Antigens of Actinobacillus actinomycetemcomitans recognized by patients with juvenile periodontitis and periodontally normal subjects

Abstract

Most juvenile periodontitis patients respond to infection by Actinobacillus actinomycetemcomitans by producing serum antibodies. Specific antigens inducing the humoral immune response have not been identified, nor has the role of the resulting antibodies in disease progression been determined. Adsorbed and unadsorbed sera from juvenile periodontitis patients and normal subjects were analyzed by enzyme-linked immunosorbent assay and Western blots (immunoblots), using digested and undigested bacterial sonicates and French pressure cell fractions to determine the biochemical class, cross-reactivity, and cellular location of the antigens in different A. actinomycetemcomitans serotypes. Antigens detected by using high-titer sera included the following: (i) serotype-specific nonprotein material located on the cell surface, (ii) soluble-fraction proteins showing highly variable antibody binding, (iii) cross-reactive proteins, and (iv) a protein present in soluble and cell wall fractions and immunopositive for all sera tested. In addition, one apparently nonprotein component that was enriched in the cell wall fraction was observed. Sera with high immunoglobulin G titers to one, two, three, or none of the three A. actinomycetemcomitans serotypes were observed. There was a high degree of variation from one patient to another in the humoral immune response to serotype-specific and cross-reactive antigens. As demonstrated by whole-cell adsorption experiments, the serotype-specific surface antigen accounted for approximately 72 to 90% of the total antibody-binding activity for sera with titers greater than 100-fold above background, while cross-reactive antigen accounted for <28%. Antibody binding the whole-cell sonicate for high-titer sera was inhibited 90% by lipopolysaccharide from the same serotype, strongly suggesting that lipopolysaccharide is the immunodominant antigen class. * Corresponding author. exceptions, the antigenic properties of these fractions have not been compared. Recently, Califano et al. (3) used Western blots (immunoblots) to establish the importance of nonprotein antigens of A. actinomycetemcomitans serotype b in patients with high serum antibody titers. However, whether this nonprotein antigen is the mannose-rich serotype-specific polysaccharide purified and described by Zambon et al. (44,45), the fucose-rhamnose serotype-specific polysaccharide described by Amano et al. (1), or LPS remains an unanswered question.The long-term goal of our research is to identify, purify, and characterize the major antigen(s) of A. actinomycetemcomitans recognized by antibodies in the sera of patients with JP and high-titer normal control subjects. Our approach has been to assess antibody binding to components of whole-cell sonicates and crude cell wall and soluble fractions by using ELISA and Western blots of whole-cell adsorbed and unadsorbed sera to determine the degree of serotype specificity and location of the major antigen(s) and subsequently to focus upon the most highly reactive fractions for further purifica...

Immunogenicity of Actinobacillus actinomycetemcomitans serotype b‐specific polysaccharide‐protein conjugate

“…Various investigators have demonstrated that patients with localized juvenile periodontitis exhibit elevated serum IgG antibody to A. actinomycetemcomitans and that the immunodominant antigen of the organism is a capsular-like polysaccharide or the polysaccharide portion of lipopolysaccharide (6,10,23,26,36). In addition, serum IgG antibody to A. actinomycetemcomitans elevated in periodontitis patients has been reported to enhance in vitro phagocytosis of the organism by human neutrophils (3,27,34).…”

“…The serotype-specific antigens of serotypes d and e have not yet characterized. Patients with localized juvenile periodontitis exhibit elevated serum antibody levels to SPA of A. actinomycetemcomitans serotype b (6,23,26,36). However, the role of serum SPA-specific antibodies in host responses to A. actinomycetemcomitans is poorly understood.…”

Shared antigens of Porphymmonas gingivalis and Bacteroides forsythus

“…ELISA Specific IgG antibody titers were determined by ELISA as previously described (49). Ultrasonically disrupted cells (10 [xg protein per ml) and purified antigen preparations (5 jig dry weight per ml) in 100 mM carbonate buffer (pH 9.6) were coated onto the surface of polystyrene microtest plate wells (Flow Laboratories) for 2 h at room temperature and overnight at 4°C respectively.…”

“…Western immunoblots of the whole-cell sonic extract and purified antigens were prepared by the method of Towbin et al (55) as described by Sims et al (49). Samples were suspended in sample buffer (100 mM Tris, 10% sucrose, 1% SDS, 1% 2-mercaptoethanol [pH 6.8]) at a dry weight concentration of 0.5 mg/ ml and heated in a boiling water bath for 4 min.…”

“…Seventy microliters (35 |ig) of each preparation was separated on 10%-polyacrylamide gels, transferred to nitrocellulose, blocked with 2% dry milk in 10 mM TES 0.1% Tween for 2 h and developed with pre-or post-immune serum. Immuno-dot blots were prepared as described by Sims et al (49). Antigen preparations were suspended in sodium carbonate buffer at a concentration of 0.25 mg/ml and were serially diluted 1:1 across 10 microtest fold increase plate wells.…”