Imaging the cell entry of the anthrax oedema and lethal toxins with fluorescent protein chimeras
Article first published online: 23 APR 2010
© 2010 Blackwell Publishing Ltd
Volume 12, Issue 10, pages 1435–1445, October 2010
How to Cite
Zornetta, I., Brandi, L., Janowiak, B., Dal Molin, F., Tonello, F., Collier, R. J. and Montecucco, C. (2010), Imaging the cell entry of the anthrax oedema and lethal toxins with fluorescent protein chimeras. Cellular Microbiology, 12: 1435–1445. doi: 10.1111/j.1462-5822.2010.01480.x
- Issue published online: 7 SEP 2010
- Article first published online: 23 APR 2010
- Received 17 December, 2009; revised 9 April, 2010; accepted 13 April, 2010.
To investigate the cell entry and intracellular trafficking of anthrax oedema factor (EF) and lethal factor (LF), they were C-terminally fused to the enhanced green fluorescent protein (EGFP) and monomeric Cherry (mCherry) fluorescent proteins. Both chimeras bound to the surface of BHK cells treated with protective antigen (PA) in a patchy mode. Binding was followed by rapid internalization, and the two anthrax factors were found to traffic along the same endocytic route and with identical kinetics, indicating that their intracellular path is essentially dictated by PA. Colocalization studies indicated that anthrax toxins enter caveolin-1 containing compartments and then endosomes marked by phoshatidylinositol 3-phoshate and Rab5, but not by early endosome antigen 1 and transferrin. After 40 min, both EF and LF chimeras were observed to localize within late compartments. Eventually, LF and EF appeared in the cytosol with a time-course consistent with translocation from late endosomes. Only the EGFP derivatives reached the cytosol because they are translocated by the PA channel, while the mCherry derivatives are not. This difference is attributed to a higher resistance of mCherry to unfolding. After translocation, LF disperses in the cytosol, while EF localizes on the cytosolic face of late endosomes.