNTHESIS OF POLYRIBONUCLEOTIDES OF KNOWN BASE-TRIPLET SEQUENCES. Nature. 1965 Apr 3;206:93–93. doi: 10.1038/206093b0. [DOI] [PubMed] [Google Scholar]
  30. Marvin H. J., ter Beest M. B., Witholt B. Release of outer membrane fragments from wild-type Escherichia coli and from several E. coli lipopolysaccharide mutants by EDTA and heat shock treatments. J Bacteriol. 1989 Oct;171(10):5262–5267. doi: 10.1128/jb.171.10.5262-5267.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mattsby-Baltzer I., Lindgren K., Lindholm B., Edebo L. Endotoxin shedding by enterobacteria: free and cell-bound endotoxin differ in Limulus activity. Infect Immun. 1991 Feb;59(2):689–695. doi: 10.1128/iai.59.2.689-695.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Munford R. S., Hall C. L., Lipton J. M., Dietschy J. M. Biological activity, lipoprotein-binding behavior, and in vivo disposition of extracted and native forms of Salmonella typhimurium lipopolysaccharides. J Clin Invest. 1982 Oct;70(4):877–888. doi: 10.1172/JCI110684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nonomura N., Nakano Y., Satoh Y., Fujioka O., Niijima H., Fujita M. Otitis media with effusion following inoculation of Haemophilus influenzae type b endotoxin. Arch Otorhinolaryngol. 1986;243(1):31–35. doi: 10.1007/BF00457904. [DOI] [PubMed] [Google Scholar]
  34. OSBORN M. J. STUDIES ON THE GRAM-NEGATIVE CELL WALL. I. EVIDENCE FOR THE ROLE OF 2-KETO- 3-DEOXYOCTONATE IN THE LIPOPOLYSACCHARIDE OF SALMONELLA TYPHIMURIUM. Proc Natl Acad Sci U S A. 1963 Sep;50:499–506. doi: 10.1073/pnas.50.3.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ophir D., Hahn T., Schattner A., Wallach D., Aviel A. Tumor necrosis factor in middle ear effusions. Arch Otolaryngol Head Neck Surg. 1988 Nov;114(11):1256–1258. doi: 10.1001/archotol.1988.01860230050021. [DOI] [PubMed] [Google Scholar]
  36. Phillips N. J., Apicella M. A., Griffiss J. M., Gibson B. W. Structural characterization of the cell surface lipooligosaccharides from a nontypable strain of Haemophilus influenzae. Biochemistry. 1992 May 12;31(18):4515–4526. doi: 10.1021/bi00133a019. [DOI] [PubMed] [Google Scholar]
  37. Rothfield L., Pearlman-Kothencz M. Synthesis and assembly of bacterial membrane components. A lipopolysaccharide-phospholipid-protein complex excreted by living bacteria. J Mol Biol. 1969 Sep 28;44(3):477–492. doi: 10.1016/0022-2836(69)90374-x. [DOI] [PubMed] [Google Scholar]
  38. Shenep J. L., Mogan K. A. Kinetics of endotoxin release during antibiotic therapy for experimental gram-negative bacterial sepsis. J Infect Dis. 1984 Sep;150(3):380–388. doi: 10.1093/infdis/150.3.380. [DOI] [PubMed] [Google Scholar]
  39. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  40. Straus D. C., Atkisson D. L., Garner C. W. Importance of a lipopolysaccharide-containing extracellular toxic complex in infections produced by Klebsiella pneumoniae. Infect Immun. 1985 Dec;50(3):787–795. doi: 10.1128/iai.50.3.787-795.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tanimura F., Tachibana M., Morioka H., Mizukoshi O. Experimental otitis media with effusion induced by intratympanic lipid A instillation. Am J Otolaryngol. 1987 Jan-Feb;8(1):23–30. doi: 10.1016/s0196-0709(87)80015-7. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Tsai C. M. The analysis of lipopolysaccharide (endotoxin) in meningococcal polysaccharide vaccines by silver staining following SDS-polyacrylamide gel electrophoresis. J Biol Stand. 1986 Jan;14(1):25–33. doi: 10.1016/s0092-1157(86)80006-3. [DOI] [PubMed] [Google Scholar]
  44. Tuyau J. E., Sims W. Aspects of the pathogenicity of some oral and other haemophili. J Med Microbiol. 1983 Nov;16(4):467–475. doi: 10.1099/00222615-16-4-467. [DOI] [PubMed] [Google Scholar]

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