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
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.