Fusions of anthrax toxin lethal factor to the ADP-ribosylation domain of Pseudomonas exotoxin A are potent cytotoxins which are translocated to the cytosol of mammalian cells.
The lethal factor (LF) and edema factor (EF) components of anthrax toxin are toxic to animal cells only if internalized by interaction with the protective antigen (PA) component. PA binds to a cell surface receptor and is proteolytically cleaved to expose a binding site for LF and EF. To study how LF and EF are internalized and trafficked within cells, LF was fused to the translocation and ADP-ribosylation domains (domains II and III, respectively) of Pseudomonas exotoxin A. LF fusion proteins containing Pseudomonas exotoxin A domains II and III were less toxic than those containing only domain III. Fusion proteins with a functional endoplasmic reticulum retention sequence, REDLK, at the carboxyl terminus of domain III were less toxic than those with a nonfunctional sequence, LDER. The most potent fusion protein, FP33, had an EC50 = 2 pM on Chinese hamster ovary cells, exceeding that of native Pseudomonas exotoxin A (EC50 = 420 pM). Toxicity of all the fusion proteins required the presence of PA and was blocked by monensin. These data suggest that LF and LF fusion proteins are efficiently translocated from acidified endosomes directly to the cytosol without trafficking through other organelles, as is required for Pseudomonas exotoxin A. This system provides a potential vehicle for importing diverse proteins into the cytosol of mammalian cells.
Arora
N
N
Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Klimpel
K R
KR
Singh
Y
Y
Leppla
S H
SH
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects*; D003001:Cloning, Molecular; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D005798:Genes, Bacterial; D007700:Kinetics; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects; D051379:Mice; D010957:Plasmids; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D005098:Exotoxins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C030325:anthrax toxin