Abstract. Endocytosed Shiga toxin is transported from the Golgi complex to the endoplasmic reticulum in butyric acid-treated A431 ceils. We here examine the extent of this retrograde transport and its regulation. The short B fragment of Shiga toxin is sufficient for transport to the ER. The
Abstract. The glycolipid-binding cytotoxin produced by Shigella dysenteriae 1, Shiga toxin, binds to MDCK cells (strain 1) only after treatment with short-chain fatty acids like butyric acid or with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. The induced binding sites were found to be functional with respect to endocytosis and translocation of toxin to the cytosol. Glycolipids that bind Shiga toxin appeared at both the apical and the basolateral surface of polarized MDCK cells grown on filters, and Shiga toxin was found to be endocytosed from both sides of the cells. This was demonstrated by EM of cells incubated with Shiga-HRP and by subcellular fractionation of cells incubated with t25I-labeled Shiga toxin. The data indicated that toxin molecules are endocytosed from coated pits, and that some internalized Shiga toxin is transported to the Golgi apparatus. Fractionation of polarized cells incubated with 125I-Shiga toxin showed that the transport of toxin to the Golgi apparatus was equally efficient from both poles of the cells. After 1-h incubation at 37°C ~10% of the internalized toxin was found in the Golgi fractions. The results thus suggest that glycolipids can be efficiently transported to the Golgi apparatus from both sides of polarized MDCK cell monolayers.
To assess the humoral immunological responses at the subclass level in shigellosis, specific antibody responses against Shigella dysenteriae 1 lipopolysaccharide (LPS), S. flexneri Y LPS, invasion plasmid-coded protein antigens (Ipa), and Shiga toxin were analyzed. Antibody responses of 41 patients with S. dysenteriae 1 infection (SDIP) and 15 patients with S. flexneri infection (SFIP) were compared with those of controls (n ؍ 40). The levels of total immunoglobulin G (IgG), IgA, IgM, and albumin in serum and stool samples were analyzed. In addition, total IgA (t-IgA), secretory IgA (s-IgA), and antigen-specific s-IgA in fecal samples were analyzed to evaluate the specificities and magnitudes of the mucosal immune responses. By comparing the relative increases in optical density for each IgG subclass separately, it was determined that the anti-LPS (homologous) response initially increased in the order IgG2 > IgG1 > IgG3 > IgG4 and that this order changed to IgG2 > IgG3 > IgG1 > IgG4 later in the disease. The IgG subclass response against protein antigens initially showed the order IgG1 > IgG3 > IgG2 > IgG4, which changed to IgG3 > IgG1 > IgG2 > IgG4 later in the disease. A significant increase in the proportion of IgA2 among t-IgA compared with that in controls was seen in both SDIP and SFIP, while significant changes in the proportions of IgG1 and IgG2 among t-IgG compared with controls was seen only in SDIP. The anti-LPS IgA2 response was more prominent in SDIP than in SFIP. We found an early peak of antigen-specific s-IgA in fecal samples, with a shorter duration than the corresponding response in serum samples. The simultaneous increase of serum IgA, fecal t-IgA, and s-IgA in SDIP compared with those in SFIP suggests that there is a massive increase in the local IgA production, giving an increase in systemic IgA concomitant with an extensive gut mucosal inflammation leading to an increased loss of albumin, IgG, and IgA with a high ratio of t-IgA to s-IgA.
Two species of Propionibucterium were analysed regarding their binding to glycosphingolipids. Bacteria were labeled with rzsI and selective interaction with glycolipids on thin-layer chromatograms was revealed by autoradiography. The carbohydrate site in common for active molecular species appeared to be lactose. The two bacteria differed, however, in the overall binding pattern on the chromatogram, probably due to recognition of separate epitopes on lactose. P. freudenreichii bound only to lactosylceramide while P. granuloszon also recognized substituted lactosylceramide: Galal+ 3Gal/31 -r4Glc/7Cer, GlcNAcj?l-r3Ga431+4Glc&Cer and Gab?l~3GlcNAcj?l~3Gal/.?l+4Glc~er were active, but Galal +4Gal/?l -Xilcj?Cer was inactive. Also, there was an interesting dependence on ceramide structure in the case of lactosylceramide. P. freudenreichii bound to lactosylceramide with sphingosine and non-hydroxy fatty acids but not to species with sphingosine and 2-hydroxy fatty acids, phytosphingosine and non-hydroxy fatty acids or phytosphingosine and 2-hydroxy fatty acids. For P. grunufosum the situation was reversed. This may be explained by an inthtence of ceramide structure on the presentation of the two lactose epitopes at the assay surface. These results were supported by curves from the binding of labeled bacteria to glycolipids coated in microtiter wells and in part by binding to glycolipid-coated chicken erythrocytes.
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