Studies Of The Intracellular Transport Of Shiga Toxin B Fragments Using Fusion Proteins

Shiga toxin (STx) is produced by shigella dysenteriae, which is the infective agent in bacillary dysentery. Shiga toxin is a protein toxin consisting of A and B subunits. The B subunits (STxB) bind to the receptor, globotriaosylceramide, on the plasma membrane of target cells and mediate the internalization of the holotoxin. It is now clear that the plasma membrane of HeLa cells expresse abundant Gb3 and mediate the endocytosis of STxB. STx and STxB follow a retrograde transport route from the plasma membrane, via the early/recyling endosome (EE/RE) and the Golgi apparatus, to the endoplasmic reticulum (ER). The mechanism and pathway of STx and STxB during their intracellular transport is unclear and controversial. Specifically, it is unkown that whether STxB is hydrolysed during transport, and whether the pathway of STxB is changed under various culture conditions. Therefore, we investigated these two issues in this paper.In the first part of the thesis, we have investigated if STxB is hydrolysed at its C-terminus during its intracellular transport. We constructed a recombinant STxB carrying a Myc-tag at its C-terminus (STxB-Myc) and then studied if STxB and Myc-tag portions were separated during transport using an in vivo transport assay. Our results showed that STxB and Myc-tag colocalized all the way from the plasma membrane to the Golgi apparatus, indicating that STxB was not hydrolysed during its transport from plasma membrane to the Golgi. However, STxB and its C-terminal Myc-tag were partially separated after the Golgi. The STxB portion was mainly found in the ER, while a fraction of Myc-tag was detected in the lysosome. Thus we concluded that some STxB molecules were hydrolysed at its C-terminus during its transport from the Golgi to the ER.In the second part of the thesis, we have investigated if STxB-(sulf)2 transport route is affected by different culture conditions. STxB-(sulf)2, a fusion protein of STxB carring two sulfations sites at its C-terminus, has been widely used as a tool for the study of STxB transport. We compared the transport of STxB-(sulf)2 in HeLa cells grown in medium containing either fetal calf serum or new born calf serum. Our results indicated that STxB-(sulf)2 followed the same route from plasma membrane to the Golgi, but segregated thereafter under these two conditions. In the case of newborn calf serum, STxB-(sulf)2 accumulated in crystal-like structures distributed throughout the whole cytoplasm, while in the case of fetal calf serum, STxB-(sulf)2 localized in the ER as reported previously.