Processing of Pseudomonas aeruginosa Exotoxin A Is Dispensable for Cell Intoxication

Abstract

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.Several bacterial toxins are cleaved by cell proteases upon uptake by mammalian cells. Site-specific processing by furinlike endoproteases is often associated with toxin activation and is therefore a key step of the intoxication process. Indeed, toxins such as anthrax protective antigen, aerolysin, Shiga toxin, and diphtheria toxin (DT) require furin-mediated activation in order to intoxicate cells (47). Exotoxin A (PE) that is secreted by Pseudomonas aeruginosa can also be cleaved by furin in vivo and in vitro (19). PE is produced as a single polypeptide chain composed of three structural and functional domains (domains I, II, and III) that are successively involved in the intoxication process (3). This process starts with the binding of domain I to the low-density receptor-related protein (LRP) (25), a huge protein synthesized in a 600-kDa precursor form that is cleaved by furin into 515-and 85-kDa subunits (16). The LRP is the only functional receptor for PE since LRP-deficient cells are resistant to PE (53). This receptor enables endocytosis of PE and various other ligands (17). Once PE is internalized, exposure to a low endosomal pH triggers major conformational changes within its structure and leads to domain II-mediated insertion into the endosome membrane (32). After this step, two pathways have been documented for continuation of the PE intoxication process. Either the entire toxin could cross the endosome membrane (2, 32), or the toxin could be processed by furin after Arg279 within an exposed loop between the...

Towards Engineering Novel PE-Based Immunotoxins by Targeting Them to the Nucleus

“…We detected only full-length toxins in the intoxicated cells and showed that the unnicked toxin (PE-furin) retains undiminished cytotoxicity. This indicates that furin cleavage is not critical for PE activity, at least in some cell types, which is consistent with several previous studies showing lack of correlation between proteolytic processing and PE toxicity [37,54]. …”

“…Interestingly, proteasome inhibition had slightly lower impact on A549 cells intoxicated with PE-NLS (37% decrease of viability), and there was only a very slight increase in vulnerability of lactacystin pre-treated HepG2 cells to PE-NLS intoxication, possibly due to strong accumulation of this mutein in the nucleus. Therefore, we conclude that proteasome degradation might play a role in both, stability of PE (as suggested previously [37]) and its cytotoxicity (similar to DT [38]).…”

The 1.8 Å Cholix Toxin Crystal Structure in Complex with NAD+ and Evidence for a New Kinetic Model

“…Low affinity for NAD ϩ in the full-length toxin is consistent with DT group toxins requiring activation by furin cleavage at an arginine-rich loop and/or reduction at disulfide bonds (46,47); however, it is clear both structurally and biochemically that full-length cholix is still capable of binding NAD ϩ , although relatively poorly. Interestingly, some recent studies claim that the toxicity of ExoA results from translocation of the full-length toxin directly across the endosome and into the cytoplasm (rather than via the retrograde pathway to the endoplasmic reticulum) and that the C-terminal fragment is unstable in the cytoplasm (48), casting some doubt on the conventional model. Notably, the K D1 for cholix c is in the same range as the…”

Reductive Activation of Type 2 Ribosome-inactivating Proteins Is Promoted by Transmembrane Thioredoxin-related Protein

“…The exotoxin A produced by the bacterium Pseudomonas aeruginosa was also used. PE is formed by a single polypeptide of ϳ60 kDa containing three functional domains, an N-terminal domain containing regions able to bind cell surface structures, a C-terminal domain able to catalytically inactivate protein synthesis of the target cell, and a domain involved in transmembrane translocation (13). An intrachain disulfide bond is also present, whose reduction leads to the activation of the toxin inside the cell (possibly in the ER), and it has been reported that PDI (or members of the PDI family) could be involved in this process (14).…”