The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide

Abstract

Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of ActinobaciUus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blueand silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas-liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low-molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the 0 antigen of the LPS.

Microbiological and immunological characteristics of young Moroccan patients with aggressive periodontitis with and without detectable Aggregatibacter actinomycetemcomitans JP2 infection

“…Antibodies to A. actinomycetemcomitans leukotoxin in the serum of patients with periodontitis have been previously assessed in gravimetric units only by Califano et al (1997) and at levels comparable to those reported here. To obtain leukotoxin free of contaminating lipopolysaccharide, which is an immunodominant antigen of A. actinomycetemcomitans (Page et al, 1991;Lu et al, 1993), these authors adopted a complicated assay based on the use of leukotoxin antigen in the form of bands in Western blots (Calif-ano et al, 1997). In the present study this problem was solved by the use of purified leukotoxin free of lipopolysaccharide as demonstrated by a sensitive immunochemical assay.…”

Altered antigenicity is seen in the lipopolysaccharide profile of non-serotypeable Actinobacillus actinomycetemcomitans strains

“…OMP pro¢les have shown antigenic heterogeneity among di¡erent subtypes of non-typeable H. in£uenzae [15], and most non-typeable H. in£uenzae strains possess unique outer membrane patterns compared with those of typeable strains [16]. The typeability of H. in£uenzae, reacting with antisera speci¢c for capsular types [31], however, di¡ers from serotypeability of A. actinomycetemcomitans, reacting with antisera against carbohydrate cellsurface antigens [7].…”

“…The serotype b-speci¢c antibody binding to A. actinomycetemcomitans is mediated through lipopolysaccharide (LPS) O-antigen [7,8]. Characterization of the LPS Opolysaccharide and its unique chemical composition in each of the reference strains representing all ¢ve A. actinomycetemcomitans serotypes support the view that serotype-speci¢c antigens of A. actinomycetemcomitans are localized in this O-antigenic polysaccharide of LPS in all serotypes [9,10].…”

Serotype‐dependent expression patterns of stabilized lipopolysaccharide aggregates in Aggregatibacter actinomycetemcomitans strains

“…Smooth LPS, which possesses O-antigen polysaccharide, is more hydrophilic than rough LPS. Some study groups think that SPA of A. actinomycetemcomitans is LPS O-antigen (21)(22)(23), and that SPA occurs as a highmolecular-weight smear that is not easily visualized by silver staining of SDS-PAGE gels. To evaluate involvement of hydrophilic O-antigen in the physical state of LPS aggregates and generation of AP-LPS, the SPAs in detergent phase and aqueous phase were compared by Western blot assay using polyclonal rabbit antiserum raised against A. actinomycetemcomitans serotype a (Fig.…”

“…Its strains are classified into seven serotypes (a-g), based on structurally and immunologically distinct SPAs (17)(18)(19)(20). Although there is a consensus that A. actinomycetemcomitans SPA is a polysaccharide, whether it is an O-antigen of LPS (21)(22)(23) or capsular polysaccharide (24,25) is controversial. Moreover, it remains unclear if any serotypes or stains of A. actinomycetemcomitans are able to produce stabilized LPS aggregates.…”