Construction of an expression system of the Pseudomonas aeruginosa cytotoxin gene and the activation mechanism of the protoxin

T. Hayashi, Y. Terawaki

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Pseudomonas aeruginosa produces cytotoxin, a protein toxin which exerts a strong cytotoxic activity especially on leukocytes (1), and it is thought to be one of the virulence factors of this important opportunistic pathogen. We have already purified the active cytotoxin from the crude extract of P. aeruginosa 158 (2), recently succeeded in cloning the gene (ctx) and determined its nucleotide sequence. The result revealed that ctx encoded a polypeptide of 286 amino acid residues with a molecular mass of 31,681 Da, a value which was slightly larger than that of the purified active toxin (29 kDa). However, the cloned ctx was not expressed in either Escherichia coli nor a cytotoxin non-producing strain of P. aeruginosa. In this study, we constructed an expression system of ctx by placing its structural gene direclty downstream of tac promotor on a broad-host-range plasmid vector. In the crude extract of E. coli or P. aeruginosa harboring the recombinant plasmid, a cytotoxin-related polypeptide with a molecular weight slightly higher than that of the purified active toxin was detected by immunodetection. The polypeptide was converted to a form with the same molecular weight as that of the purified toxin by trypsin treatment, resulting in a marked increase in the leukocytotoxic activity. Similar findings were obtained with the extract of the parent strain P. aeruginosa 158. This confirms that ctx encodes the procytotoxin and suggests that the protoxin could be activated by proteolytic removal of only a few amino acid residues. Therefore, we determined the N- and C-terminal amino acid sequence of the purified active toxin. The N-terminal amino acid sequence was identical with that of the protoxin. On the other hand, the C-terminus of the active toxin was Arg-266 and 20 amino acid residues present at the C-terminus of the protoxin were processed. The results mean that the procytotoxin is converted into the active form by proteolytic removal of 20 amino acid residues from the C-terminus and that processing at the N-terminus of the protoxin is not required for its activation. Although this active toxin was obtained by trypsin treatment in vitro, a similar processing of the protoxin seems to occur in vivo at the loci of infection.

Original languageEnglish
Pages (from-to)198-199
Number of pages2
JournalJapanese Journal of Medical Science and Biology
Issue number5-6
Publication statusPublished - 1989
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General Biochemistry,Genetics and Molecular Biology


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