Cytotoxic activity of a recombinant chimaeric protein between Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin.
A segment of the exotoxin A gene of Pseudomonas aeruginosa, coding for the N-terminal end of domain I and domain II of the toxin (ETA), was genetically fused to the diphtheria toxin gene of Corynebacterium diphtheriae, coding for the N-terminal end of A fragment of diphtheria toxin (DT). The resulting hybrid protein (termed CED1) was produced in large amounts and exported to the periplasm in Escherichia coli. This chimaeric protein reacted with both anti-ETA and anti-DT antisera. Furthermore, the chimaeric protein displayed ADP-ribosylation activity and exhibited cytotoxicity to mouse 3T6 fibroblasts. These results demonstrated that the chimaeric protein is cytotoxic, and that the toxic potential of DTA can be selectively internalized and translocated via domains I and II of exotoxin A, which are thus sufficient to direct and translocate an enzymatically active heterologous polypeptide segment into the cytosol of sensitive cells.
Guidi-Rontani
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Unité des Toxines Microbiennes, CNRS URA557, Institut Pasteur, Paris, France.
D016475:3T3 Cells / Q000187:drug effects*; D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D003353:Corynebacterium diphtheriae / Q000235:genetics*; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D004926:Escherichia coli; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D051379:Mice; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000633:toxicity*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004167:Diphtheria Toxin; D005098:Exotoxins; D011065:Poly(ADP-ribose) Polymerases; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A