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Infection and Immunity, August 2002, p. 4433-4440, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4433-4440.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Immunochemical Properties of the Staphylococcal Poly-N-Acetylglucosamine Surface Polysaccharide

Tomás Maira-Litrán,1* Andrea Kropec,1 C. Abeygunawardana,2 Joseph Joyce,3 George Mark III,3 Donald A. Goldmann,4 and Gerald B. Pier1

Channing Laboratory, Department of Medicine, Brigham and Women's Hospital,1 Children's Hospital, Harvard Medical School, Boston, Massachusetts,4 Department of Bioprocess and Bioanalytical Research,2 Department of Virus and Cell Biology, Merck Research Laboratories, Merck and Co., Inc., West Point, Pennsylvania3

Received 20 February 2002/ Returned for modification 8 April 2002/ Accepted 19 April 2002

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 ~28 kDa soluble linear ß(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 ß(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 µg) 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-µg dose. When we purified a small fraction of PNAG with a molecular mass of ~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.

* Corresponding author. Mailing address: Channing Laboratory, 181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-2667. Fax: (617) 525-2510. E-mail: tmaira{at}rics.bwh.harvard.edu.

Editor: E. I. Tuomanen

Infection and Immunity, August 2002, p. 4433-4440, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4433-4440.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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