Identification and characterisation of a cytotoxic porin-lipopolysaccharide complex 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 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)/microg of protein for HEp-2 cells, 7.49 TCD50/microg of protein for HeLa cells and 1.87 TCD50/microg 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 degrees 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.
Bacon
David J
DJ
Johnson
Wendy M
WM
Bureau of Microbiology, Laboratory Centre for Disease Control, Tunney's Pasture, Ottawa, Ontario K1A OL2, Canada.
Rodgers
Frank G
FG
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D016123:Campylobacter jejuni / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003603:Cytotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D004269:DNA, Bacterial / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D027801:Galanthus; D006367:HeLa Cells; D006801:Humans; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction; D018272:Porins / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid