Toxin a secretion in Pseudomonas aeruginosa

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Texas Tech University

Toxin A (ToxA), an ADP-ribosyl transferase, is the most toxic among the secreted virulence factors of Pseudomonas aeruginosa. The determination of mechanisms by which ToxA reaches the extracellular space is of practical and fundamental importance. Evidence suggests that ToxA crosses the inner and outer membranes of P. aeruginosa in two separate stages according to the general secretory pathway (GSP) model of secretion. Both steps require recognition of determinants within the toxin molecule (intragenic factors) by components of the bacterial secretion machinery (extragenic factors). We have used a series of subcloning and deletion analyses to localize the intragenic signals within ToxA which are required to target the molecule to the extracellular environment. Restriction endonuclease sites within the gene for ToxA (toxA) were used to generate several toxA deletions which were expressed from the E. coli lac promoter. Bal3^ exonuclease deletion analyses were used to further examine the role of the ToxA leader peptide and the amino terminal regions in ToxA secretion. In-frame Sa/31-generated deletions were identified by colony blotting assays using ToxA-speclfIc antiserum and confirmed by DNA sequencing analyses. Localizations of the various toxA subclone products were determined by enzyme assays and Immunoblotting using ToxAspeclflc antiserum. Processing of the ToxA derivatives was examined using in wYro transcription and translation assays. Our results suggest that (1) the first 13 amino adds of the ToxA leader peptide are sufficient to direct the toxin across the cytoplasmic membrane to the periplasm in the first step of the GSP; (2) there are two secretion signals, one within the amino- and one within the carboxy-terminal regions of the mature toxin, either of which is sufficient to direct the molecule across the outer membrane in the second step of the GSP; and, (3) processing of the ToxA precursor (cleavage of the leader peptide) may not be required for release of the toxin to the periplasm or to the extracellular environment.

Pseudomonas aeruginosa