TOXIN-A SECRETION IN PSEUDOMONAS-AERUGINOSA - THE VOLE OF THE FIRST 30 AMINO-ACIDS OF THE MATURE TOXIN

Toxin A, one of several virulence factors secreted by the gram-negativ e bacterium Pseudomonas aeruginosa, is synthesized as a 71 kDa precurs or with a typical prokaryotic leader peptide (LP), and is secreted as a 68 kDa mature protein. Evidence from a previous study suggested that a signal required for toxin A secretion in P. aeruginosa may reside w ithin the region defined by the toxin A LP and the first 30 amino acid s (aa) of mature toxin A. In the present study, we have used exonuclea se Bal31 deletion analysis to examine the specific role of the first 3 0 aa in toxin A secretion. Four toxA subclones, which encode products containing the toxin A LP and different segments of the 30-residue reg ion fused to a toxin A carboxy-terminal region, were identified. In ad dition, a gene fusion encoding a hybrid protein consisting of the LP o f P. aeruginosa elastase and the final 305 residues of toxin A, was ge nerated. The cellular location of the toxA subclone products in P. aer uginosa was determined by immunoblotting analysis. Toxin A CRMs (cross -reacting material) encoded by different subclones were detected in di fferent fractions of P. aeruginosa including the periplasm and the sup ernatant. Results from these studies suggest that (1) mature toxin A c ontains two separate secretion signals one within the N-terminal regio n and one within the C-terminal region; and (2) the first 30 residues of the mature toxin A form part of the N-terminal secretion signal.