Identification and characterisation of a cytotoxic porin-lipopolysaccharide complex from Campylobacter jejuni

Citation
Dj. Bacon et al., Identification and characterisation of a cytotoxic porin-lipopolysaccharide complex from Campylobacter jejuni, J MED MICRO, 48(2), 1999, pp. 139-148
Citations number
45
Categorie Soggetti
Microbiology
Journal title
JOURNAL OF MEDICAL MICROBIOLOGY
ISSN journal
00222615 → ACNP
Volume
48
Issue
2
Year of publication
1999
Pages
139 - 148
Database
ISI
SICI code
0022-2615(199902)48:2<139:IACOAC>2.0.ZU;2-7
Abstract
A clinical isolate of Campylobacter jejuni, previously found to produce a t oxin active in cell culture assays, was used for identification and charact erisation of a cytotoxic porin-lipopolysaccharide (LPS) complex. This cytot oxic 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)/mu g of protein for HEp-2 cells, 7.49 TCD50/mu g of protein for HeLa cells and 1.8 7 TCD50/mu g of protein for Chinese hamster ovary cells. Analysis by SDS-PA GE 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 speci ficity for the lectins Galanthus nivalis agglutinin, Maackia amurensis aggl utinin and Datura stramonium agglutinin, The cytotoxic activity associated with the complex was heat-labile at 70 degrees C, resistant to inactivation with trypsin and retained activity after treatment with sodium metaperioda te and the glycosidases neuraminidase and N-glycosidase F, Sequencing of th e N-terminus of the protein component of the complex revealed 97% homology with the major outer-membrane porin protein from C, jejuni. The cytotoxic a ctivity of the complex was neutralised by a polyclonal, homologous antiseru m, which reacted on Western blot with the 45-kDa protein, but not by polycl onal antisera raised against a number of other bacterial toxins.