Skip to content
1887

Microbiology Society logo Microbiology

Volume 138, Issue 7

Demonstration of peptidoglycan-associated outer-membrane proteins by use of monoclonal antibodies Free

Abstract

A monoclonal antibody (3D6) was produced which reacted only with sonicated cell extracts that had been lysozyme-treated after sonication. The monoclonal antibody (mAb) reacted with the three major outer-membrane proteins (OMPs) of B115 in Western blots. A large number of reactive bands ranging from 12 to 43 kDa were present in lysozyme-treated and sonicated cell extracts. In a latex agglutination inhibition immunoassay, mAb 3D6 showed better reactivity with purified peptidoglycan (PG) of B115 than with that of . This mAb was also used in immunogold electron microscopy with whole cells and sections. No binding was observed on whole cells and immunogold labelling in sections was observed close to the outer membrane, in the periplasmic space and in the cytoplasm. These findings indicate that mAb 3D6 is specific for PG subunits. Immunoblot analysis of B115 rough sonicated cell extracts after SDS-PAGE, with or without lysozyme treatment, was performed using mAbs specific for OMPs of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa, for PG and for rough lipopolysaccharide (R-LPS) and smooth lipopolysaccharide (S-LPS). mAbs specific for the 25-27, 31-34 and 36-38 kDa OMPs reacted with three to six bands. All of them except the band of lowest molecular mass reacted with the PG-specific mAb and not with R-LPS- and S-LPS-specific mAbs. Therefore we propose that variation in the apparent molecular mass of the major OMPs (25-27, 31-34, and 36-38 kDa) is due to varying numbers of PG subunit residues rather than attached R-LPS or S-LPS molecules. Our results suggest a very strong, possibly covalent, interaction of the major OMPs (25-27, 31-34 and 36-38 kDa) with PG. There is no evidence of association of the minor OMPs (10, 16.5, 19 and 89 kDa) with PG, since mAbs specific for these proteins reacted with single bands that were not apparent when reacted with the anti-PG mAb. Furthermore, lysozyme treatment did not affect the electrophoretic mobility of these proteins.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology, 1992
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-138-7-1543
1992-07-01
2025-04-30
Loading full text...

Full text loading...

/deliver/fulltext/micro/138/7/mic-138-7-1543.html?itemId=/content/journal/micro/10.1099/00221287-138-7-1543&mimeType=html&fmt=ahah

