Immunochemical properties of the staphylococcal poly-N-acetylglucosamine surface polysaccharide.
Staphylococcus aureus and Staphylococcus epidermidis often elaborate adherent biofilms, which contain the capsular polysaccharide-adhesin (PS/A) that mediates the initial cell adherence to biomaterials. Biofilm cells produce another antigen, termed polysaccharide intercellular adhesin (PIA), which is composed of a approximately 28 kDa soluble linear beta(1-6)-linked N-acetylglucosamine. We developed a new method to purify PS/A from S. aureus MN8m, a strain hyperproducing PS/A. Using multiple analytical techniques, we determined that the chemical structure of PS/A is also beta(1-6)-N-acetylglucosamine (PNAG). We were unable to find N-succinylglucosamine residues in any of our preparations in contrast to previously reported findings (D. McKenney, K. Pouliot, Y. Wang, V. Murthy, M. Ulrich, G. Doring, J. C. Lee, D. A Goldmann, and G. B. Pier, Science 284:1523-1527, 1999). PNAG was produced with a wide range of molecular masses that could be divided into three major fractions with average molecular masses of 460 kDa (PNAG-I), 100 kDa (PNAG-II), and 21 kDa (PNAG-III). The purified antigens were not soluble at neutral pH unless first dissolved in 5 M HCl and then neutralized with 5 M NaOH. PNAG-I was very immunogenic in rabbits, but the responses of individual animals were variable. Immunization of mice with various doses (100, 50, or 10 microg) of PNAG-I, -II, and -III demonstrated that only PNAG-I was able to elicit an immunoglobulin G (IgG) immune response with the highest titers obtained with 100-microg dose. When we purified a small fraction of PNAG with a molecular mass of approximately 780 kDa (PNAG-780) from PNAG-I, significantly higher IgG titers than those in mice immunized with the same doses of PNAG-I were obtained, suggesting the importance of the molecular mass of PNAG in the antibody response. These results further clarify the chemical structure of PS/A and help to differentiate it from PIA on the basis of immunogenicity, molecular size, and solubility.
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA. tmaira@rics.bwh.harvard.edu
Tomás
T
Maira-Litrán
Andrea
A
Kropec
C
C
Abeygunawardana
Joseph
J
Joyce
George
G
Mark
Donald A
DA
Goldmann
Gerald B
GB
Pier
D000117:Acetylglucosamine / Q000737:chemistry / Q000145:classification / Q000276:immunology* / Q000302:isolation & purification; D018829:Adhesins, Bacterial / Q000737:chemistry / Q000145:classification / Q000276:immunology* / Q000302:isolation & purification; D000818:Animals; D005260:Female; D007120:Immunochemistry; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009895:Opsonin Proteins / Q000276:immunology; D010587:Phagocytosis / Q000276:immunology; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000145:classification / Q000276:immunology* / Q000302:isolation & purification; D011817:Rabbits; D013211:Staphylococcus aureus / Q000737:chemistry / Q000276:immunology*
D018829:Adhesins, Bacterial; D009895:Opsonin Proteins; D011135:Polysaccharides, Bacterial; C055239:bacterial adhesins, polysaccharide; C113831:poly-N-acetyl glucosamine; D000117:Acetylglucosamine