Immunochemical properties of the staphylococcal poly-N-acetylglucosamine surface polysaccharide

Abstract

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.

FIG. 1.

Fractionation of S. aureus MN8m crude extract by size exclusion chromatography. Column chromatography was performed by using Sephacryl S-300 as described in Materials and Methods. The material of fractions 32 to 39, 40 to 50, and 51 to 65 were pooled and are referred to as PNAG-I, PNAG-II, and PNAG-III, respectively.

FIG. 2.

¹H-NMR spectrum (500 MHz) of PNAG-I in D₂O.

FIG. 3.

(A) Mean IgG titers of five mice immunized IP with 100 μg (▪), 50 μg (▨), and 10 μg (░⃞) of PNAG-I. (B) Mean IgG titers of five mice immunized with 10 μg (▪) or 1 μg (▨) of PNAG-780. The bars represent means, and error bars indicate the standard deviation. All preimmune titers were <25.

FIG. 4.

Mean IgM titers of five mice immunized with 100 μg (▪), 50 μg (▨), and 10 μg (░⃞) of PNAG-I, PNAG-II, and PNAG-III. Bars represent the means, and the error bars indicate the standard deviation.

FIG. 5.

Opsonophagocytosis of S. epidermidis strain indicated in the legend by immune serum from rabbit 1 (upper graph) or rabbit 2 (lower graph). Each point represents the mean of two to three assays, and the error bars indicate the SEM. Killing by preimmune sera at a dilution of 1:10 was always <15%.