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Infection and Immunity, July 2009, p. 3090-3099, Vol. 77, No. 7
0019-9567/09/$08.00+0     doi:10.1128/IAI.01390-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Processing of Pseudomonas aeruginosa Exotoxin A Is Dispensable for Cell Intoxication{triangledown}

Juliette Morlon-Guyot, Jocelyn Méré, Anne Bonhoure, and Bruno Beaumelle*

UMR 5236 CNRS-Université Montpellier 2, 34095 Montpellier Cedex 05, France

Received 13 November 2008/ Returned for modification 7 January 2009/ Accepted 13 April 2009

Exotoxin A is a major virulence factor of Pseudomonas aeruginosa. This toxin binds to a specific receptor on animal cells, allowing endocytosis of the toxin. Once in endosomes, the exotoxin can be processed by furin to generate a C-terminal toxin fragment that lacks the receptor binding domain and is retrogradely transported to the endoplasmic reticulum for retrotranslocation to the cytosol through the Sec61 channel. The toxin then blocks protein synthesis by ADP ribosylation of elongation factor 2, thereby triggering cell death. A shorter intracellular route has also been described for this toxin. It involves direct translocation of the entire toxin from endosomes to the cytosol and therefore does not rely on furin-mediated cleavage. To examine the implications of endosomal translocation in the intoxication process, we investigated whether the toxin required furin-mediated processing in order to kill cells. We used three different approaches. We first fused to the N terminus of the toxin proteins with different unfolding abilities so that they inhibited or did not inhibit endosomal translocation of the chimera. We then assayed the amount of toxin fragments delivered to the cytosol during cell intoxication. Finally we used furin inhibitors and examined the fate and intracellular localization of the toxin and its receptor. The results showed that exotoxin cytotoxicity results largely from endosomal translocation of the entire toxin. We found that the C-terminal fragment was unstable in the cytosol.

* Corresponding author. Mailing address: UMR 5236 CNRS, Case 100, Université Montpellier II, 34095 Montpellier Cedex 05, France. Phone: 33-46714-3398. Fax: 33-46714-3338. E-mail: beaumel{at}univ-montp2.fr

{triangledown} Published ahead of print on 20 April 2009.

Editor: S. R. Blanke

Infection and Immunity, July 2009, p. 3090-3099, Vol. 77, No. 7
0019-9567/09/$08.00+0     doi:10.1128/IAI.01390-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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