Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal 125 I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after Ͼ 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4-and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocationcompetent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect 125 I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when 125
Pseudomonas aeruginosa is a gram-negative bacterium that secretes many proteins into the extracellular medium via the Xcp machinery. This pathway, conserved in gram-negative bacteria, is called the type II pathway. The exoproteins contain information in their amino acid sequence to allow targeting to their secretion machinery. This information may be present within a conformational motif. The nature of this signal has been examined for P. aeruginosa exotoxin A (PE). Previous studies failed to identify a common minimal motif required for Xcp-dependent recognition and secretion of PE. One study identified a motif at the N terminus of the protein, whereas another one found additional information at the C terminus. In this study, we assess the role of the central PE domain II composed of six ␣-helices (A to F). The secretion behavior of PE derivatives, individually deleted for each helix, was analyzed. Helix E deletion has a drastic effect on secretion of PE, which accumulates within the periplasm. The conformational rearrangement induced in this variant is predicted from the three-dimensional PE structure, and the molecular modification is confirmed by gel filtration experiments. Helix E is in the core of the molecule and creates close contact with other domains (I and III). Deletion of the surface-exposed helix F has no effect on secretion, indicating that no secretion information is contained in this helix. Finally, we concluded that disruption of a structured domain II yields an extended form of the molecule and prevents formation of the conformational secretion motif.
SummaryPseudomonas exotoxin A (PE) is a cytotoxin composed of three structural domains. Domain I is responsible for cell binding, domain II for membrane translocation enabling access to the cytosol, and domain III for the catalytic inactivation of protein synthesis, which results in cell death. To investigate the role of the six a-helices (A±F) that form the translocation domain, we deleted them successively one at a time. All mutants showed native cell-binding and catalytic activities, indicating that deletions speci®cally affected translocation activity. This step of the intoxication procedure was examined directly using a cell-free translocation assay, and indirectly by monitoring cytotoxicity. Translocation activity and log(cytotoxicity) were highly correlated, directly indicating that translocation is rate limiting for PE intoxication. Deletion of B, C and D helices resulted in non-toxic and non-translocating molecules, whereas mutants lacking the A or E helix displayed signi®cant cytotoxicity albeit 500-fold lower than native PE. We concluded that B, C and D helices, which make up the core of domain II, are essential, whereas the more peripheral A and E helices are comparatively dispensable. The last helix (F) is inhibitory for translocation because its deletion produced a mutant displaying a translocation activity 60% higher than PE, along with a three-to sixfold increase in cytotoxicity in all tested cell lines. This toxin is the most in vitro active PE mutant obtained until now. Finally, partial duplication of domain II did not give rise to a more actively translocated PE, but rather to a threefold less active molecule.
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