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 surroundi ng medium, The presence of a putative extracellular targeting signal w ithin one such protein, exotoxin A, was investigated, A series of exot oxin A truncates, fused to beta-lactamase, was constructed, Hybrid pro teins, 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 an d 1-60 of the mature exotoxin A were unstable; however, they could be detected entirely within the cells after a short labeling period, A hy brid with beta-lactamase was constructed which carried only the N-term inal residues 1-3 and region 60-120 of exotoxin A, It was also secrete d into the culture medium, suggesting that a specific 60 amino acid do main contains the necessary targeting information for translocation of exotoxin A across the outer membrane, The secretion of the hybrid pro teins is independent of the passenger protein, since a similar exotoxi n A-murine interleukin 4 hybrid protein was also secreted, The extrace llular 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 e xtracellular secretion machinery.