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

Department of Biological Sciences¹ and Department of Microbiology and Infectious Diseases², University of Calgary, Calgary, Alberta, Canada T2N 1N4
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA³

Author for correspondence: Douglas G. Storey. Tel: +1 403 220 5274. Fax: +1 403 289 9311. e-mail: storey{at}ucalgary.ca

The role of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in the lung infections of cystic fibrosis (CF) patients is not well understood. Transcript-accumulation studies of bacterial populations in sputum reveal high levels of transcription of toxA, which encodes ETA, in some patients 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 active 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 isolate, strain 4384, transcribed toxA at levels similar to the hypertoxigenic strain PA103 but produced an ETA with reduced ADP-ribosyltransferase (ADPRT) activity. Complementation in trans of strain 4384 with the wild-type toxA and a mixed toxin experiment suggested the absence of inhibitory accessory factors within this strain. The toxA gene from strain 4384 was cloned and sequenced, revealing only three mutations in the gene, all within the enzymic domain. The first mutation changed Ser-410 to Asn. The second mutation was located within an -helix, altering Ala-476 to Glu. The third mutation, Ser-515 to Gly, was found at the protein surface. To date, Ser-410, Ala-476 and Ser-515 have not been reported to play a role in the ADPRT activity of ETA. However, it may be the combination of these mutations that reduces 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 10-fold less ADPRT activity than wild-type ETA. ETA purified from strain 4384 also demonstrated 10-fold less ADPRT activity as compared to wild-type ETA. 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 reduction in ADPRT activity to that seen in strain 4384. Sequence analysis of the enzymic domain of toxA from the five CF strains identified a number of mutations that could account for the reduction in ADPRT activity. These results suggest that some CF isolates produce an ETA with reduced enzymic activity and this may partially explain the pathogenesis of chronic lung infections of CF due to P. aeruginosa.

Keywords: cystic fibrosis, Pseudomonas aeruginosa, exotoxin A, ADPRT, cytotoxicity

Abbreviations: ADPRT, ADP-ribosyltransferase; CF, cystic fibrosis; EF-2, elongation factor 2; ETA, exotoxin A; TSBDC, trypticase soy broth dialysate chelexed

The GenBank accession numbers for the toxA sequences are: strain 4384, AF227419; strain 5154, AF227420; strain 5166, AF227421; strain 5552, AF227422; strain 5585, AF227423; strain 5588, AF227424.

^a Present address: Department of Molecular Biology, Princeton University, Princeton, NJ, USA.

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