f Identification and characterisation of a cytotoxic porin-lipopolysaccharide complex from Campylobacter jejuni
- Authors: DAVID J. BACON*, WENDY M. JOHNSON, FRANK G. RODGERS1
- 1Corresponding author: Professor F. G. Rodgers.
- J. Med. Microbiol., February 1999 48: 139-148, doi: 10.1099/00222615-48-2-139
- Subject: Bacterial Pathogenicity
- Published Online:
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 porin-lipopolysaccharide (LPS) complex. This cytotoxic complex was isolated by high-performance 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)/μg of protein for HEp-2 cells, 7.49 TCD50/μg of protein for HeLa cells and 1.87 TCD50/μg 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 heat-labile 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.
Present address: Enteric Disease Program, Naval Medical Research Institute Annex, 12300 Washington Avenue, Rock-ville, MD 20852, USA.
© 1999 The Pathological Society of Great Britain and Ireland | Published by the Microbiology Society
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