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Imaging the cell entry of the anthrax oedema and lethal toxins with fluorescent protein chimeras.

Abstract
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.
AuthorsIrene Zornetta, Lucia Brandi, Blythe Janowiak, Federica Dal Molin, Fiorella Tonello, R John Collier, Cesare Montecucco
JournalCellular microbiology (Cell Microbiol) Vol. 12 Issue 10 Pg. 1435-45 (Oct 2010) ISSN: 1462-5822 [Electronic] India
PMID20438574 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Copyright© 2010 Blackwell Publishing Ltd.
Chemical References
  • Antigens, Bacterial
  • Bacterial Toxins
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • anthrax toxin
  • enhanced green fluorescent protein
  • red fluorescent protein
  • Green Fluorescent Proteins
Topics
  • Animals
  • Antigens, Bacterial (metabolism)
  • Bacterial Toxins (metabolism)
  • Cells, Cultured
  • Cricetinae
  • Cytosol (chemistry)
  • Endosomes (chemistry)
  • Genes, Reporter
  • Green Fluorescent Proteins (genetics, metabolism)
  • Luminescent Proteins (genetics, metabolism)
  • Protein Binding
  • Protein Transport
  • Recombinant Fusion Proteins (metabolism)
  • Staining and Labeling (methods)
  • Time Factors

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