Binding of Actinobacillus pleuropneumoniae Lipopolysaccharides to Glycosphingolipids Evaluated by Thin-Layer Chromatography

Abstract

ABSTRACT 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 (Glcβ1Cer), galactosylceramide (Galβ1Cer) with hydroxy and nonhydroxy fatty acids, sulfatide (SO 3 -3Galβ1Cer), lactosylceramide (Galβ1-4Glcβ1Cer), gangliotriaosylceramide GgO 3 (GalNAcβ1-4Galβ1-4Glcβ1Cer), and gangliotetraosylceramide GgO 4 (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1Cer) 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, and GT1b. The A. pleuropneumoniae strains tested also failed to detect phosphatidylethanolamine 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 glycosphingolipids, was inhibited after incubation of the bacteria with monoclonal antibodies against LPS O antigen. These findings indicate the involvement of LPS in recognition of three groups of glycosphingolipids: (i) GlcCer and LacCer, where glucose is probably an important saccharide sequence required for LPS binding; (ii) GalCer and sulfatide glycosphingolipids, where the sulfate group is part of the binding epitope of the isoreceptor; and (iii) GgO 3 and GgO 4 , where GalNacβ1-4Gal disaccharide represents the minimal common binding epitope. Taken together, our results indicate that A. pleuropneumoniae LPS recognize various saccharide sequences found in different glycosphingolipids, which probably represents a strong virulence attribute.