| Cholera toxin (CT), the toxin produced by the bacteria Vibrio cholerae, is the causative agent of tropical diarrhoea. High morbidity and mortality rates are related to V. cholerae infection, most victims being young children, elderly people or travellers.;Before CT can exert its toxic effect, internalization via the host cell machinery is a prerequisite, followed by intracellular transport along the retrograde pathway, which guides CT via the Golgi apparatus to the endoplasmic reticulum. Here, CT is subjected to a reduction, an essential event in the generation of the catalytically active/toxic CT fragment. Hereafter, the active subunit forces its transport to the target enzyme, the adenylate cyclase, resulting in a permanent over-stimulation.;Although the overall CT intoxication process by CT is rather well understood, details about individual processes, including the internalization are still a matter of debate. The uptake of the toxin by the host cell can occur via distinct internalization routes. Historically, caveolae/lipid raft-mediated endocytosis was suggested to be the main internalization pathway. However, recent findings and some contradictory internalization characteristics also point the to clathrin-mediated endocytic pathway as a potential uptake route. Straightforward experimental approaches to distinguish between the different cellular uptake mechanisms are not evident, e.g. most drugs never specifically affect one internalization route and other biochemical procedures suffer even more from cross-reactivity. Therefore, the need for a selective blocking system was high. As a first part of this PhD project, a genetically modified permanent cell line was engineered, exploiting the technique of RNA interference. This cell line, characterized by a severe epsin knock-down, is impaired in clathrin-mediated endocytosis and therefore an excellent tool to investigate CT internalization (self evidently also the internalization of other ligands) via this pathway.;In a second part of this research project, internalization of CT was investigated using biochemical procedures (intracellular acidification and potassium depletion), drugs (chloropromazin) and finally the epsin-deficient cell line. All experimental data collected sustain that the functional internalization of CT occurs via the clathrin-mediated endocytic pathway. However, exclusive internalization via the latter pathway could not be demonstrated, rather pointing to a synergistic or complementary action of different internalization routes. |
