1887

Abstract

It has been shown that the autotransporter plasmid-encoded toxin (Pet) of enteroaggregative (EAEC) produces cytotoxic and enterotoxic effects. Both effects can be explained by the proteolytic activity of Pet on its intracellular target -fodrin (II spectrin). In addition, Pet cytotoxicity and enterotoxicity depend on Pet serine protease activity, and on its internalization into epithelial cells. However, the mechanisms of Pet uptake by epithelial cells are unknown. Here, we show that Pet interacts with the plasma membrane of epithelial cells, and afterwards is detected inside the cells. Furthermore, Pet was internalized via clathrin-mediated endocytosis, since its internalization was inhibited by monodansylcadaverine and sucrose, but not by filipin or methyl--cyclodextrin, which are drugs that interfere with protein entry via a clathrin-independent pathway. Additionally, Pet was immunoprecipitated by anti-clathrin antibodies, but not by anti-caveolin antibodies. Moreover, small interfering RNA (siRNA), designed to knock out clathrin gene expression in HEp-2 cells, prevented Pet internalization, and thereby the Pet-induced cytotoxic effect. However, the use of siRNA to knock out caveolin expression had no effect on Pet internalization, and the cytotoxic effect was clearly observed. Together, these data indicate that Pet secreted by EAEC binds to the cell surface via an unknown receptor, to be taken up by clathrin-mediated endocytosis, and exert its toxic effect in the cytoplasm.

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2007-09-01
2019-10-18
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vol. , part 9, pp. 2828-2838

Inhibition of CT and transferrin internalization by filipin or cadaverine. HEp-2 cells were preincubated for 30 min with drugs that inhibit the endocytic process. After the incubation, cells were treated with CT (0.5 μg ml ) or transferrin (37 μg ml ) for 3 h. The cells were fixed, permeabilized, and stained with rhodamine-phalloidin (red) and anti-CT or anti-transferrin antibodies, followed by a secondary fluorescein-labelled goat anti-rabbit antibody (green). Slides were observed by using confocal microscopy. Surface (a) and vertical (b) sections of filipin-preincubated cells treated with CT. Surface (c) and vertical (d) sections of cadaverine-preincubated cells treated with transferrin.



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