Identification and characterisation of a cytotoxic pori n -I i po po I ysacc h a r id e co m p I ex from Campylobacter jejuni

A clinical isolate of Campylobacter jejuni, previously found to produce a toxin active in cell culture assays, was used for identification and characterisation of a cytotoxic porinlipopolysaccharide (LPS) complex. This cytotoxic complex was isolated by highperformance liquid chromatography of crude concentrated culture supernate and DEAE-anion exchange chromatography. The complex had a toxic activity of 20.1 tissue culture dose50 (TCD50)lpg of protein for HEp-2 cells, 7.49 TCD5O/pg of protein for HeLa cells and 1.87 TCD5Olpg of protein for Chinese hamster ovary cells. Analysis by SDS-PAGE revealed a single protein band of 45 kDa and a high mol. wt carbohydrate moiety. The complex gave a positive result in the Limulus amoebocyte lysate test, indicating that the co-purifying carbohydrate was LPS, and had specificity for the lectins Galanthus nivalis agglutinin, Maackia amurensis agglutinin and Datura stramonium agglutinin. The cytotoxic activity associated with the complex was heatlabile at 70°C, resistant to inactivation with trypsin and retained activity after treatment with sodium metaperiodate and the glycosidases neuraminidase and N-glycosidase F. Sequencing of the N-terminus of the protein component of the complex revealed 97% homology with the major outer-membrane porin protein from C. jejuni. The cytotoxic activity of the complex was neutralised by a polyclonal, homologous antiserum, which reacted on Western blot with the 45-kDa protein, but not by polyclonal antisera raised against a number of other bacterial toxins.