Imaging the cell entry of the anthrax oedema and lethal toxins with fluorescent protein chimeras.
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.
Zornetta
Irene
I
Dipartimento di Scienze Biomediche dell'Università di Padova and Istituto di Neuroscienze del CNR, Via G. Colombo 3, 35100 Padova, Italy.
Brandi
Lucia
L
Janowiak
Blythe
B
Dal Molin
Federica
F
Tonello
Fiorella
F
Collier
R John
RJ
Montecucco
Cesare
C
D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D003600:Cytosol / Q000737:chemistry; D011992:Endosomes / Q000737:chemistry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D000097573:Red Fluorescent Protein
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C030325:anthrax toxin; C485184:enhanced green fluorescent protein