References

  1. Amano K.-I., Williams J. C. 1983; Peptidoglycan of Legionella pneumophila: apparent resistance to lysozyme hydrolysis correlates with a high degree of peptide cross-linking. Journal of Bacteriology 153 520 526
    [Google Scholar]
  2. Blundell J. K., Smith G. J., Perkins H. R. 1980; The peptidoglycan of Neisseria gonorrhoeae: O-acetyl groups and lysozyme sensitivity. FEMS Microbiology Letters 9 259 261
    [Google Scholar]
  3. Butler C. A., Hoffman P. S. 1990; Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila. Journal of Bacteriology 172 2401 2407
    [Google Scholar]
  4. Cloeckaert A., de Wergifosse P., Dubray G., Limet J. N. 1990; Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay. Infection and Immunity 58 3980 3987
    [Google Scholar]
  5. Dougherty J. T. 1983; Synthesis and modification of the peptidoglycan in Neisseria gonorrhoeae. FEMS Microbiology Letters 17 51 53
    [Google Scholar]
  6. Douglas J. T., Rosenberg E. Y., Nikaido H., Verstraete D. R., Winter A. J. 1984; Porins of Brucella species. Infection and Immunity 44 16 21
    [Google Scholar]
  7. Dubray G. 1973; Le peptidoglycane des Brucella: mise en évidence d’une structure à triple feuillet. Comptes Rendus de l’ Académie des Sciences Paris 277 2281 2283
    [Google Scholar]
  8. Dubray G. 1981 Etude ultrastructurale et biochimique des enveloppes des bactéries du genre Brucella PhD thesis Université de Paris-Sud;
    [Google Scholar]
  9. Dubray G. 1987; Protective antigens in brucellosis. Annales de l’ Institut Pasteur/Microbiologie 138 84 86
    [Google Scholar]
  10. Dubray G., Bezard G. 1980; Isolation of three protective cell-wall antigens of Brucella abortus in experimental brucellosis in mice. Annales de Recherches Vétérinaires 11 367 373
    [Google Scholar]
  11. Dubray G., Charriaut C. 1983; Evidence of three major polypeptide species and two major polysaccharide species in the Brucella outer membrane. Annales de Recherches Vétérinaires 14 311 318
    [Google Scholar]
  12. Dubray G., Limet J. 1987; Evidence of heterogeneity of lipopolysaccharides among Brucella biovars in relation to A and M specificities. Annales de l’ lnstitut Pasteur/Microbiologie 138 27 37
    [Google Scholar]
  13. Dubray G., Plommet M. 1976; Structure et constituants des Brucella. Caracterisation des fractions et propériétes biologiques. Developments in Biological Standardization 31 68 91
    [Google Scholar]
  14. Fischetti V. A., Jones K. F., Scott J. R. 1985; Size variation of the M protein in group A streptococci. Journal of Experimental Medicine 161 1384 1401
    [Google Scholar]
  15. Gmeiner J., Kroll H.-P. 1981; Murein biosynthesis and O-acetylation of N-acetylmuramic acid during the cell-division cycle of Proteus mirabilis. European Journal of Biochemistry 117 171 177
    [Google Scholar]
  16. Gomez-Miguel M. J., Moriyon I. 1986; Demonstration of a peptidoglycan-linked lipoprotein and characterization of its trypsin fragment in the outer membrane of Brucella spp. Infection and Immunity 53 678 684
    [Google Scholar]
  17. Gomez-Miguel M. J., Moriyon I., Lopez J. 1987; Brucella outer membrane lipoprotein shares antigenic determinants with Escherichia coli Braun lipoprotein and is exposed on the cell surface. Infection and Immunity 55 258 262
    [Google Scholar]
  18. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227 680 685
    [Google Scholar]
  19. Lam J. S., Lam M. Y. C., MacDonald L. A., Hancock R. E. W. 1987; Visualization of Pseudomonas aeruginosa O antigens by using a protein A-dextran-colloidal gold conjugate with both immunoglobulin G and immunoglobulin M monoclonal antibodies. Journal of Bacteriology 169 3531 3538
    [Google Scholar]
  20. Lear A. L., Perkins H. R. 1983; Degrees of O-acetylation and cross-linking of the peptidoglycan of Neisseria gonorrhoeae during growth. Journal of General Microbiology 129 885 888
    [Google Scholar]
  21. Limet J. N., Berbinschi A., Cloeckaert A., Cambiaso C. L., Masson P. L. 1988; Longitudinal study of brucellosis in mice by immunoassay of lipopolysaccharide-related antigens in blood and urine. Journal of Medical Microbiology 26 37 45
    [Google Scholar]
  22. Lopez-Merino A., Asselineau J., Serre A., Roux J., Bascoul S., Lacave C. 1976; Immunization by an insoluble fraction extracted from Brucella melitensis: immunological and chemical characterization of the active substances. Infection and Immunity 13 311 321
    [Google Scholar]
  23. de Maagd R. A., de Rijk R., Mulders I. H. M., Lugtenberg A.J. J. 1989a; Immunological characterization of Rhizobium leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies. Journal of Bacteriology 171 1136 1142
    [Google Scholar]
  24. de Maagd R. A., Wientjes F. B., Lugtenberg B. J. 1989b; Evidence for divalent cation (Ca2+)-stabilized oligomeric proteins and covalently bound protein-peptidoglycan complexes in the outer membrane of Rhizobium leguminosarum. Journal of Bacteriology 171 3989 3995
    [Google Scholar]
  25. Mardarowicz C. 1966; Isolierung und Charakterisierung des Murein-Sacculus von Brucella. Zeitschrift fur Naturforschung 21b 1006 1007
    [Google Scholar]
  26. Moriyon I., Berman D. T. 1983; Isolation, purification, and partial characterization of Brucella abortus matrix protein. Infection and Immunity 39 394 402
    [Google Scholar]
  27. Nakane P. K., Kawaoi A. 1974; Peroxidase-labelled antibody, a new method of conjugation. Journal of Histochemistry and Cytochemistry 22 1084 1091
    [Google Scholar]
  28. Pancholi V., Fischetti V. A. 1988; Isolation and characterization of the cell-associated region of group A streptococcal M6 protein. Journal of Bacteriology 170 2618 2624
    [Google Scholar]
  29. Pellon G. 1990; Biosynthesis of peptidoglycans from eubacterial cell envelopes. Bulletin de l’Institut Pasteur 88 203 236
    [Google Scholar]
  30. Rosenthal R. S., Blundell J. K., Perkins H. R. 1982; Strain-related differences in lysozyme sensitivity and extent of Oacetylation of gonococcal peptidoglycan. Infection and Immunity 37 826 829
    [Google Scholar]
  31. Rosenthal R. S., Folkening W. J., Miller D. R., Swim S.C. 1983; Resistance of O-acetylated gonococcal peptidoglycan to human peptidoglycan-degrading enzymes. Infection and Immunity 40 903 911
    [Google Scholar]
  32. Santos J. M., Verstraete D. R., Perera V. Y., Winter A.J. 1983; Outer membrane proteins from rough strains of four Brucella species. Infection and Immunity 46 188 194
    [Google Scholar]
  33. Swim S. C., Gfell M. A., III Wilde C. E., Rosenthal R. S. 1983; Strain distribution in extents of lysozyme resistance and O-acetylation of gonococcal peptidoglycan determined by high-performance liquid chromatography. Infection and Immunity 42 446 452
    [Google Scholar]
  34. Verstraete D. R., Winter A. J. 1984; Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains. Infection and Immunity 46 182 187
    [Google Scholar]
  35. Verstraete D. R., Creasy M. T., Caveney N. T., Baldwin C. L., Blab M. W., Winter A. J. 1982; Outer membrane proteins of Brucella abortus: isolation and characterization. Infection and Immunity 35 979 989
    [Google Scholar]
  36. Wensink J., Without B. 1981; Identification of different forms of the murein-bound lipoprotein found in isolated outer membranes of Escherichia coli. European Journal of Biochemistry 113 349 357
    [Google Scholar]
/content/journal/micro/10.1099/00221287-138-7-1543
Loading
Demonstration of peptidoglycan-associated Brucella outer-membrane proteins by use of monoclonal antibodies
Microbiology 138, 1543 (1992); https://doi.org/10.1099/00221287-138-7-1543
/content/journal/micro/10.1099/00221287-138-7-1543
/content/journal/micro/10.1099/00221287-138-7-1543
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error