A specific targeting domain in mature exotoxin A is required for its extracellular secretion from Pseudomonas aeruginosa.
A number of Gram-negative bacteria, including Pseudomonas aeruginosa, actively secrete a subset of periplasmic proteins into their surrounding medium. The presence of a putative extracellular targeting signal within one such protein, exotoxin A, was investigated. A series of exotoxin A truncates, fused to beta-lactamase, was constructed. Hybrid proteins, which carry at their N- termini 120, 255, 355 or the entire 613 residues of the mature exotoxin A, were stable and were secreted into the extracellular medium. Hybrid proteins which carry residues 1-30 and 1-60 of the mature exotoxin A were unstable; however, they could be detected entirely within the cells after a short labeling period. A hybrid with beta-lactamase was constructed which carried only the N-terminal residues 1-3 and region 60-120 of exotoxin A. It was also secreted into the culture medium, suggesting that a specific 60 amino acid domain contains the necessary targeting information for translocation of exotoxin A across the outer membrane. The secretion of the hybrid proteins is independent of the passenger protein, since a similar exotoxin A-murine interleukin 4 hybrid protein was also secreted. The extracellular targeting signal between amino acids 60 and 120 is rich in anti-parallel beta-sheets. It has been shown previously to be involved in the interaction of the exotoxin A with the receptors of the eukaryotic cells. In the three- dimensional view, the targeting region is on the toxin surface where it is easily accessible to the components of the extracellular secretion machinery.
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Department of Microbiology, School of Medicine, University of Washington, Seattle, 98195, USA.
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D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000378:metabolism; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005098:Exotoxins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D015847:Interleukin-4 / Q000096:biosynthesis / Q000378:metabolism; D007739:L Cells; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D021382:Protein Sorting Signals; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D037521:Virulence Factors; D001618:beta-Lactamases / Q000096:biosynthesis / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D015847:Interleukin-4; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D001618:beta-Lactamases; D000097668:Pseudomonas aeruginosa Exotoxin A