Binding of Actinobacillus pleuropneumoniae lipopolysaccharides to glycosphingolipids evaluated by thin-layer chromatography

The binding profile of Actinobacillus pleuropneumoniae serotypes 1 and 2 to various glycosphingolipids was evaluated by using thin-layer chromatogram overlay. A. pleuropneumoniae whole cells recognized glucosylceramide (Glc b eta 1Cer), galactosylceramide (Gal beta 1Cer) with hydroxy and nonhydroxy f atty acids, sulfatide (SO3-Gal beta 1Cer), lactosylceramide (Gal beta 1-4Gl c beta 1Cer), gangliotriaosylceramide GgO(3) (GalNAc beta 1-4Gal beta 1-4Gl c beta 1Cer), and gangliotetraosylceramide GgO(4) (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1Cer) glycosphingolipids. We observed no binding to globoseries, globotriaosylceramide Gb(3), globoside Gb(4), or Forssman Gb( 5) glycosphingolipids or to gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, an d GT1b, The A. pleuropneumoniae strains tested also failed to detect phosph atidylethanolamine or ceramide, Interestingly, extracted lipopolysaccharide (LPS) of serotype 1 and serotype 2 as well as detoxified LPS of serotype 1 showed binding patterns similar to that of whole bacterial cells. Binding to GlcCer, GalCer, sulfatide, and LacCer, but not to GgO(3) and GgO(4) glyc osphingolipids, was inhibited after incubation of the bacteria with monoclo nal antibodies against LPS O antigen. These findings indicate the involveme nt of LPS in recognition of three groups of glycosphingolipids: (i) GlcCer and LacCer, where glucose is probably an important saccharide sequence requ ired for LPS binding; (ii) GalCer and sulfatide glycosphingolipids, where t he sulfate group is part of the binding epitope of the isoreceptor; and (ii i) GgO(3) and GgO(4), where GalNac beta 1-4Gal disaccharide represents the minimal common binding epitope, Taken together, our results indicate that A , pleuropneumoniae LPS recognize various saccharide sequences found in diff erent glycosphingolipids, which probably represents a strong virulence attr ibute.