Pseudomonas aeruginosa cystic fibrosis clinical isolates produce exotoxin A with altered ribosyltransferase activity and cytotoxicity

The role of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor i n the lung infections of cystic fibrosis (CF) patients is not well understo od. Transcript-accumulation studies of bacterial populations in sputum reve al high levels of transcription of toxA, which encodes ETA, in some patient s with CF. However, in general, tissue damage in the lungs of patients with CF does not seem to be consistent with a high level of expression of activ e ETA. To address this discrepancy the authors analysed the production and activity of ETA produced by a number of P. aeruginosa CF isolates. One CF i solate, strain 4384 transcribed toxA at levels similar to the hypertoxigeni c strain PA103 but produced an ETA with reduced ADP-ribosyltransferase (ADP RT) activity. Complementation in trans of strain 4384 with the wild-type to xA and a mixed toxin experiment suggested the absence of inhibitory accesso ry factors within this strain. The toxA gene from strain 4384 was cloned an d sequenced, revealing only three mutations in the gene, all within the enz ymic domain. The first mutation changed Ser-410 to Asn. The second mutation was located within an alpha-helix, altering Ala-476 to Glu. The third muta tion, Ser-515 to Cry, was found at the protein surface. To date, Ser-410, A la-476 and Ser-515 have not been reported to play a role in the ADPRT activ ity of ETA. However, it may be the combination of these mutations that redu ces the enzymic activity of ETA produced by strain 4384. Expression of 4384 toxA and wild-type toxA in an isogenic strain revealed that 4384 ETA had 1 0-fold less ADPRT activity than wild-type ETA. ETA purified from strain 438 4 also demonstrated 10-fold less ADPRT activity as compared to wild-type ET A, Cytotoxicity assays of purified ETA from strain 4384 indicated that the cytotoxicity of 4384 ETA is not reduced; it may be slightly more toxic than wild-type ETA. Analysis of five other CF isolates revealed a similar reduc tion in ADPRT activity to that seen in strain 4384. Sequence analysis of th e enzymic domain of toxA from the five CF strains identified a number of mu tations that could account for the reduction in ADPRT activity. These resul ts suggest that some CF isolates produce an ETA with reduced enzymic activi ty and this may partially explain the pathogenesis of chronic lung infectio ns of CF due to P, aeruginosa.