1887

Abstract

A clinical isolate of , 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 amoebocyte lysate test, indicating that the co-purifying carbohydrate was LPS, and had specificity for the lectins agglutinin, agglutinin and agglutinin. The cytotoxic activity associated with the complex was heat-labile at 70C, 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 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.

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/content/journal/jmm/10.1099/00222615-48-2-139
1999-02-01
2019-10-13
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http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/00222615-48-2-139
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